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Hemangioblasts: From Hematopoietic Stem Cells to Endothelial Progenitor Cells and Their Effector Molecules

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PAGE 1

H E M A N G I O B L A S T S : F R O M H E M A T O P O I E T I C S T E M C E L L S T O E N D O T H E L I A L P R O G E N I T O R C E L L S A N D T H E I R E F F E C T O R M O L E C U L E S B y S T E V E N M I T C H E L L G U T H R I E A D I S S E R T A T I O N P R E S E N T E D T O T H E G R A D U A T E S C H O O L O F T H E U N I V E R S I T Y O F F L O R I D A I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y U N I V E R S I T Y O F F L O R I D A 2005

PAGE 2

C opyr i ght 2005 by S t e ve n M i t c he l l G ut hr i e

PAGE 3

T hi s w or k i s de di c a t e d t o m y m ot he r B e r na de t t e G ut hr i e a nd m y f a t he r E dw i n G ut hr i e

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i v A C K N O W L E D G M E N T S I w oul d f i r s t l i ke t o t ha nk m y m e nt or D r E dw a r d S c ot t f or t he e xc e l l e nt t r a i ni ng a nd oppor t uni t i e s I r e c e i ve d dur i ng m y s t a y i n hi s l a b. I w oul d a l s o l i ke t o t ha nk a l l of m y c om m i t t e e m e m be r s D r M a r i a G r a nt D r J or g B unge r t D r B r yon P e t e r s e n, a nd D r N a ohi r o T e r a da f o r t he i r t i m e e ne r gy a nd gui da nc e I t ha nk D r C hr i s C ogl e a l s o m y good f r i e nd f o r hi s c ons t a nt s ha r i ng of i de a s a nd d i s c us s i on s i nc l udi ng but not l i m i t e d t o s c i e nc e M y de e pe s t t ha nks a l s o a r e e xt e nde d t o c ur r e nt a nd pa s t m e m be r s of t he S c ot t l a b, e s pe c i a l l y G a r y B r ow n f or ha s va s t a ni m a l kn ow l e dge a nd e xpe r t i s e ; D oug S m i t h f or hi s ve r y c a pa bl e F A C S a na l ys i s a nd c onf oc a l i m a gi ng; C hr i s C ul l e r a nd D us t i n H a r t f or m a i nt a i ni ng a n o r ga ni z e d a nd e f f i c i e nt l a b; J e n T a r ga c f or a l w a ys c l e a ni ng up a f t e r m e ; a nd J a s on B ut l e r w i t h w hom I w or ke d s i de by s i de on m a ny e xpe r i m e nt s I n a ddi t i on, I w oul d l i ke t o t ha nk J e f f H a r r i s D r R ob e r t F i s he r D r R on S a nde r s a nd e s pe c i a l l y D r B i l l S l a yt on, C hr i s B r a y, a nd G r e g M a r s ha l l f or c ons t a nt a nd e nga gi ng di s c us s i on. O ut s i de t he l a b, a nd m os t i m po r t a nt l y, I w oul d l i ke t o t ha nk m y pa r e nt s a nd m y s i s t e r A l i s a i n P e nns yl va ni a A l t hough w e w e r e s e pa r a t e d by a t hous a nd m i l e s I c oul d a l w a ys he a r t he i r e nc our a ge m e nt a nd f e e l t he i r c a r i ng. F i na l l y I w oul d l i ke t o t ha nk m y f i a nc D r C hr i s t i na C ove l l i w ho ha s be e n t hr oug h t hi c k a nd t h i n dur i ng m y s c i e nc e c a r e e r a nd ha s pr ovi de d s t r e ngt h w i s dom m ot i va t i on, a nd l ove

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v T A B L E O F C O N T E N T S pa ge A C K N O W L E D G M E N T S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i v L I S T O F F I G U R E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi i A B S T R A C T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi i i C H A P T E R 1 I N T R O D U C T I O N A N D B A C K G R O U N D I N F O R M A T I O N . . . . . . . . . . . . . . . . . 1 H e m a t opoi e s i s a nd V a s c ul oge ne s i s dur i ng E m br yo ni c D e ve l opm e nt . . . . . . . . . . . . 2 F or m a t i on o f B l ood V e s s e l s i n A dul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 R e gul a t i on of N e ova s c ul a r i z a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 S t e m C e l l T r a ns pl a nt a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 E ndot he l a i l P r oge ni t or C e l l s f or N e o va s c ul a r i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . 9 N i t r i c O xi de a s P ot e nt i a l R e gul a t or o f V a s c ul a r F o r m a t i on . . . . . . . . . . . . . . . . . . 11 R ol e of N i t r i c O xi de S ynt ha s e i n V e s s e l F or m a t i on . . . . . . . . . . . . . . . . . . . . . . . . 13 2 G E N E R A L M E T H O D S A N D M A T E R I A L S . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 G e ne r a t i ng t he G F P / B L 6 C hi m e r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 H a r ve s t i ng B one M a r r ow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 I ni t i a l P u r i f i c a t i on of H e m a t opoi e t i c S t e m C e l l s b y M a gne t i c A c t i va t e d C e l l S or t i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 F i na l P ur i f i c a t i on o f H e m a t opoi e t i c S t e m C e l l s by F l our e s c e nc e A c t i va t e d C e l l S or t i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 H a r ve s t i ng of B L 6 R e s c ue M a r r ow w i t h H e m a t op oi e t i c S t e m C e l l s D e pl e t i o n, a nd I r r a di a t i on of R e c i pi e nt A ni m a l s . . . . . . . . . . . . . . . . . . . . . 19 P ur i f i e d G r e e n F l uor e s c e nc e P r ot e i n H e m a t opoi e t i c S t e m C e l l s a nd D e pl e t e d R e s c ue M a r r ow T r a ns pl a nt a t i on a nd E ns ui ng A ni m a l H us ba ndr y C onc e r ns 20 V e r i f i c a t i on of M ul t i l i ne a ge R e c ons t i t ut i on . . . . . . . . . . . . . . . . . . . . . . . . . . 21 I nduc t i on of R e t i na l I s c he m i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 E ye R e m ova l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3 T H E H E M A T O P O I E T I C S T E M C E L L H A S H E M A N G I O B L A S T A C T I V I T Y . 25 A dul t H e m a t opoi e t i c S t e m C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 D i a be t i c R e t i nopa t hy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

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vi A ngi oge ne s i s vs N e ova s c ul a r i z a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 T he C 57B L 6. G F P C hi m e r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 A s s e s s m e nt of G r e e n F l uor e s c e nc e P r ot e i n R e t i na l B l ood V e s s e l E ndot he l i a l C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 T he H e m a t opoi e t i c S t e m C e l l s ha s H e m a ngi obl a s t F unc t i on . . . . . . . . . . . . . 38 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 4 M O D U L A T O R S O F H S C / H E M A N G I O B L A S T A C T I V I T Y . . . . . . . . . . . . . . . . 42 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 I nduc i bl e N i t r i c O xi de S ynt ha s e a nd E ndot he l i a l N i t r i c O xi de S ynt ha s e G r e e n F l uor e s c e nc e P r ot e i n C hi m e r a s D e m ons t r a t e d R obus t H e m a t opoi e t i c S t e m C e l l s E ngr a f t m e nt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 T he N i t r i c O xi de P a t hw a y A f f e c t s B l ood V e s s e l F or m a t i on . . . . . . . . . . . . . . 51 T he N i t r i c O xi de S ynt ha s e P a t hw a y A f f e c t s B l ood V e s s e l B r a nc hi ng C ha r a c t e r i s t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 N i t r i c O xi de P r oduc t i on E f f e c t on V a s c ul a t ur e i n N on oc ul a r T i s s ue . . . . . . . 57 Q ua nt i t a t i on a nd L oc a t i on of N i t r i c O xi de S ynt ha s e P r oduc e d i n K noc kout A ni m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 5 L I M I T A T I O N S O F S T E M C E L L R E S E A R C H A N D E T H I C A L C O N S I D E R A T I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 B i ol ogi c a l L i m i t a t i ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 E t hi c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 C onc e r ns O ve r S t e m C e l l U s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 6 G E N E R A L C O N C L U S I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 L I S T O F R E F E R E N C E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 B I O G R A P H I C A L S K E T C H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

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vi i L I S T O F F I G U R E S F i gur e pa ge 2 1 F l uor e s c e nc e a c t i va t e d c e l l s or t i ng ga t e s f or i s ol a t i ng H S C . . . . . . . . . . . 19 3 1 R e a na l ys i s of H S C pos t e nr i c hm e nt us e d f or t r a ns pl a nt a t i on . . . . . . . . . . 32 3 2 H S C c a n e ngr a f t m u l t i pl e l i ne a ge s l ong t e r m a nd s e l f r e ne w . . . . . . . . . . 33 3 3 H S C c a n pr oduc e a l l he m a t opoi e t i c l i ne a ge s c l ona l l y . . . . . . . . . . . . . . . 34 3 4 D onor de r i ve d H S C c ont r i but e t o e ndot he l i a l c e l l s of bl ood ve s s e l s i n t he e ye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3 5 D onor de r i ve d H S C p r oduc e f unc t i ona l e ndot he l i a l c e l l s s ur r oundi ng bl ood ve s s e l l um e ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3 6 T he H S C i s s e l f r e ne w i ng a nd c a n c l ona l l y f or m e ndot he l i a l c e l l s . . . . . . 40 4 1 N O S knoc kout a ni m a l s e xhi bi t l ong t e r m m ul t i l i ne a ge donor G F P pe r i phe r a l bl ood e ngr a f t m e nt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4 2 T he i N O S pa t hw a y m odul a t e s he m a ngi obl a s t ne ova s c ul a r i z a t i on . . . . . . 52 4 3 T he e N O S pa t hw a y m odul a t e s he m a ngi obl a s t ne o va s c ul a r i z a t i on . . . . . . 54 4 4 T he ni t r i c oxi de pa t hw a y a l t e r s he m a ngi obl a s t bl o od ve s s e l f or m e d br a nc hi ng c ha r a c t e r i s t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 4 5 C hr oni c va s c ul a r i nj ur y i n e N O S G F P c hi m e r a s i n duc e s w i de s pr e a d he m a ngi obl a s t a c t i vi t y f r om a dul t H S C . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4 6 D onor de r i ve d c e l l s l i ni ng va s c ul a r l um e ns i n e N O S G F P a ni m a l s a r e M E C A 32 pos i t i ve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4 7 N i t r i c oxi de p r oduc t i on i s dys r e gul a t e d i n e N O S k noc kout a ni m a l s . . . . . 62 5 1 P r opi di um i odi de s t a i ni ng of c i r c ul a t i ng E P C doe s not i ndi c a t e a bnor m a l pl oi dy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

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vi i i A bs t r a c t of D i s s e r t a t i on P r e s e nt e d t o t he G r a dua t e S c hool of t he U ni ve r s i t y of F l or i da i n P a r t i a l F u l f i l l m e nt o f t he R e qui r e m e nt s f or t he D e g r e e of D oc t o r of P hi l os o phy H E M A N G I O B L A S T S : F R O M H E M A T O P O I E T I C S T E M C E L L S T O E N D O T H E L I A L P R O G E N I T O R C E L L S A N D T H E I R E F F E C T O R M O L E C U L E S B y S t e ve n M i t c he l l G ut hr i e M a y 2005 C ha i r : E dw a r d S c ot t M a j or D e pa r t m e nt : M ol e c ul a r G e ne t i c s a nd M i c r obi ol ogy R e s e a r c h i n t he f i e l d o f s t e m c e l l ha s r e c e i ve d m uc h a t t e nt i on i n t he pa s t f e w ye a r s S t e m c e l l s hol d t r e m e ndous pot e nt i a l f o r t r e a t i ng m a ny de bi l i t a t i ng c ondi t i ons a nd di s e a s e s M y s t udy de s c r i be s how t he he m a t opoi e t i c s t e m c e l l i s pl a s t i c or c a pa bl e o f pr oduc i ng non he m a t opoi e t i c t i s s ue i n a ddi t i on t o a l l of t he e xpe c t e d bl ood l i ne a ge s S pe c i f i c a l l y, t he he m a t opoi e t i c s t e m c e l l i s c a pa bl e of p r oduc i ng e ndot he l i a l c e l l s of bl ood ve s s e l s I de s c r i be t h i s t hr ough a s e r i e s of e xpe r i m e nt s w he r e I t r a ns pl a nt e d a s i ngl e he m a t opoi e t i c s t e m c e l l i nt o a l e t ha l l y i r r a di a t e d r e c i pi e nt a nd r e c ons t i t ut e d a l l of t he bl ood l i ne a ge s T hi s s i ngl e c e l l w a s t he n a bl e t o pr oduc e e ndot he l i a l c e l l s unde r c ondi t i ons of i nj u r y a nd i s c he m i a i n a n a t t e m pt t o r e l i e ve t he i s c he m i c pr e s s ur e I f ound t ha t t he he m a t opoi e t i c s t e m c e l l c a n f unc t i on a s a h e m a ngi obl a s t c a pa bl e of pr odu c i ng not a l l of t he b l ood l i ne a ge s a nd a l s o bl ood ve s s e l s T hi s a c t i vi t y s ugge s t s t he pos s i bi l i t y of m odul a t i ng t hi s he m a ngi obl a s t a c t i vi t y.

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i x I de t e r m i ne d t ha t t w o ge ne s pl a y a r ol e i n bl ood pr e s s ur e m a i nt e na nc e a nd i m m une r e s pons e s i n t he N i t r i c O xi de S ynt ha s e pa t hw a y. T he s e ge ne s a r e a l s o a bl e t o m odul a t e he m a ngi obl a s t f unc t i on i n m i c e T hi s a bi l i t y t o a l t e r bl ood ve s s e l f or m a t i on w oul d be e xt r e m e l y us e f ul i n c ondi t i ons of pa t hol ogi c bl ood ve s s e l gr ow t h s uc h a s di a be t i c r e t i nopa t hy, t he l e a di ng c a us e of b l i ndne s s w or l dw i de or t um or bl ood ve s s e l gr ow t h w he r e de c r e a s i ng t he bl ood s uppl y c oul d s t a r ve t he c a nc e r c e l l s C onve r s e l y, w ound he a l i ng, a nd t he r a py f or c ondi t i ons s uc h a s s t r oke or c a r di a c i s c he m i a w oul d be ne f i t f r om i nc r e a s e d bl ood ve s s e l gr ow t h. T hi s know l e dge c a n be di r e c t l y a ppl i e d by us i ng pha r m a c ol ogi c a l a ge nt s t ha t e i t he r i nhi bi t o r up r e gul a t e t he N i t r i c O xi de S ynt ha s e ge ne s t o m odul a t e bl ood ve s s e l f or m a t i on f or t he r a pi e s u s e f ul i n hum a n pa t i e nt s

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1 C H A P T E R 1 I N T R O D U C T I O N A N D B A C K G R O U N D I N F O R M A T I O N T he di s c ove r y of t he a bi l i t y of s t e m c e l l s t o di f f e r e nt i a t e a l ong a l t e r na t i ve de ve l opm e nt a l f a t e s he r a l de d a ne w t ool f o r t he t r e a t m e nt of m a ny de bi l i t a t i ng di s e a s e s T he a bi l i t y of e xoge nous c e l l s t o hom e t o a r e a s of i nj ur y, t a ke up r e s i de nc e a nd r e pr ogr a m t he m s e l ve s t o ne w t i s s ue t ype s a l l ow s f or f unc t i ona l r e pa i r of dys f unc t i ona l t i s s ue s W hi l e i n s om e t i s s ue t ype s t hi s ha s be e n k now n t o oc c ur s uc h a s va s c ul a t ur e r e pe r f us i on i n w ound he a l i ng, t he e xa c t c e l l s c ont r i but i ng t o t he e ndot he l i a l t i s s ue w e r e i de nt i f i e d onl y r e c e nt l y. E l uc i da t i on of t he c ont r i b ut i ng c e l l t o c e r t a i n t ype s o f va s c ul a r r e pa i r v i z he m a t opoi e t i c s t e m c e l l s now a l l ow s e xpl or a t i on of t he m o l e c ul e s t ha t pl a y pa r a l l e l r ol e s i n bot h he m a t opoi e s i s a nd bl ood ve s s e l f or m a t i on I nde e d, t a i l or i ng of t he he m a t opoi e t i c s t e m c e l l s he m a ngi obl a s t a c t i vi t y c oul d i m pr ove c ur r e nt l y l i m i t e d pa l l i a t i ve c a r e f or c ondi t i ons s uc h a s di a be t i c r e t i n opa t hy or c oul d pr ovi de a n i m pr ove d t a r ge t e d a ppr oa c h f o r t um o r gr ow t h s uppr e s s i on a nd e l i m i na t i on. T he pot e nt i a l f or c l i ni c a l t he r a pi e s i s pr of ound T he uni f yi ng goa l o f m y s t udy w a s t o f ur t he r de s c r i be t he c ha r a c t e r i s t i c s of t he he m a t opoi e t i c s t e m c e l l i n r e l a t i on t o i t s pl a s t i c a bi l i t y t o p r oduc e t he e ndot he l i a l t i s s ue l i ni ng t he bl ood ve s s e l w a l l s I do t hi s i m m e r s e d i n t he c ur r e nt e nvi r onm e nt of s a ngui ne s ke pt i c i s m t ow a r ds s t e m c e l l pl a s t i c i t y hi ghl i ght i n g how t hi s w or k a ddr e s s e s t he c ont r ove r s y. I be gi n by out l i ni ng t he ba c kdr op f or c ur r e nt r e s e a r c h a nd pr ovi de a ba r om e t e r t o m e a s ur e t he c ur r e nt s t e m c e l l c l i m a t e I out l i ne t he l i m i t a t i ons t o s t e m c e l l r e s e a r c h i n r e l a t i on t o t he he m a t opoi e t i c e xpl or a t i o n a l ong w i t h t he m e t hods by w hi c h

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2 t he y w e r e a ddr e s s e d. C ha pt e r 2 de s c r i be s t he de ve l opm e nt of a nove l r obus t a nd r e pr oduc i bl e m ode l f or i nduc i ng he m a t opoi e t i c s t e m c e l l he m a ngi obl a s t a c t i vi t y t he r e by pr om ot i ng a n a l t e r na t i ve de ve l opm e nt a l f a t e a l ong t he e ndot he l i a l l i ne a ge C ha pt e r 3 unde r s c or e s how t hi s m ode l w a s a ppl i e d t o t he c r i t i que s of s t e m c e l l pl a s t i c i t y a nd how t he he m a t opoi e t i c s t e m c e l l f unc t i ons i n c ondi t i ons of i n j ur y. T he r e a r e m a ny bi ol og i c a l m ol e c ul e s t ha t c a n m odul a t e he m a t opoi e s i s a nd ne ova s c ul a r i z a t i on. I n c ha pt e r 4 I de s c r i be how ni t r i c oxi de ha s t he a bi l i t y t o p l a y a s i gni f i c a nt r ol e i n he m a t opoi e t i c s t e m c e l l de r i ve d he m a ngi obl a s t a c t i vi t y. F i na l l y i n c ha pt e r 5 I w i l l i de nt i f y s om e of t he l i m i t a t i ons of s t e m c e l l ba s e d r e s e a r c h a nd t he r a py i nc l udi ng bot h bi ol ogi c a l a nd e t hi c a l i m pl i c a t i ons H e m at op oi e s i s an d V as c u l oge n e s i s D u r i n g E m b r yon i c D e ve l op m e n t T he r a pi d gr ow t h o f t he e a r l y e m b r yo ne c e s s i t a t e s c onve r s i on f r om a m e c ha ni s m w he r e s i m pl e di f f us i on pr ovi de s t he ne c e s s a r y nut r i e nt s a nd r e m ove s m e t a bol i c bypr oduc t s f or t he e ve r i nc r e a s i ng c e l l num be r t o a m e c ha ni s m of c i r c u l a t e d t r a ns por t T he de ve l opi ng bl ood a nd va s c ul a t ur e pr ovi de t hi s c i r c ul a t i on. D u r i ng m u r i ne de ve l opm e nt he m a t opoi e s i s a nd va s c ul oge ne s i s b e gi n a s e a r l y a s D a y 7 i n t he r e gi on o f t he yol k s a c 1 2 E ndot he l i a l c e l l s a r e de r i ve d f r o m m e s ode r m a l pr e c ur s or s i n t he yol k s a c a nd be gi n t o c ons t i t ut e t he pr i m a r y va s c ul a r s ys t e m i n pa r a l l e l w i t h i ni t i a t i on o f pr e m a t ur e he m a t opoi e s i s 3 5 T h i s va s c ul oge ne s i s b e gi ns w i t h a c l us t e r of c e l l s c a l l e d bl ood i s l a nds c om pos e d of a nuc l e us c ont a i ni ng he m a t opoi e t i c s t e m c e l l s ( H S C ) s ur r ounde d by m o r e di f f e r e nt i a t e d a ngi obl a s t s t he c e l l s w hi c h w i l l f o r m bl ood ve s s e l s on t he pe r i phe r y 6 T he c l os e pr oxi m i t y of t he t w o pr e c ur s or c e l l s a nd t he de ve l opm e nt a l r e l a t i ons hi p be t w e e n t he f o r m a t i on o f bl ood a nd bl ood ve s s e l s s ugge s t a s ha r e d pa r e nt c e l l f r om w hi c h bot h a r e de r i ve d: t he he m a ngi obl a s t

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3 U nt i l t he D a y 10 of de ve l opm e nt t he yol k s a c r e m a i ns t he pr i m a r y s i t e of he m a t opoi e s i s A r ound D a y 12 t he l i ve r w hi c h t he n be c om e s t he pr i m a r y s i t e of he m a t opo i e s i s 7 H ow e ve r t he r e a r e ot he r r e gi ons of pot e nt i a l he m a t opoi e s i s i n t he pa r a a or t i c s pl a nc hnopoe ur a ( P A S ) f r om D a y 8 5 t o 10 a nd t he a or t a gona d m e s one phr ous ( A G M ) r e gi on f r om D a y 10. 5 t hr ough D a y 12 7 1 1 T he pot e nt i a l o f t he s e a r e a s w a s de t e r m i ne d t hr ough a s e r i e s of t r a ns pl a nt a t i on s t ud i e s w he r e c e l l s i s ol a t e d f r om t he s e r e gi ons a r e a bl e t o r e s c ue l e t ha l l y i r r a di a t e d r e c i pi e nt s 1 2 1 4 T hi s he m a t opoi e t i c r e s c ue c a pa bi l i t y de f i ne s t he f i r s t l oc a t i on f r om w he r e f un c t i ona l l y de f i ne d H S C a r i s e E ndot he l i a l c e l l s on t he ve nt r a l s ur f a c e of t he a or t a a r e de r i ve d f r om t he P A S / A G M r e gi ons a nd H S C a r e a l s o f ound ne s t l e d i n t he e ndot he l i a l f l oor of t he a or t a a ga i n s ugge s t i ng t he t ha t t hi s a r e a c ont a i ns c e l l s w hi c h a r e ha ve t he c a pa bi l i t i e s of t he he m a ngi obl a s t 1 2 F or m at i on of B l ood V e s s e l s i n A d u l t s V a s c ul oge ne s i s a nd a ngi oge ne s i s a r e t w o di s t i nc t r ol e s of t he he m a ngi obl a s t V a s c ul oge ne s i s i s de f i ne d a s t he de nov o ge ne r a t i o n of bl ood ve s s e l s vi a t he r e c r ui t m e nt of undi f f e r e nt i a t e d pr oge ni t o r c e l l s t o t he s i t e o f ve s s e l f or m a t i on w he r e t he y di f f e r e nt i a t e i nt o va s c ul a r e ndot he l i um 1 1 D ur i ng e m br yoni c de ve l opm e nt t he va s c ul a r s ys t e m i s f or m e d t h r ough va s c ul oge ne s i s A f t e r de ve l opm e nt i s c om pl e t e ne w bl ood ve s s e l f or m a t i on i s a t t r i but e d t o t he p r oc e s s of a ngi oge ne s i s w he r e ve s s e l s a r e f or m e d by s pr out i ng f r om t he p r e e xi s t i ng va s c ul a t ur e 1 5 U nt i l 1991, a ngi oge ne s i s w a s t hought t o oc c ur by t he p r ol i f e r a t i on of r e s i de nt e ndot he l i a l c e l l s a t t he s i t e w he r e ne w ve s s e l s a r e f or m i ng, but G e or ge e t al 1 6 s how e d t ha t e ndot he l i a l c e l l s c i r c ul a t e i n t he bl ood. T he y f ound t ha t pe r i phe r a l bl ood c ont a i ne d e ndot he l i a l c e l l s by s t a i ni ng bl ood s a m pl e s w i t h t he

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4 e ndot he l i a l c e l l s pe c i f i c a nt i body S E ndo 1 a nd a na l yz i ng t he s e c e l l s by F l uor e s c e nc e A c t i va t e d C e l l S or t i ng ( F A C S ) T he di s c ove r y of c i r c ul a t i ng e ndot he l i a l c e l l s unva r yi ngl y l e a ds us t o que s t i on w he r e t he s e c e l l s a r e de r i ve d. T he r e a r e t w o pos s i bi l i t i e s of c i r c ul a t i ng e ndot he l i a l c e l l pa r e nt a ge : t he e xi s t i ng va s c ul a t ur e w he r e c e l l s e xt r ude t he m s e l ve s f r om b l ood ve s s e l w a l l s a nd e nt e r t h e c i r c ul a t i on; t he bone m a r r ow i t s e l f vi a a n e ndot he l i a l c e l l pr oge ni t o r ( E P C ) i nt e r m e di a t e S e ve r a l s t udi e s de s c r i be e ndot he l i a l c e l l s w hi c h de r i ve d f r o m t he bone m a r r ow 1 7 2 2 I f t hi s i s t he c a s e t he H S C a nd E P C popul a t i ons c oul d pos s i bl e be di s t i ngui s he d t hr ough t he i r c e l l s ur f a c e m a r ke r e xp r e s s i on, or t hr ough t a ggi ng of t he pa r e nt c e l l N o s t udi e s ha ve ye t di r e c t l y a dd r e s s e d t hi s que s t i on; how e ve r t he r e i s s i gni f i c a nt i ndi r e c t e vi de nc e l i nki ng e ndot he l i a l c e l l s t o t he E P C a nd i t s i nvol ve m e nt i n a dul t ne ova s c ul a r i z a t i on. O ne s u c h s t udy de s c r i be d s e ve r a l c e l l s ur f a c e a nt i ge ns pr e s e nt on t he E P C s uc h a s C D 133 a nd C D 34, t ha t a r e a l s o pr e s e nt on t he H S C 2 3 H o w e ve r t he r e a r e di f f e r e nc e s i n t he t w o popul a t i ons na m e l y t ha t f e t a l l i ve r ki na s e 2 ( V E G F R 2) e xpr e s s i on i s onl y f ound on c om m i t t e d pr oge ni t or s 2 4 T hi s i s one o f t he f i r s t h i nt s t ha t E P C m a y be a m or e di f f e r e nt i a t e d o r c om m i t t e d H S C da ught e r c e l l C D 34 pos i t i ve c e l l s c a n phe not ypi c a l l y f unc t i on a s e ndot he l i a l c e l l s a f t e r s e ve r a l da ys of c ul t ur e on f i br one c t i n T he y a r e c a pa bl e of i nc or por a t i ng a c e t yl a t e d L D L pr oduc i n g ni t r i c oxi de w he n s t i m ul a t e d w i t h V E G F a nd e xpr e s s of P E C A M 1 a nd T i e 2, bot h of w hi c h a r e s pe c i f i c t o e ndot he l i a l c e l l s 2 5 C D 133 pos i t i ve c e l l s a ppe a r t o be a m o r e i m m a t ur e s ubgr oup o f t he C D 34 popul a t i on. T he C D 133 pos i t i ve c e l l s a r e a bl e t o r e popul a t e t he bone m a r r ow c om pa r t m e nt of r a di oa bl a t e d s he e p, a nd e vi de nc e s how s t ha t a s ubs e t of c e l l s w hi c h a r e C D 34, C D 133 a nd V E G F R 2 pos i t i ve m a y be E P C 2 6 2 8 C D 133 a nd C D 34 pos i t i ve c e l l s

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5 a r e be l i e ve d t o be m or e p r i m i t i ve E P C be c a us e t he y l a c k V E c a dhe r i n or V on W i l l e br a nd e xpr e s s i on. O nl y 3 % of t he s e c e l l s e xpr e s s V E G F R 2. 2 7 C D 34 ne ga t i ve C D 133 po s i t i ve a nd V E G F R 2 pos i t i ve c e l l s m a y r e pr e s e nt a m or e m a t ur e o r f ur t he r di f f e r e nt i a t e d popul a t i on of e ndot he l i a l c e l l s T he e xa c t m a r ke r s a nd phe not ype of E P C a r e not k now n, a nd t he c ondi t i ons unde r w hi c h t he s e c e l l s a r e s t i m ul a t e d t o p r ol i f e r a t e c i r c ul a t e a nd hom e t o s i t e s of i nj ur y a r e poor l y unde r s t ood. T he r e i s d i s pa r i t y i n t he a m ou nt of ne ova s c ul a r i z a t i on oc c ur i ng i n c e r t a i n va s c ul a r be ds w i t h s om e t i s s ue t ype s e xpe r i e nc i ng s i gni f i c a nt l y m or e ve s s e l f or m a t i on i n r e l a t i on t o o t he r s I n a ddi t i on, t he w i de r a nge of i s c he m i a m ode l s ut i l i z e d f or s t udy ha ve be e n f ound t o i nduc e di f f e r e nt l e ve l s of ne ova s c ul a r i z a t i on. C r os by e t al 2 9 ha ve s how n t ha t up t o 11% o f e ndot he l i a l c e l l s c ont r i but i ng t o ne ova s c ul a r i z a t i on a r e E P C de r i ve d. T hi s c ont r i but i on oc c ur r e d du r i n g i nj ur y a nd w a s not obs e r ve d unde r nor m a l phys i ol ogi c c ondi t i ons G r a nt e t a l 3 0 de m ons t r a t e d t ha t c i r c ul a t i ng e ndot he l i a l c e l l s s pe c i f i c a l l y e ndot he l i a l c e l l s w hi c h c ont r i bu t e t o t he f or m a t i on of bl ood ve s s e l s dur i ng i nj u r y r e pa i r a r i s e f r om t he H S C t hr ough a n E P C i n t e r m e di a t e T h i s f i ndi ng l e nds t o t he pos s i bi l i t y o f r e gu l a t i ng ve s s e l f or m a t i on a t a pr e c ur s or l e ve l t hr ough a m ol e c ul a r m e di a t or T he a bi l i t y t o or c he s t r a t e f or m a t i on of b l ood ve s s e l s i s hi ghl y de s i r e d f or c ondi t i ons i n w hi c h pa t hol ogi c a l va s c ul a r gr ow t h, or l a c k of gr ow t h a nd l e a ds t o da m a gi ng c ondi t i ons ul t i m a t e l y de c r e a s i ng t he qua l i t y of l i f e R e gu l at i on o f N e ovas c u l ar i z at i on V a s c ul a r e ndot he l i a l c e l l s m a i nt a i n a t i ght bor de r b e t w e e n t he c i r c ul a t i ng bl ood a nd t he out s i de t i s s ue T hi s m onol a ye r o f c e l l s a c t s a s a non a dhe r e nt s ur f a c e w he r e c i r c ul a t i ng c e l l s c a nnot i nt e r a c t a nd a dhe r e w i t hout t he pr e s e nc e of c e r t a i n s ur f a c e m a r ke r s s uc h a s t he i nt e g r i ns or s e l e c t i ns of t he c e l l ul a r a dhe s i on m ol e c ul e f a m i l y.

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6 W hi l e t hi s bounda r y m us t ne c e s s a r i l y r e m a i n i nt a c t m e c ha ni s m s e xi s t i n w hi c h c e l l s w i t hi n t he bl o od c a n e xt r a va s a t e i nt o t he s ur r oundi ng t i s s ue i n or de r t o f i ght i nf e c t i on o r pr ovi de r e pa i r C onve r s e l y, m e c ha ni s m s e xi s t by w hi c h c e l l s i n t i s s ue c a n e nt e r t he bl oods t r e a m i l l us t r a t e d by bone m a r r ow c e l l s a bi l i t y t o pr o l i f e r a t e i n t he bone m a r r ow c om pa r t m e nt m i g r a t e t o t he i nne r m a r r ow ve s s e l s a nd e nt e r t he c i r c ul a t i on E ndot he l i a l c e l l s ge ne r a l l y ha ve a ve r y l ow l e ve l o f a popt os i s a nd t hus a l ow t ur nove r r a t e C e l l s i n c e r t a i n or ga ns s uc h a s t he e ye c a n l i ve f or ye a r s w i t hout be i ng r e pl a c e d. 3 1 A s a r e s ul t t he r e a r e i n f r e que nt e ndot he l i a l c e l l s c i r c ul a t i ng i n he a l t hy a dul t s us ua l l y num be r i ng 1 3 pe r m i l l i l i t e r o f bl ood 3 2 T hi s e m pha s i z e s how t he s t e a dy s t a t e of e ndot he l i a l c e l l s i s non di vi di ng unl e s s s t i m ul a t e d by i nj u r y w he n m e c ha ni s m s t o upr e gul a t e e ndot he l i a l m i t os i s s t i m ul a t e pr ol i f e r a t i on P os i t i ve r e gul a t or s a r e g r ow t h f a c t or s f r e que nt l y d e t e c t e d i n a dul t t i s s ue s i n w hi c h t he r e i s a ppa r e nt a ngi oge ne s i s a nd i nc l ude V a s c ul a r E ndot he l i a l G r ow t h F a c t or ( V E G F ) a nd ba s i c F i br obl a s t G r ow t h F a c t or ( bF G F ) 3 3 I n v i t r o i t ha s be e n f ound t ha t V E G F a nd bF G F upr e gul a t e m a ny e ndot he l i a l c e l l f unc t i ons i nc l udi ng pr ol i f e r a t i on, m i gr a t i on e xt r a c e l l ul a r pr ot e ol yt i c a c t i vi t y, a nd t ube f or m a t i o n. 3 4 T hi s ha s l e d t o t he not i on t ha t t he s e f a c t or s a c t di r e c t l y on e ndot he l i a l c e l l s t o upr e gul a t e t he i r a c t i vi t y I nde e d, V E G F i s i nc r e a s e d i n t um or s w he n t he t r a ns f or m e d c e l l s be gi n t o r e c r ui t bl ood ve s s e l s f or gr ow t h. 3 4 C onve r s e l y, a m e t hod m us t e xi s t t ha t c a n l i m i t t he a m ount of ne ova s c ul a r i z a t i on oc c ur r i ng s o a s t o not pr oduc e pa t hol ogi c va s c ul a t ur e E ndot he l i a l qui e s c e nc e i s t hought t o be m a i nt a i ne d by t he p r e s e nc e of e ndoge nous dow nr e gul a t or s s uc h a s T um or G r ow t h F a c t or be t a ( T G F ) a nd T um or N e c r os i s F a c t or a l pha ( T N F ) 3 5 U nl i ke t o V E G F a nd b F G F a ngi oge ni c dow nr e gul a t or s m a y a c t di r e c t l y on

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7 e ndot he l i a l c e l l s or i ndi r e c t l y by i nduc i ng t he pr o duc t i on of i n f l a m m a t or y a nd ot he r non e ndot he l i a l c e l l r e gul a t or s 3 6 3 7 T G F a nd T N F A i nhi bi t e ndot he l i a l c e l l g r ow t h i n v i t r o a nd ha ve t he r e f or e be e n c ons i de r e d a s di r e c t a c t i ng ne ga t i ve r e gul a t or s 3 5 U ne xpe c t e dl y, T G F a nd T N F a r e a ngi oge ni c i n v i v o a nd i t h a s be e n de m ons t r a t e d t ha t t he s e c yt oki ne s i nduc e a ngi oge ne s i s i ndi r e c t l y by s t i m ul a t i ng t he pr oduc t i on of s t r om a l a nd c he m oa t t r a c t e d i nf l a m m a t or y c e l l pos i t i ve r e gul a t o r s 3 8 O t he r c yt oki ne s t ha t ha ve be e n r e por t e d t o r e gul a t e a ngi oge ne s i s i n v i v o i nc l ude H G F E G F / T G F P D G F B B i nt e r l e uki ns ( I L 1 I L 6, a nd I L 12 ) i nt e r f e r ons G M C S F P l G F p r ol i f e r i n, a nd pr ol i f e r i n r e l a t e d pr ot e i n 3 9 4 1 C he m oki ne s t ha t r e gul a t e a ngi oge ne s i s i n v i t r o ha ve a l s o be e n i de nt i f i e d i nc l udi ng I L 8, pl a t e l e t f a c t or I V a nd gr o. 4 1 4 3 A ngi oge ne s i s c a n a l s o be r e gul a t e d by a va r i e t y of nonc yt oki ne or nonc he m oki ne f a c t or s i nc l udi ng e nz ym e s ( a ngi og e ni n a nd P D E C G F / T P ) i nhi bi t or s o f m a t r i x de gr a di ng p r ot e ol yt i c e nz ym e s ( T I M P s ) pl a s m i noge n a c t i va t or i nhi bi t or 1 ( P A I s ) e xt r a c e l l ul a r m a t r i x c om pone nt s c oa gul a t i on f a c t or s or f r a gm e nt s ( t hr om bos pondi n, a ngi os t a t i n, hya l u r ona n, a nd i t s ol i gos a c c ha r i de s ) s ol ubl e c yt oki ne r e c e pt or s pr os t a gl a ndi ns a di poc yt e l i pi ds a nd c oppe r i ons 3 9 4 2 4 5 T hi s pl e t hor a o f c yt oki ne s de m ons t r a t e s t he c om pl e xi t y of r e gul a t i n g of t he a ngi oge ni c pr oc e s s a nd j us t i f i e s a s s e s s i ng t he i r r ol e i n s t e m a nd p r oge ni t or c e l l gove r na nc e of ne ova s c ul a r i z a t i on. T he s e pos i t i ve a nd ne ga t i ve r e gul a t or s o f t e n c oe xi s t i n t i s s ue s i n w hi c h e ndot he l i a l c e l l t ur nove r i s i nc r e a s e d. A l t hough t hi s ha s ye t t o be pr ove n i n v i v o t he c u r r e nt w o r ki ng hypot he s i s i s t ha t t he a ngi oge ni c s w i t c h of t um or s i nvol ve s e i t he r t he i nduc t i on of a pos i t i ve r e gul a t or a nd/ o r t he l os s of a ne ga t i ve r e g ul a t or

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8 S t e m C e l l T r an s p l an t at i on T he a dul t bone m a r r ow ( B M ) i s a r i c h r e s e r voi r o f t i s s ue s pe c i f i c s t e m a nd pr oge ni t or c e l l s B M c e l l s m a y be a s our c e of E P C T h e r e f or e t a ppi ng i nt o B M i n c om bi na t i on w i t h ne ova s c ul a r i z a t i on r e gul a t o r s m a y pr ovi de s i gni f i c a nt a nd m a na ge a bl e t he r a py. S t i m ul a t i on of a ngi oge ne s i s m a y be of be ne f i t i n w ound he a l i ng a nd f r a c t ur e r e pa i r T he r a pe ut i c gr ow t h w i l l a l s o be be ne f i c i a l i n t h e t r e a t m e nt of i s c he m i a a nd s ubs t a nt i a t e d by e xt e ns i ve e xpe r i m e nt a l da t a 4 6 4 9 P e s c e e t al 4 9 de m ons t r a t e d t ha t unde r i s c he m i c c ondi t i ons t r a ns pl a nt e d um bi l i c a l c o r d c e l l s ga ve r i s e t o e nha nc e d a r t e r i o l e l e ngt h a nd de ns i t y a l ong w i t h s ke l e t a l m us c l e f i be r s A not he r gr oup t r a ns pl a nt e d e a r l y bone m a r r ow c e l l s i nt o noni r r a di a t e d, a ge d m i c e a nd f ound a c ont r i but i on t o va s c ul a t ur e f r om s ubs e que nt l y t r a ns pl a nt e d ne ona t a l m yoc a r di um 4 8 I n a ddi t i on O r l i c e t al 5 0 de m ons t r a t e d t ha t bone m a r r ow c e l l s c a n di f f e r e nt i a t e i nt o m yoc yt e s a nd va s c ul a r s t r uc t ur e s T he y a l s o m obi l i z e d bo ne m a r r ow c e l l s w i t h s t e m c e l l f a c t or a nd gr a nul oc yt e c ol ony s t i m ul a t i ng f a c t or a nd f ound t ha t m a r r ow c e l l s c oul d hom e t o i nf a r c t e d r e gi ons of t he he a r t r e pl i c a t e d i f f e r e nt i a t e a nd u l t i m a t e l y p r om ot e m yoc a r di a l r e pa i r 5 1 T hi s c oul d l e a d t o s i g ni f i c a nt a l t e r a t i ons a nd i m pr ove m e nt s i n t r e a t m e nt f or c a r d i a c i s c he m i a C ur r e nt t he r a py f or m yoc a r di a l i s c he m i a r e l i e s on dr ugs t ha t r e duc e m yoc a r di a l oxyge n de m a nd, m e c ha ni c a l e ndova s c ul a r r e va s c ul a r i z a t i on pr oc e dur e s ( a ngi opl a s t y) o r bypa s s s ur ge r y. 5 2 H ow e ve r c om pe ns a t or y ne ova s c ul a r i z a t i on i s a n i m po r t a nt phys i ol ogi c a l pr oc e s s t ha t oc c ur s i n c hr oni c m yoc a r di a l i s c he m i a 5 3 I t ha s r e c e nt l y be e n de m ons t r a t e d i n e xpe r i m e nt a l m ode l s o f m yoc a r di a l i s c he m i a a nd i nf a r c t i on i n t he pi g a nd r a t t ha t V E G F a nd V E G F r e c e pt or s 1 a nd 2 a r e i nc r e a s e d i n c hr oni c a l l y i s c he m i c m yoc a r di um a nd a l s o i n r e gi ons of i s c he m i a s ur r o undi ng a n a r e a o f i n f a r c t i on. 5 4 5 6 T hos e s t udi e s de m ons t r a t e d t ha t t he V E G F l i ga nd i s up r e gul a t e d i n c a r di om yoc yt e s a nd i t s

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9 c ogna t e r e c e pt or s e xhi bi t e d i nc r e a s e d e xpr e s s i on i n e ndot he l i a l c e l l s F ur t he r s t udi e s ha ve r e ve a l e d t ha t hypoxi a i s a pot e nt i nduc e r of V E G F i n c ul t ur e d c a r di a c m yoc yt e s 5 7 C or r e s pondi ngl y, e s c a l a t e d bF G F a c t i vi t y ha s be e n s how n i n m yoc a r di um a f t e r c or ona r y a r t e r y l i ga t i on 5 8 T hi s oc c ur s i n pa r a l l e l w i t h a n i n c r e a s e i n c ol l a t e r a l bl ood f l ow i n dogs a nd e l e va t e d l e ve l s of bF G F ( but not V E G F ) ha ve be e n de t e c t e d i n t he pe r i c a r d i a l f l ui d of pa t i e nt s w i t h uns t a bl e a ngi na 5 2 T he s e obs e r va t i on s on t he m ol e c ul a r m e c ha ni s m s of phys i ol ogi c a l a ngi oge ne s i s i n i s c he m i c m yoc a r di um l e d t o t he not i on t ha t c e l l ba s e d t he r a py or pha r m a c ol ogi c a l s t i m ul a t i on o f a ngi oge ne s i s m a y a ugm e nt or e ve n r e pl a c e m or e c onve nt i ona l f o r m s of t he r a py. A s w i l l be de s c r i be d ne xt t hi s not i on ha s r e c e nt l y r e c e i ve d c ons i de r a bl e e xpe r i m e nt a l s uppor t i n a ni m a l m ode l s V a s c ul a r he a l i ng m a y be m e di a t e d i n pa r t by t he r e c r ui t m e nt of E P C I n s e ve r a l s t u di e s ge ne t i c a l l y m a r ke d bone m a r r ow de r i ve d E P C w e r e r e c r ui t e d t o t he i s c he m i c l i m bs of m i c e 1 1 1 7 I n a ddi t i on, t r a ns pl a nt a t i on of m a t ur e e ndot he l i a l c e l l s ( E C ) de r i ve d f r om i n v i t r o ge ne r a t e d, hum a n bone m a r r ow de r i v e d, m ul t i pot e nt a dul t p r oge ni t or c e l l s ha s f a c i l i t a t e d r e va s c ul a r i z a t i on of va r i ous t i s s ue s 5 9 T he phys i ol ogi c s i gni f i c a nc e of E P C s a nd E C i n ne ova s c ul a r i z a t i on w a s f ur t he r un de r s c or e d w he n t hor a c i c a or t a f r om a dul t dogs pr e vi ous l y t r a ns pl a nt e d w i t h ha pl oi de nt i c a l bone m a r r ow w e r e r e pl a c e d w i t h D a c r on gr a f t s i m pe r v i ous t o t he i ngr ow t h of e s t a bl i s he d E C I n 3 m ont h ol d g r a f t s t he ne w l y e s t a bl i s he d E C l a ye r w e r e de t e r m i ne d t o a r i s e f r om dono r de r i ve d c e l l s f r om t he bone m a r r ow 5 8 T he s e f i ndi ngs i ndi c a t e t ha t E C d e r i ve d f r om t he E P C of bone m a r r ow or i gi n c a n c ont r i but e t o ne w bl ood ve s s e l f or m a t i o n. E P C f or N e ovas c u l ar i z at i on T hi s l ow num be r of E P C i n t he c i r c ul a t i on i nc r e a s e s dr a m a t i c a l l y unde r c ondi t i ons s uc h a s a c ut e s t r e s s or i nj ur y t o va s c ul a t ur e w a l l s w he r e t he r e i s a l a r ge a popt ot i c e ve nt of

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10 E C N or m a l r e pl a c e m e nt of t he E C i s us ua l l y a c c om pl i s he d by t he s ur r ound i ng l oc a l e ndot he l i a l c e l l s w hi c h i nc r e a s e t he i r p r ol i f e r a t i on a nd m i gr a t e t o t he a r e a s of i s c he m i a T he t e r m i na l l y di f f e r e nt i a t e d E C how e ve r a r e not a bl e t o p r ol i f e r a t e c ons i de r a bl y a nd m a y not ha ve t he c a pa c i t y t o p r ovi de f o r t he de m a nd f or ne w ve s s e l s A s de s c r i be d i n num e r ous s t ui di e s r e s e a r c he r s ha ve i s ol a t e d c i r c ul a t i ng c e l l s t ha t a r e bone m a r r ow de r i ve d ye t ha ve e ndot he l i a l pot e nt i a l t he E P C T he s e E P C a r e c a pa bl e o f l e s s e ni ng t he i s c he m i c pr e s s ur e of i nj ur e d o r ga ns by r e va s c ul a r i z i ng i nj ur e d a r e a s a nd r e s t or i ng o r ga n f unc t i on. O ur c ur r e nt unde r s t a ndi ng of t he ne ova s c ul a r i z a t i on pr oc e s s i s f ounde d on t he c l a s s i c a l l i ght m i c r os c opy obs e r va t i ons m a de by C l a r k a nd C l a r k i n 1953 6 0 T he y w e r e a m ong t he f i r s t t o r e ve a l t he s e que nc e of e ve nt s l e a di ng t o t he f or m a t i on of ne w c a pi l l a r y bl ood ve s s e l s i n t he t r a ns l uc e nt t a i l s of a m phi bi a n l a r va e T he s e a nd l a t e r obs e r va t i ons i n nonde ve l opm e nt a l s e t t i ngs pr ovi de d a de t a i l e d hi s t ol ogi c a l a c c ount of ne w bl ood ve s s e l f or m a t i on. 6 1 6 2 O n t he s e pi one e r i ng r e s ul t s our c ur r e nt know l e dge w a s f ounde d. C l a r k a nd C l a r k de s c r i be d a l oc a l a ngi oge ni c s t i m ul us t h a t c a us e s e ndot he l i a l c e l l s of pr e e xi s t i ng c a pi l l a r i e s or pos t c a pi l l a r y ve nul e s t o b e c om e a c t i va t e d. A l t hough t he p r e c i s e m ol e c ul a r c ons e que nc e s of t h i s a c t i va t i on pr oc e s s r e m a i n t o be c l e a r l y de f i ne d a c t i va t e d bl ood ve s s e l s a r e va s odi l a t e d, ha ve i nc r e a s e d va s c ul a r pe r m e a bi l i t y a nd e xpe r i e nc e a c c um ul a t i on of e xt r a va s c ul a r f i br i n a s w e l l a s pr o t e ol yt i c de gr a da t i on of t he ba s e m e nt m e m br a ne of t he pa r e nt ve s s e l 4 6 4 8 T he e ndot he l i a l c e l l s t he n e xt e nd t hi n c yt opl a s m i c a r m s w h i c h di r e c t m i gr a t i on i nt o t he s ur r oundi ng m a t r i x t ow a r ds t he a ngi oge ni c s t i m ul us M i gr a t i ng e ndot he l i a l c e l l s e l onga t e a nd a l i gn w i t h one a not he r t o f o r m a c a pi l l a r y s pr out a nd e ndot he l i a l c e l l di vi s i on, w hi c h oc c ur s p r oxi m a l t o t he m i gr a t i ng t i p, f u r t he r

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11 i nc r e a s e s t he l e ngt h of t he s pr out T he s ol i d s pr out gr a dua l l y de ve l ops a l um e n p r oxi m a l t o t he r e gi on of pr ol i f e r a t i on. C ont i guous t ubul a r s pr out s f us e a t t he i r t i ps t o f o r m a f unc t i ona l c a pi l l a r y l oop i n w hi c h bl ood f l ow i s s oon e s t a bl i s he d. V e s s e l m a t ur a t i on i s a c c om pl i s he d by r e c ons t i t ut i on of t he ba s e m e nt m e m br a ne a nd r e c r ui t m e nt of m ur a l c e l l s 4 9 T he s e c e l l ul a r f unc t i ons c ont r i but e t o t he f or m a t i on of pa t e nt e ndot he l i um l i ne d bl ood ve s s e l s t r uc t ur e s N i t r i c O xi d e as P ot e n t i al R e gu l a t or of V as c u l ar F or m at i on T he pr oc e s s of a ngi oge ne s i s i n t he a dul t i s a c om pl e x s e que nc e of gr ow t h f a c t or r e l e a s e va s odi l a t i on, a nd r e c r ui t m e nt o r p r ol i f e r a t i on of e ndot he l i a l c e l l s t o bui l d t he ve s s e l s T he s e e ve nt s a r e he r a l de d by E C a c t i va t i o n, m os t not a bl y va s odi l a t i on, w hi c h f a c i l i t a t e s gr ow t h by gr a nt i ng a c c e s s f or c e l l s t o e n t e r t he a r e a a nd r e m ove a ny da m a ge d a nd de a d c e l l s / de br i s i nc r e a s e s nut r i e nt de pos i t i ng a nd br e a kdow n o f e xi s t i ng e xt r a c e l l ul a r m a t r i x a nd a l l ow s c e l l s t o e s t a bl i s h p e r m a ne nt r e s i d e nc e O ne of t he m ol e c ul e s w hi c h ha s be e n s how n t o pl a y a n e xt e ns i ve r ol e i n va s odi l a t i on i s N i t r i c O xi de ( N O ) N O ha s be e n us e d i n na t ur e f or ove r 250 m i l l i on y e a r s l onge r t ha n m a m m a l s ha ve e xi s t e d. T he hor s e s hoe c r a b us e s N O t o pr e ve nt bl ood c e l l a ggr e ga t i on, a nd t hi s f unc t i on i s s t i l l r e t a i ne d i n m a m m a l s O t he r ki ngdom a nd p hyl a a l s o ut i l i z e N O i nc l udi ng f i r e f l i e s f or t he i r f l a s he s a nd pl a nt s t ha t us e N O s c yt ot oxi c e f f e c t s t o f i ght i n f e c t i on. V i c t or i a n phys i c i a ns r e c ogni z e d i t s va s odi l a t or y e f f e c t e ve n i f t he y di d not unde r s t a nd i t s m e c ha ni s m a nd i t s m e di c i na l va l ue w a s w r i t t e n i n a S he r l oc k H ol m e s s t or y 1 3 0 T he m e di c a l us e s f or N O c ont i nue d i nt o W or l d W a r I w he r e doc t or s not i c e d t ha t f a c t or y w or ke r s i n a m m uni t i on pl a nt s ha d l ow e r bl ood pr e s s ur e s T hi s l e d di r e c t l y t o t he ni t r ogl yc e r i ne t a bl e t s t i l l us e d t oda y t o t r e a t a ngi na T he ga s m ol e c ul e i t s e l f how e ve r

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12 w a s c ons i de r e d onl y a pol l ut a nt un t i l r e c e nt l y. I n t he e a r l y 1990s t he j ou r na l Sc i e nc e na m e d i t m ol e c ul e o f t he ye a r D ur i ng t hi s t i m e ov e r 250 a r t i c l e s pe r m ont h w e r e w r i t t e n f ur t he r c ha r a c t e r i z i ng N O a nd i t s e f f e c t s R obe r t F F u r c hgot t L oui s J I gna r r o, a nd F e r i d M ur a d r e c e i ve d t he N obe l P r i z e i n M e di c i ne i n 1998 f o r t he i r w or k on ni t r i c oxi de a s a s i gna l i ng m ol e c ul e i n t he c a r di ova s c ul a r s ys t e m O ne h i s t or i c i r ony i s t ha t A l f r e d N obe l m a de hi s f or t une by m a ki ng dyna m i t e f r om ni t r ogl yc e r i ne a know n N O donor N O i s uni que a m ong phys i ol ogi c s ubs t a nc e s i n t he body a s i t i s t he onl y ga s pr oduc e d i n m a m m a l s t ha t ha s a bi ol ogi c a l e f f e c t T hi s s i ngul a r m e s s e nge r m ol e c ul e i s i nvol ve d i n t he r e gul a t i on o f di ve r s e phys i ol ogi c f unc t i ons i nc l udi ng c e nt r a l a nd pe r i phe r a l ne r ve c e l l ne ur ot r a ns m i s s i on, pr o m ot i on of t he c yt ot oxi c a c t i ons of i m m une c e l l s a nd pr e ve nt i ng/ i nc r e a s i ng l e ukoc yt e a dhe s i o n. 6 3 6 7 I t a l s o ha s pr of ound va s om ot or r e gul a t or y a f f e c t on va s c ul a r be ds s pe c i f i c a l l y t he r e gul a t i on of s m oot h m us c l e c ont r a c t i l i t y a nd t hus va s odi l a t i on 6 3 6 4 T hr e e di s t i nc t i s of o r m s of t he e nz ym e t ha t s ynt he s i z e s N O ( N O S ) ha ve be e n i de nt i f i e d, a l l of w hi c h s ha r e a 50 60% hom ol ogy. 6 7 T w o i s of o r m s a r e c ons t i t ut i ve l y a c t i ve : t he f or m e xpr e s s e d pr i m a r i l y i n ne u r ona l t i s s ue ( nN O S ) a nd t he f or m f i r s t f ound i n va s c ul a r e ndot he l i a l t i s s ue ( e N O S ) T he t h i r d f o r m s a c t i vi t y c a n be i nduc e d i n a va r i e t y of c e l l t ype s us ua l l y i n r e s pons e t o i nf l a m m a t or y s i gna l s a nd ba c t e r i a l p r oduc t s a nd ha s be e n na m e d i nduc i bl e N O S ( i N O S ) E a c h of t he t hr e e i s of o r m s r e qui r e ho m odi m e r i z a t i on f o r a c t i vi t y T he C t e r m i na l po r t i on of t he N O S p r ot e i n c l os e l y r e s e m bl e s t he c yt oc hr om e P 450 r e duc t a s e pos s e s s i ng m a ny of t he s a m e c of a c t or bi ndi ng s i t e s 6 8 T he e xt r e m e C t e r m i nus c ont a i ns a n N A P D H bi ndi ng r e gi on c ons e r ve d i n a l l t hr e e i s of or m s t ha t e xa c t l y a l i gns w i t h t he b i ndi ng r e gi on o f t he c yt oc hr om e P 450. 6 8

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13 F ol l ow i ng t hi s i s a f l a vi n a de ni ne di nuc l e ot i de a nd f l a vi n m ono nuc l e ot i de c ons e ns us s e que nc e t ha t i s s e l f s uf f i c i e nt unl i ke t he P 450 e n z ym e i n t ha t t he oxyge na t i on of i t s s ubs t r a t e L a r gi ni ne oc c ur s a t t he he m e s i t e i n t he N t e r m i na l r e gi on 6 9 N O i s ge ne r a t e d vi a a 5 e l e c t r on oxi da t i on o f a t e r m i na l gua n i di ni u m ni t r oge n on L a r gi ni ne 6 8 M os t of t he phys i ol ogi c a c t i ons of N O a r e br ought a bout by t he a c t i va t i on of s ol ubl e gua nyl a t e c yc l a s e B i ndi ng of N O t o t he h e m e m oi e t y of t he e nz ym e c a us e s a c onf or m a t i ona l c ha nge t ha t upr e gul a t e s t he a c t i vi t y ove r 400 f ol d r e s ul t i ng i n t h e f or m a t i on o f t he i nt r a c e l l ul a r s e c ond m e s s e nge r c yc l i c G M P 7 0 N O ha s num e r ous a ngi oge ni c e f f e c t s i nc l udi ng ( but not l i m i t e d t o ) i n c r e a s i ng m a t r i x m e t a l l opr ot i na s e ( M M P ) e xpr e s s i on a nd t yr os i ne phos phor yl a t i on o f pr ot e i n s i n s pr out i ng t i ps of c a pi l l a r i e s 6 5 I nhi bi t i ng N O p r oduc t i on ha s be e n s how n t o de c r e a s e c a pi l l a r y f o r m a t i on i n r a t s w i t h por t a l hype r t e ns i on. 6 6 I n a ddi t i on D N A s ynt he s i s c a n be i m pa i r e d by t he i nhi bi t or y e f f e c t of N O on r i bonuc l e ot i de r e duc t a s e w hi c h a ddr e s s e s t he c yt ot oxi c a nd c yt os t a t i c e f f e c t of N O dur i ng a n i m m une r e s pons e I n t he a que ous e nvi r onm e nt o f t he c yt os ol N O i nt e r a c t s w i t h w a t e r t o f or m t he f r e e r a di c a l pe r oxyni t r a t e 6 7 P e r oxyni t r a t e i nt e r a c t s w i t h D N A l e a di ng t o oxi da t i on a nd i ni t i a t i on of a c om pl e x s e r i e s of t r a ns f or m a t i ons i nvol vi ng ba s e da m a ge or s t r a nd b r e a ks a s w e l l a s r e a c t i ons w i t h t he de oxyr i bos e por t i on o f t he D N A 7 1 T he D N A da m a ge i t s e l f a l ong w i t h t he c e l l c yc l e a r r e s t a s r e pe a t e d a nd c os t l y D N A r e pa i r oc c ur s ul t i m a t e l y l e a ds t o a popt os i s R ol e of N O S i n ve s s e l f or m at i on T he pr oc e s s of a ngi oge ne s i s c a n be di vi de d i nt o t w o c om pone nt s : e ndot he l i a l c e l l pr ol i f e r a t i on a nd bl ood ve s s e l t ube f or m a t i on T he pot e nt a ngi oge ni c a ge nt V E G F s t i m ul a t e s N O r e l e a s e f r om e ndot he l i a l c e l l s 7 2 V E G F i nduc e d N O r e l e a s e ha s be e n s how n t o m odul a t e a ngi oge ne s i s bot h i n v i t r o a nd i n v i v o 7 3 7 4 T he a dul t m ous e m ode l w e

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14 ha ve de ve l ope d ut i l i z e s t he a ngi oge ni c i nf l ue nc e o f V E G F a s w e a r t i f i c i a l l y i nc r e a s e l oc a l e xpr e s s i on of t hi s g r ow t h f a c t or i n t he r e t i na m i m i c ki ng t he pa t hophys i ol ogy t ha t oc c ur s i n di s e a s e s a s s oc i a t e d w i t h r e t i na l ne ova s c ul a r i z a t i on s uc h a s D i a be t i c R e t i nopa t hy a nd R e t i nopa t hy of P r e m a t ur i t y T he e s t a bl i s he d r e s i de nt va s c ul a r e ndot he l i a l c e l l s t he e ndot he l i a l c e l l s f ound i n t he c i r c ul a t i on, a nd t hos e de r i ve d f r om H S C a l l r e s pond t o V E G F a nd i nf l ue nc e l oc a l N O c onc e nt r a t i on. N O i s c r uc i a l f or t he m yr i a d of phys i ol ogi c a l va s c ul a r f unc t i ons a nd i t s i na ppr opr i a t e p r oduc t i on a nd r e l e a s e ha s be e n l i nke d t o s e ve r a l pa t hol ogi e s 7 5 C ons e que nt l y, a ge nt s w hi c h m odu l a t e N O a c t i vi t y c oul d f i nd be ne f i c i a l us e i n a t he r a pe ut i c s e t t i ng. A s ha s be e n s how n, N O pl a ys a n i nt e gr a l r o l e i n bl ood ve s s e l f or m a t i on a nd c ons e que nt l y m a ke s a good s t a r t i ng c a ndi da t e f or m a ni pul a t i ng he m a ngi obl a s t f unc t i on T he t w o i s of o r m s w hi c h ha ve a di r e c t i n f l ue nc e ov e r e ndot he l i a l c e l l s a r e t he i N O S a nd e N O S i s of or m s a s nN O S i s f ound on l y i n ne ur ona l t i s s ue 6 7 T he r ol e o f e N O S i n a ngi oge ne s i s i s c om pl e x. B r ooks e t al ha ve de m ons t r a t e d t ha t e N O S de f i c i e nc y, e i t he r t hr ough ge ne di s r upt i on or t hr ough pha r m a c ol ogi c a l i nhi bi t i on, s i gni f i c a nt l y pr ot e c t s t he de ve l opi ng r e t i na f r om oxyge n i nduc e d r e t i nopa t h y. 7 6 T he f a c t t ha t nons pe c i f i c i nhi bi t or s of N O S a c t i vi t y pr o duc e d qua nt i t a t i ve l y s i m i l a r l e ve l s of va s o obl i t e r a t i on c om pa r e d t o e N O S ge ne d i s r upt i on a l s o s ugge s t s t ha t e N O S m a y be a n i s of or m i nvol ve d i n bl ood ve s s e l r e gul a t i on. E vi de nc e s ugge s t s t ha t N O a nd V E G F a r e r e c i pr oc a l l y r e gul a t e d s uc h t ha t s t i m ul a t i on o f V E G F R 2 a c t i va t e s e N O S l e a di ng t o N O f or m a t i on 7 6 N O i nhi bi t s V E G F pr oduc t i on i n a dj a c e nt c e l l s by a pa r a c r i ne f e e dba c k m e c ha ni s m i nvol vi ng i nhi bi t i on of A P 1 bi ndi ng t o t he V E G F pr om ot e r 7 7

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15 i N O S ha s c ons e ns us s e qu e nc e s i n i t s pr om ot e r f or t he t r a ns c r i pt i on f a c t or s hypoxi a i nduc i bl e f a c t or ( H I F ) a nd N F ka ppa B b ot h of w hi c h a r e a c t i va t e d unde r c ondi t i ons of i s c he m i a 7 8 C o ns e que nt l y, i N O S i s t hought t o be i nduc e d unde r c ondi t i ons of i s c he m i a S e nnl a ub e t al pe r f us e d r e t i na s o f w i l d t ype a nd i N O S knoc kout ( i N O S / ) m i c e e xpos e d t o hypoxi c c ondi t i ons T he y f ound t ha t i N O S / a ni m a l s ha d no r m a l i nt r a r e t i na l va s c ul a t ur e pa t t e r ni ng w he r e a s w i l d t y pe a ni m a l s ha d pe r s i s t e nt a va s c ul a r a r e a s 7 9 I nt e r e s t i ngl y, t he r e w a s a r e duc t i on i n pr e r e t i na l ne ova s c ul a r i z a t i on i n i N O S / m i c e i ndi c a t i ng a dua l r ol e o f i N O S i n di s t i nc t r e t i na l a ye r s T he y c or r obor a t e d t he s e obs e r va t i ons w i t h p ha r m a c ol ogi c a l i nhi bi t i on of i N O S w hi c h i nc r e a s e d r e t i na l ne ova s c ul a r i z a t i on a nd de c r e a s e d pr e r e t i na l ne ova s c ul a r i z a t i on. T he y f ound t ha t pa t hol ogi c a l i nt r a r e t i na l ne ova s c ul a r i z a t i on w a s m or e s e ve r e i n i N O S e xpr e s s i ng a ni m a l s 8 0 T he s e s t udi e s s ugge s t t ha t N O c a n be a n i m por t a nt m odul a t or of a ngi oge ne s i s i n t he r e t i na a nd t ha t l oc a l l e ve l s of N O c a n i nf l ue nc e t he l oc a t i on a nd de g r e e of ne ova s c ul a r i z a t i on. T o our know l e dge our m ode l i s t he onl y one w hi c h a l l ow s f or t he s i m ul t a ne ous e xa m i na t i on of pr e r e t i na l a nd i nt r a r e t i na l ne ova s c ul a r i z a t i on a t t he s a m e t i m e i n a n a dul t a ni m a l W e w i l l us e t h i s m ode l t o unde r s t a nd t he r e qui r e m e nt of be ne f i c i a l i nt r a r e t i na l ne ova s c ul a r i z a t i on c om pa r e d t o pa t hol ogi c a l pr e r e t i na l ne ova s c ul a r i z a t i on a l l ow i ng f or t he di s s e c t i on of N O a nd ot he r m ol e c ul e s w hi c h a f f e c t va s c ul a r gr ow t h.

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16 C H A P T E R 2 G E N E R A L M E T H O D S A N D M A T E R I A L S T he m e t hods de t a i l e d be l ow a r e us e d e xt e ns i ve l y i n e a c h c ha pt e r A ny m odi f i c a t i ons m a de t o t hi s f r a m e w or k dur i ng a n e xpe r i m e nt a r e not e d i n t he s pe c i f i c c ha pt e r M e t hods w i l l be de s c r i be d i n t hi s ba s i c o ut l i ne : ( 1) t he ge ne r a t i on of t he G F P / B L 6 c hi m e r a ( 2 ) t he i nduc t i on o f t he r e t i na l ne ova s c ul a r i z a t i on, ( 3) t he e nuc l e a t i on of t he e ye f or m ount i ng, ( 4 ) e xa m i na t i on of ne ova s c ul a r i z a t i on vi a c onf oc a l m i c r os c opy a nd ( 5) i m m unohi s t oc he m i s t r y s t a i ni ng o f s e r i a l s e c t i ons G e n e r at i n g T h e G F P / B L 6 C h i m e r a T he ge ne r a t i on o f t he c hi m e r i c G F P / B L 6 a ni m a l w i l l be de s c r i be d be l ow T hi s i nc l ude s t he ha r ve s t i ng of bone m a r r ow f r o m t he G F P donor a ni m a l t he pur i f i c a t i on a nd pr e pa r a t i on of t he m a r r ow f or F A C S s or t i ng o f H S C t he pr e p a r a t i on of t he C 57B L 6 r e s c ue m a r r ow a nd r e c i pi e nt a ni m a l s a nd t he H S C t r a ns pl a nt a nd c om m e ns ur a t e a ni m a l hus ba ndr y c onc e r ns H ar ve s t i n g B on e M ar r ow T he ge ne r a t i on o f t he G F P / B L 6 c hi m e r a a ni m a l s r e qui r e s e xt e ns i ve a ni m a l us e a nd c e l l m a ni pul a t i on. T he t r a n s ge ni c m ous e us e d a s t he donor s t r a i n w a s obt a i ne d f r o m A ndr a s N a gy a t m ount S a na i i n T or ont o C a na da 8 1 T he s t r a i n c a r r i e s gr e e n f l uor e s c e nt pr ot e i n ( G F P ) d r i ve n by c hi c ke n be t a a c t i n p r om ot e r a nd C M V i nt e r m e di a t e e a r l y e nha nc e r a nd i s ubi qui t ous l y e xpr e s s e d T he B L 6 f e m a l e s w e r e obt a i ne d f r o m J a c ks on L a bor a t or i e s ( B a r H a r bor M a i ne ) a nd w e r e a t l e a s t 5 w e e ks ol d a t t he t i m e of bone m a r r ow t r a ns pl a nt a t i on. R e c e nt c ont r ove r s y c onc e r n i ng t he e ve nt s dur i ng s t e m c e l l

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17 t r a ns di f f e r e nt i a t i on f or r e pa i r ha s l e d t o t he pos s i bi l i t y t ha t t h i s m a y not be a n i nhe r e nt a bi l i t y s t e m c e l l s but r a t he r a f us i on e ve nt oc c ur r i ng be t w e e n t he s t e m c e l l a nd t a r ge t t i s s ue T he t r a ns pl a nt a t i on of m a l e H S C i n t o f e m a l e r e c i pi e nt s di r e c t l y a dd r e s s e s t hi s i s s ue by a l l ow i ng f or f l uo r e s c e nt i n s i t u hybr i di z a t i on of t i s s ue s a m pl e s l ooki ng f or t he Y c hr om os om e a nd de t e r m i na t i on i f a f us i on e ve nt h a s oc c ur r e d. A f t e r f ul l y g r ow n G F P m a l e s a r e e ut ha ni z e d a nd s a c r i f i c e d t he l ong bone s i n t he l e gs w e r e i m m e di a t e l y r e m ove d. A l l m us c l e t e ndon, a nd l i ga t ur e w a s di s s e c t e d f r om t he bone w hi c h w a s i m m e di a t e l y pl a c e d i n i c e c ol d P B S E a c h bone e nd w a s t he n pr une d ba c k a bout 1 2 m i l l i m e t e r s t o e xpos e t he hol l ow c or e of t he m a r r o w s pa c e T he bone m a r r ow w a s f l us he d out i nt o a t i s s ue c ul t ur e t r e a t e d pl a t e by i ns e r t i ng a 26 ga uge ne e dl e i nt o one e nd of t he bone a nd w a s hi ng 1 2 m i l l i l i t e r s of D ul be c c o s M odi f i e d E a gl e s M e di um ( G i bc o) t hr ough t he ho l l ow bone c o r e T he c e l l s w e r e ke pt on i c e a t a l l t i m e s T he l i be r a t e d m a r r ow w a s t he n t r i t ur a t e d w i t h a 26 ga uge ne e dl e t o br e a k up t he c e l l c l um ps a nd a l l ow e d t o a dhe r e t o a t i s s ue c ul t ur e t r e a t e d pl a t e ( G i bc o) f or 120 m i nut e s T hi s s t e p a l l ow s f or a n i ni t i a l e n r i c hm e nt of H S C f r om ot he r a dhe r e nt pr oge ni t o r c e l l s s uc h a s m e s e nc hym a l s t e m c e l l s ( M S C ) s i nc e he m a t opoi e t i c pr oge ni t or a nd s t r om a l c e l l s a dhe r e t o t he t i s s ue c ul t ur e t r e a t e d pl a s t i c w hi l e H S C w i l l r e m a i n s us pe nde d i n t he m e di a T he c om pl e t e vol um e of m e di a c ont a i ni ng t he nona dhe r e nt H S C w a s t he n ge nt l y dr a w n up w a s he d i n > 10m L vol um e of c ol d m e di a a nd pe l l e t e d by c e nt r i f uga t i on a t 1000 x g pe r f or m e d a t 4 de gr e e s C e l s i us T he c e l l s w e r e r e s us pe nde d a nd s t a i ne d a s out l i ne d by t he pr ot oc ol o f t he M i l t e ny M A C S s ys t e m i n t he f o l l ow i ng s e c t i on. I n i t i al P u r i f i c at i on of H S C b y M A C S I ni t i a l H S C pu r i f i c a t i on w a s done t hr ough s or t i ng of t he c e l l s by m a gne t i c be a ds us i ng t he M i l t e ny M a gne t i c A c t i va t e d C e l l S or t i ng ( M A C S ) s ys t e m B r i e f l y c e l l s w e r e

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18 s t a i ne d w i t h a n a nt i body c onj uga t e d t o a m a gne t i c be a d. T he a nt i body, a nd s ubs e que nt l y t he be a d, i s bound t o t he c e l l W he n t he s e c e l l s a r e t he n r un ove r a c ol um n i n t he pr e s e nc e of a m a gne t i c f i e l d, t hos e c e l l s w hi c h ha ve t he s pe c i f i c s ur f a c e a nt i ge ns a nd t hus t he a nt i body be a d bound t o t he m w i l l a dhe r e t o t he c ol um n ( t e r m e d pos i t i ve f r a c t i on) C e l l s w hi c h do not pr e s e nt t ha t s ur f a c e m a r ke r ( ne ga t i ve f r a c t i on) w i l l pa s s di r e c t l y t hr ough t he m a gne t i c f i e l d a nd be r e m ove d f r om t he pos i t i ve f r a c t i on o f c e l l s T he m a gne t i c f i e l d c a n t h e n be r e m ove d a nd t he p os i t i ve f r a c t i on c ol l e c t e d f r om t he c ol um n. T o be gi n t he M A C S e nr i c hm e nt c e l l num be r a nd vi a bi l i t y w e r e de t e r m i ne d f r om t he t ot a l m a r r ow f l us he d f r om t he l ong bone s t o e n s ur e t ha t t he c or r e c t a m ount o f a nt i body, be a ds a nd s t a i ni n g vol um e w i l l be us e d. T o de t e r m i ne t he c e l l num be r I r e s us pe nde d t he w a s he d c e l l s i n t r ypa n bl ue a nd c o unt e d br i ght c e l l s us i ng a he m a c yt om e t e r unde r a pha s e c ont r a s t m i c r os c ope T he e num e r a t e d c e l l s w e r e t he n w a s he d i n > 10m L c ol d P B S a nd s t a i ne d w i t h S c a 1 m i c r obe a ds ( M i l t e ny ) i n a ppr op r i a t e vol um e T he c e l l s w e r e r un ove r 2 s e pa r a t e c ol um n s t o i ns ur e e nr i c hm e nt a nd t he f l ow t hr ough w a s di s c a r de d a nd t he pos i t i ve f r a c t i on r e t a i ne d. A t t hi s t i m e a > 90% S c a 1 pos i t i ve pur i t y t yp i c a l l y ha s be e n a c hi e ve d A f t e r e nr i c hm e nt c e l l s w e r e i m m e di a t e l y pe l l e t e d a nd pl a c e d ba c k on i c e f or f l uo r e s c e nt a nt i body s t a i ni ng f or F A C S s or t i ng F i n al P u r i f i c a t i on o f H S C b y F A C S A ga i n a l l a nt i body c onc e nt r a t i ons a nd i nc uba t i on t i m e s w e r e f ol l ow e d a c c or di ng t o t he pa r a m e t e r s de s c r i be d by t he m a nuf a c t ur e r gui d e l i ne s F or H S C pu r i f i c a t i on I us e d t hr e e di f f e r e nt f l uo r oc hr om e s : C K I T c onj uga t e d t o A P C bi ot yn yl a t e d S c a 1 ( w i t h S t r e pt a vi di n P ha r R e d s e c onda r y a nt i body ) a nd t he l i ne a ge m a r ke r s B 220, C D 3, C D 4, C D 8, C D 11B G R 1, a nd T E R 119 a l l di r e c t l y c on j uga t e d t o P E ( P ha r m i nge n) T he

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19 F A C S va nt a ge S E i s a bl e t o i s ol a t e s i ngl e c e l l s ba s e d on t he s ur f a c e a nt i ge n bound by a nt i bodi e s a nd he nc e t he s pe c t r um of a bs or ba nc e a nd f l uor e s c e nc e e m i t t e d by t ha t c e l l T w o r ounds o f pur i f i c a t i on a r e ne e de d t o e ns ur e c o m pl e t e r e m ova l o f a l l non H S C c e l l s S e e F i gur e 2 1 f o r o f a n e xa m pl e of t he ga t e s us e d t o e nr i c h a nd i s ol a t e s i ngl e H S C F i gur e 2 1 F l uor e s c e nc e a c t i va t e d c e l l s or t i ng ga t e s f or i s ol a t i ng H S C H S C w e r e r e m ove d f r om bone m a r r ow e nr i c he d by M A C S a nd s t a i ne d f or S K L s ur f a c e e xpr e s s i on. F i r s t pa ne l : F o r w a r d a nd S i de S c a t t e r of M A C S e nr i c he d c e l l s w i t h ga t e R 1 d r a w n. S e c ond pa ne l : C e l l s a r e e nr i c he d f or G F P a nd L i ne a ge pos i t i ve c e l l s ( B 220, C D 3, C D 4, C D 11b, G r 1, T e r 119) a r e de pl e t e d e xc l udi ng ga t e R 2. T hi r d pa ne l : S c a 1 a nd c ki t pos i t i ve c e l l s f r om ga t e R 1 a nd R 2 a r e e nr i c he d i n ga t e R 3. C e l l s a r e t he n f u r t he r e nr i c he d by ga t e R 4 ba s e d on t he s a m e pa r a m e t e r s P a ne l 4 : R e a na l ys i s of c e l l s ba s e d on S c a 1 a nd c ki t e xpr e s s i on. T he s e d oubl y s or t e d e nr i c he d c e l l s w e r e us e d f or t r a ns pl a nt a t i on. T he f l ow r a t e i s s e t a t 10, 000 e ve nt s pe r s e c ond w i t h no gr e a t e r t ha n a 10% a bo r t pr opor t i on. T he c e l l s w e r e t he n c ol l e c t e d i n m e di a i m m e di a t e l y a f t e r c om pl e t i on of t he s or t i s ol a t e d, a nd i nj e c t e d i nt o t he r e c i pi e nt a ni m a l s f ol l ow i ng r e s c ue m a r r ow i s ol a t i on a nd r e c i pi e nt pr e pa r a t i on ke pt on i c e a t a l l t i m e s H ar ve s t i n g of B L 6 R e s c u e M ar r ow w i t h H S C D e p l e t i on an d I r r ad i at i o n of R e c i p i e n t A n i m al s T he ha r ve s t i ng of non G F P f e m a l e B L 6 m a r r ow w a s pe r f or m e d i n t he s a m e m a nne r a s t he H S C e xc e pt t he s e c e l l s w e r e not gi ve n t i m e t o a dhe r e t o t he t i s s ue c ul t ur e t r e a t e d pl a t e O nc e t he m a r r ow w a s f l us he d, w a s he d, a nd c ount e d, a S c a 1 de pl e t i on w a s done t o r e m ove a ny H S C f r om t he r e s c ue m a r r ow w hi c h w oul d c om pe t e w i t h t he donor

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20 G F P H S C T hi s r e s c ue dos e i s a dm i ni s t e r e d f o r t w of ol d r e a s ons T he i m m une s ys t e m of t he i r r a di a t e d a ni m a l w i l l e xpe r i e nc e a n i nt e r r upt i o n a nd of t e n t he a ni m a l w i l l be c om e a ne m i c U nt i l t he H S C c a n e ngr a f t a nd r e pop ul a t e he m a t opoi e s i s t he s e s hor t t e r m r e s c ue pr oge ni t or s w i l l he l p t he a ni m a l m ount a n i m m une r e s pons e a nd pr ovi de t he ne c e s s a r y bl ood pr oduc t s a s ne e de d. A ga i n c e l l s w e r e s t a i ne d a s de s c r i be d i n t he M A C S m a gne t i c be a d s e c t i on, but t hi s t i m e t he c e l l s w e r e S c a 1 de p l e t e d t hr e e t i m e s t o e ns ur e t ha t t he r e s c ue m a r r ow w a s de voi d of H S C R e c i pi e nt B L 6 m i c e w e r e f i na l l y i r r a di a t e d w i t h 950 R A D S of ga m m a r a di a t i on t o pr e pa r e t he bone m a r r ow f o r t r a ns pl a nt a t i on. P u r i f i e d G F P H S C an d D e p l e t e d R e s c u e M ar r ow T r an s p l an t at i on an d E n s u i n g A n i m al H u s b an d r y C on c e r n s T he H S C de pl e t e d r e s c ue m a r r ow w a s c ount a s a b ove a nd 1 x 10 6 c e l l s i n a 100 m i c r ol i t e r vo l um e w e r e a l i quot e d i nt o a f r e s h E ppe ndor f t ube T he h i ghl y e nr i c he d H S C w e r e t he n s i ngl y i s ol a t e d i n t he f o l l ow i ng m a nne r A vol um e o f t he s or t e d s a m pl e w a s pl a c e d on a gl a s s dr op s l i de a nd e xa m i ne d unde r a pha s e c ont r a s t m i c r os c ope T he c e l l s w e r e di l ut e d t o a c onc e nt r a t i on w he r e s i ngl e c e l l s c a n be vi s ua l i z e d, i s ol a t e d, a nd c a pt ur e d one a t a t i m e w i t h a m i c r op i pe t t e U nde r t he s c ope a s i ngl e r ound b r i ght vi a bl e c e l l w a s i s ol a t e d a nd dr a w n up i nt o a pul l e d gl a s s m i c r opi pe t t e by m out h pi pe t t i ng w i t h a s uc t i on t ube T he ne e dl e w a s e xa m i ne d t o v i s ua l i z e t he c e l l t o e ns ur e t ha t onl y one c e l l w a s dr a w n. T he c e l l w a s t he n pl a c e i n t o t he 100 m i c r ol i t e r a l i quot c ont a i ni ng t he H S C de pl e t e d r e s c ue dos e T he r e s c ue / s i ngl e H S C m i xt ur e w a s dr a w n i nt o a f r e s h i ns ul i n ne e dl e a nd s yr i nge t o e ns ur e no c ont a m i na t i on of o t he r s a m pl e s F i na l l y a n a na e s t he t i z e d, i r r a di a t e d B L 6 a n i m a l w a s i nj e c t e d i n t he r e t r o or bi t a l s i nus c a vi t y. T he a ni m a l s w e r e m oni t or e d un t i l t he y ove r c om e t he e f f e c t s of t he a ne s t he t i c a nd t he n be

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21 pl a c e d on a r e gi m e of a nt i b i ot i c s f or t he ne xt m ont h unt i l m u l t i l i ne a ge e ngr a f t m e nt ha d be e n ve r i f i e d. V e r i f i c at i on of M u l t i l i n e age R e c on s t i t u t i on T he r e c i pi e nt a ni m a l s w e r e gi ve n one m ont h f or t h e H S C t o hom e t o t he bone m a r r ow ni c he a nd be gi n t o di vi de t o pr oduc e p r og e ni t or c e l l s w hi c h w i l l c ont r i but e t o t he va r i ous he m a t opoi e t i c c e l l l i ne a ge s D e t e r m i na t i o n of e ng r a f t m e nt w a s r e s ol ve d by pe r i phe r a l bl ood s a m pl i ng a nd F A C S a na l ys i s t o d e t e r m i ne w he t he r t he m a r r ow w a s r e popul a t e d or i f t he a ni m a l s na t i ve m a r r ow r e c ove r e d. E a c h a ni m a l ha d a pe r i phe r a l bl ood s a m pl e dr a w n t h r ough a t a i l ve i n b l e e d a nd t he bl ood w a s c ol l e c t i n a t ube c ont a i ni ng P B S a nd 5m M E D T A t o a c t a s a n a nt i c oa gul a nt T he e r yt h r oc yt e s w e r e r e m ove d w i t h a F I C O L L P L A Q U E ( A m e r s ha m B i os c i e nc e s ) pur i f i c a t i on B r i e f l y, t he bl ood/ P B S s a m pl e w a s l a ye r e d on t op of t w o t i m e s gr e a t e r vol um e of F I C O L L T he e m ul s i on w a s c e nt r i f uge d a nd t he buf f y l a ye r c o nt a i ni ng t he nuc l e a t e d c e l l s a t t he i nt e r f a c e w a s r e m ove d. T he l ym phoc yt e l a ye r c on t a i ni ng t he nuc l e a t e d c e l l s w a s w a s he d i n 5X vol um e s of P B S a nd s t a i ne d w i t h t he va r i ous l i ne a ge m a r ke r a nt i bodi e s c onj uga t e d t o P E S a m pl e s w e r e a na l yz e d by F A C S c a l i be r a nd a ni m a l s e xhi bi t i ng G F P pos i t i ve c e l l s of t he va r i ous l i ne a ge s w e r e s c or e d pos i t i ve f or e ngr a f t m e nt T he pos i t i ve a ni m a l s w e r e t he n m oni t o r e d a n a ddi t i ona l t hr e e m on t hs w he r e m ul t i l i ne a ge r e c ons t i t ut i on i s r e c onf i r m e d t o de m ons t r a t e l ong t e r m e ng r a f t m e nt by H S C E xoge nous gr ow t h f a c t or w a s t he n a dm i ni s t e r e d a s de s c r i be d be l ow I n d u c t i on of R e t i n al I s c h e m i a T he ne xt s t e p i nvol ve s a dm i ni s t r a t i on of a n e ndoge nous gr ow t h f a c t or a nd ve s s e l da m a ge i n or de r t o p r om ot e bl ood ve s s e l gr ow t h i n t he r e t i na F ul l y a nd r obus t l y e ngr a f t e d a ni m a l s w e r e s e l e c t e d a nd a na e s t he t i z e d. V E G F w a s a dm i ni s t e r e d di r e c t l y i nt o

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22 t he vi t r e ous us i ng a 36 ga uge ne e dl e a nd H a m i l t on s yr i nge E i t he r pu r i f i e d ( 40ug/ kg) V E G F pr ot e i n ( S i gm a ) o r ( 2 x 10 8 pa r t i c l e s ) A A V V E G F ( V e c t or C or e U F ) w he r e C M V pr om ot e r d r i ve s e xpr e s s i on of V E G F i n a n A de no A s s oc i a t e d V e c t or w a s us e d. V E G F i s a n e ndot he l i a l c e l l s pe c i f i c m i t oge n w hi c h i s t r a ns c r i pt i ona l l y r e gul a t e d by t he c yt om e ga l ovi r us pr om ot e r / e nha nc e r w he n pa c ka ge d i n A A V A A V m e di a t e s l ong t e r m e xpr e s s i on i n nondi vi di ng c e l l s w hi c h a l l ow s f o r s t a bl e e xpr e s s i on a nd c ons t a nt a m ount s of V E G F t o r e a c h t he a r e a o f i s c he m i a t o pr om ot e ne ova s c ul a r i z a t i on. 3 0 T he s t ud y of c l i ni c a l di s e a s e s s uc h a s D i a be t i c R e t i nopa t hy a nd R e t i nopa t hy of P r e m a t ur i t y ha s l e d t o a n unde r s t a ndi ng of t he pa t h ol ogy w hi c h oc c ur s i n t he s e di s e a s e s I n t he s e c ondi t i ons t he e ye de t e c t s a l a c k of oxyg e n, e i t he r due t o t he di a be t i c c ondi t i on l e a di ng t o l e a ky ve s s e l s or t he r e m ova l of a pr e m a t ur e l y bor n ba by f r om a n i nc uba t or s oxyge n r i c h e nvi r onm e nt T he m ode l t a ke s a dva nt a ge of t hi s ne ova s c ul a r i z a t i on by c r e a t i ng a l oc a l r e gi on of i s c he m i a i n t he e ye t hr ou gh c a ut e r i z i ng of l a r ge bl oo d ve s s e l s w i t h a l a s e r A s a r e s ul t t he c e l l s s i gna l ne w bl oo d ve s s e l gr ow t h i n t he r e gi on i n a n a t t e m pt t o r e l i e ve t he i s c he m i c pr e s s ur e P e a k e xpr e s s i on of V E G F by A A V ha s be e n de t e r m i ne d t o be a t 3 6 w e e ks t he r e f or e t he phys i c a l di s r upt i on of t he b l ood ve s s e l s i s done dur i ng t hi s t i m e ( unpubl i s he d da t a ) F i r s t m i c e w e r e a na e s t he t i z e d nor m a l l y w i t h a ge ne r a l a ne s t he t i c a nd c onc ur r e nt l y a 10% s odi um f l uor e s c e i n ( A ko r n) s ol ut i on w a s a dm i ni s t e r e d i nt r a pe r i t i ne a l l y. T hi s dye l a be l s bl ood ve s s e l s f a c i l i t a t i ng v i s ua l i z a t i on dur i ng phot oc oa gul a t i on. T he e ye s w e r e di l a t e d w i t h 1 % a t r opi ne ( A kor n ) f or 5 m i nut e s w a s he d w i t h P B S ( G i bc o) a nd s ubs e que nt l y di l a t e d w i t h 2 5% phe nyl e phr i n ( A kor n) f or 5 m i nut e s I m m e di a t e l y a f t e r t he t w o 5 m i nut e t r e a t m e nt s t he m i c e und e r w e nt l a s e r

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23 t r e a t m e nt A n A r gon G r e e n l a s e r s ys t e m ( H G M C or por a t i on) w a s us e d f or r e t i na l ve s s e l phot oc oa gul a t i on w i t h t he a i d of a 78 d i opt e r l e ns T he bl ue gr e e n a r gon l a s e r ( w a ve l e ngt h 488 514 nm ) w a s a ppl i e d t o va r i ous v e nous s i t e s j uxt a pos e d t he op t i c ne r ve T he ve nous oc c l us i on w e r e a c c om pl i s he d w i t h > 6 0 bur ns of 1 s e c dur a t i on 50 m i l l i m e t e r s pot s i z e a nd 50 100 m i l l i w a t t i nt e ns i t y. A ga i n t h e a ni m a l s w e r e a l l ow e d t o r e c ove r f or 30 da ys w hi l e t he t r a ns pl a nt e d H S C di r e c t e d by t h e i s c he m i a a nd i n duc e d by t he V E G F c ont r i but e d t o t he ne ova s c ul a r i z a t i on i n o r de r t o r e l i e ve t he hypoxi a p r oduc e d by t he c a ut e r i z i ng of t he e xi s t i ng ve s s e l s E ye R e m oval O ne m ont h a f t e r i s c he m i c i nj ur y t he e ye s w e r e r e a dy t o be e nuc l e a t e d a nd ne ova s c ul a r i z a t i on i m a ge d by c onf oc a l m i c r os c op y. M i c e w e r e f i r s t a ne s t he t i z e d a nd t he n pe r f us e d w hi l e s e da t e d. P e r i phe r a l bl ood a nd bone m a r r ow w a s c ol l e c t e d t o c onf i r m donor c ont r i but i on a na l ys i s by F A C S w i t h l i ne a ge s pe c i f i c a nt i bodi e s c onj uga t e d t o P E ( B D B i oS c i e nc e s ) s i m i l a r l y t o t he pr oc e dur e out l i n e d a bove F i r s t t he c he s t c a vi t y w a s ope ne d a nd t he r i bs c ut a w a y t o e xpos e t he he a r t c om pl e t e l y. T he l e f t a t r i a w a s punc t ur e d w i t h a 26 ga uge ne e dl e a nd i nj e c t e d w i t h > 3 m L of 50 m g/ m L t e t r a m e t hyl r hoda m i ne i s ot hi oc ya na t e ( T R I T C ) c onj uga t e d de xt r a n ( 160 000 a vg. M W S i gm a C he m i c a l ) i n phos pha t e buf f e r e d f or m a l de hyde p H 7. 4. T he pe r f us i on w a s pe r f or m e d s l ow l y i nt o t he l e f t ve nt r i c l e a nd i s i n t e gr a l f o r t he f unc t i ona l a s s a y. I m m e di a t e l y a f t e r w a r ds t he e ye s w e r e r e m ove d by s l i di n g a c ur ve d f or c e ps unde r ne a t h t he e ye ba l l a nd pul l i ng t he gl obe out T he e ye w a s punc t ur e d w i t h a 26 ga uge ne e dl e t o a l l ow c om pl e t e pe r f us i on. T he e ye w a s pl a c e d i n f r e s h 4% P F A a nd s ha ke n a t r oom t e m pe r a t ur e f or 30 m i nut e s T he gl obe w a s t he n t r a ns f e r r e d t o 1X P B S a nd w a s he d by s ha ki ng a t r oom t e m pe r a t ur e f or 30 m i nut e s t o ove r n i ght A f t e r w a s hi ng w i t h P B S t he e ye s w e r e

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24 di s s e c t e d. T o do t hi s I pl a c e d t he e ye unde r a s ur g i c a l m i c r os c ope a nd m a de a n i n i t i a l i nc i s i on i n t he c or ne a T he ope ni ng w a s e nl a r ge d unt i l i t c oul d a c c om m oda t e t he l e ns o f t he e ye T he l e ns w a s ge nt l y pus he d f or w a r d unt i l i t e xi t e d t h r ough t he ho l e c ut i n t he c or ne a T he r e m a i ni ng c or ne a w a s t he n t r i m m e d t o w he r e t he s c l e r a a nd c or ne a m e e t T he r e t i na w a s di s s e c t e d a w a y f r om t he r e t i na pi g m e nt e pi t he l i a l ( R P E ) T o do t hi s I ge nt l y pus he d dow n on t he pos t e r i or por t i on of t he R P E a nd r ol l e d t he f o r c e ps f or w a r d T he r e t i na t he n de t a c he d a nd w a s r e a di l y m ount e d. T he t hi c kne s s of t he r e t i na ( > 200um ) pr e ve nt s a de qua t e pe r f us i on of a n t i body, t he r e f or e t he r e t i na w a s pl a c e d on a gl a s s s l i de a nd 5 6 c ut s w e r e m a de a r ound t he pe r i phe r y s o t h a t t he r e t i na l i e s f l a t w he n m ount e d. T he t i s s ue w a s pl a c e d i n V e c t a s hi e l d m ount i ng m e di um ( V e c t or L a bor a t or i e s ) t o i nhi bi t phot o bl e a c hi ng. T he r e t i na s w e r e i m m e di a t e l y i m a ge d. I us e d a n O l ym pus I X 70, w i t h i nve r t e d s t a ge a t t a c he d t o t he B i o R a d C onf oc a l 1 024 E S s ys t e m f or f l uor e s c e nc e m i c r os c opy. A K r ypt on A r gon l a s e r w i t h e m i s s i on de t e c t or w a ve l e ngt hs of 598n m a nd 522nm di f f e r e nt i a t e d t he r e d a n d g r e e n f l uo r e s c e nc e T he l e ns e s us e d i n our s ys t e m w e r e t he ( O l ym pus ) 10X / 0 4 U pl a n A po, 20X / 0. 4 L C P l a n A po, 40X / 0 85 U pl a n A po, 60X / 1. 40 oi l P l a n A po a nd 100X / 1 35 oi l U pl a n A po. T he s of t w a r e w a s O S / 2 L a s e r S ha r p.

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25 C H A P T E R 3 T H E H E M A T O P O I E T I C S T E M C E L L H A S H E M A N G I O B L A S T A C T I V I T Y D ur i ng de ve l opm e nt t he r e a r e s e ve r a l t ype s of s t e m c e l l s br oa dl y c l a s s i f i e d ba s e d on t he i r a bi l i t y f o r f or m s pe c i f i c t i s s ue t ype s A f t e r f e r t i l i z a t i on du r i ng t he f i r s t f e w da ys of di vi s i on, t he e m br yon i c c e l l s a r e de s c r i be d a s t o t i pot e nt T he y ha ve t he c a pa c i t y t o pr oduc e a l l t he c e l l s t i s s ue s a nd or ga ns t ha t m a ke up t he body a l ong w i t h a l l of t he e xt r a e m br yoni c t i s s ue of t he t r ophe c t ode r m A f t e r t he f i r s t f ou r t o f i ve c e l l di vi s i ons t he e m br yo f or m s a hol l ow s phe r e c a l l e d t he bl os t oc ys t T he b l a s t oc ys t c ont a i ns a popul a t i on of c e l l s l oc a t e d i n t he i nne r w a l l w hi c h a r e c a pa bl e of p r oduc i ng e a c h of t he ove r t w o hundr e d di f f e r e nt c e l l t ype s of a n or ga ni s m T he s e di f f e r f r om t he t ot i pot e nt c e l l s i n t ha t no one of t he m c a n p r oduc e a n e nt i r e or ga ni s m no r c a n t he y p r oduc e t he c e l l s o f t he t r ophe c t ode r m F i na l l y a f t e r bi r t h a nd i nt o a dul t h ood, s e ve r a l t ype s of t i s s ue s ha ve c e l l s r e s i di ng w i t hi n t he m w hi c h a r e a bl e t o p r oduc e t he t i s s ue t ype w he r e t he y r e s i de T hi s c a n oc c ur c ons t a nt l y, s uc h a s t he he m a t opoi e t i c s t e m c e l l pr oduc i ng a l l o f t he bl ood c e l l s or onl y i n t i m e s o f s t r e s s or i nj ur y s uc h a s t he ova l c e l l s pr oduc i ng he pa t oc yt e s T he s e s t e m c e l l s a r e c a l l e d m ul t i pot e nt a nd i n m os t c a s e s unde r nor m a l c ondi t i ons t he s e c e l l s a r e t hought t o pr oduc e onl y one c e l l t ype I n t he a dul t s t e m c e l l s a r e be l i e ve d t o de f i ne uns p e c i a l i z e d c e l l s t ha t c a n s e l f r e ne w ( or p r ol i f e r a t e ) f o r e xt e nde d pe r i ods o f t i m e w i t ho ut di f f e r e nt i a t i ng. T hi s pr oc e s s i s not w e l l unde r s t ood, but i s be l i e ve d t o i nvol ve a s ym m e t r i c c e l l di vi s i on w he r e a c opy o f i t s e l f i s pr oduc e d a l ong w i t h a f ur t he r di f f e r e nt i a t e d da ught e r c e l l T he s e s t e m c e l l s e xhi bi t a s t a bl e no r m a l c hr o m os om e c om pl e m e nt a nd c a nnot pe r f or m a ny s pe c i a l i z e d

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26 f unc t i ons H ow e ve r t he y do ha ve t he pot e nt i a l t o gi ve r i s e t o c e l l s w i t h s pe c i a l i z e d f unc t i ons a p r oc e s s know n a s di f f e r e nt i a t i on I t i s s ugge s t e d t ha t s om e of t he s e c e l l s m a y be a bl e t o di f f e r e nt i a t e i nt o m ul t i pl e non r e l a t e d c e l l t ype s a c ha r a c t e r i s t i c c a l l e d pl a s t i c i t y. A d u l t H e m at op oi e t i c S t e m C e l l s A dul t he m a t opoi e t i c s t e m c e l l s a r e de f i ne d by t he i r a bi l i t y t o bot h s e l f r e ne w a nd pr ovi de a l l o f t he he m a t opoi e t i c c e l l s ne c e s s a r y t o r e pl a c e t hos e l os t e a c h da y. T he bone m a r r ow pr oduc e s a n e s t i m a t e d 2 3 m i l l i on c e l l s pe r s e c ond or ove r 200 bi l l i on pe r da y T he t r e m e ndous pr o l i f e r a t i ve po t e nt i a l of t he s e c e l l s w oul d qui c kl y be e xha us t e d t hr oughout a l i f e t i m e i f t he r e w e r e not s om e s e l f r e ne w i ng pa r e nt c a l l t o m a i nt a i n he m a t opoi e t i c a nd l ym p h s ys t e m pr oge ni t o r c e l l s T hi s pr ol i f e r a t i ve a nd s e l f r e ne w i ng c a pa c i t y m a ke H S C e xc e l l e nt c l i ni c a l t ool s f or t he t r e a t m e nt of he m a t ol ogi c a l m a l i gna nc i e s s uc h a s l e uke m i a s a nd l ym phom a s I n t he s e c ondi t i ons t he bone m a r r ow popul a t i on, m os t no t a bl y t he H S C i s r e pl a c e d by c e l l s w hi c h a r e non m a l i gna nt a nd he a l t hy t o r e c ons t i t ut e no r m a l he m a t opoi e s i s of a n i ndi vi dua l I n r e s e a r c h, our a bi l i t y t o e nr i c h f or H S C c oupl e d w i t h t he i r e a s y t r a ns pl a nt a bi l i t y ope ns up l a r ge r e a l m s o f e xpl or a t i on. S i m i l a r l y t o o t he r m ul t i pot e nt s t e m c e l l s H S C a nd be l i e ve d t o r e t a i n a s i gni f i c a nt a bi l i t y t o t r a ns di f f e r e nt i a t e T he s e t w o c ha r a c t e r i s t i c s m a ke t he H S C i de a l f o r i de nt i f yi ng t he pot e nt i a l of H S C t o r e ge ne r a t e o r c ont r i but e t o non he m a t opoi e t i c t i s s ue s f ol l ow i ng i nj u r y or s t r e s s T hi s da t a ha s yi e l de d a l a r ge a m ount of i ni t i a l e xc i t e m e nt how e ve r t he r e ha s s i nc e be e n a c ool i ng i n t he e nt h us i a s m due t o t he i nc r e a s e d, t hough w a r r a nt e d, s c r ut i ny I n o r de r f or c e l l ba s e d t he r a p y t o ha ve c l i ni c a l a ppl i c a t i ons ba s i c c r i t e r i a a nd s t a nda r d m us t be e s t a bl i s he d t o de t e r m i ne i f t he phe nom e non r e s e a r c he r s a r e c ha r a c t e r i z i ng i s t r ue H S C pl a s t i c i t y a nd c a nnot be a t t r i but e d t o a r t i f a c t A s a r e s ul t

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27 s e ve r a l s t r i nge nt c r i t e r i a ha ve be e n out l i ne d w hi c h m us t be f ul f i l l e d i n or de r t o d e m ons t r a t e t r ue pl a s t i c i t y. T he c r i t e r i a de m ons t r a t i ng H S C p l a s t i c i t y i s t hr e e f ol d. F i r s t t he c e l l m us t be c a pa bl e of s e l f r e ne w i ng a nd hom i ng t o t he bone m a r r ow t he r e by r e c ons t i t ut i ng he m a t opoi e s i s f or t he l i f e t i m e of t he o r ga ni s m T h i s i s ne c e s s a r y s o t ha t s hor t t e r m pr oge ni t or s a r e not us e d a s t he r a py w hi c h m a y s l o w l y di e of f a s pr oge ni t o r s di f f e r e nt i a t e a nd a r e not r e pl a c e d. L ong t e r m r e popul a t i ng s e l f r e ne w i ng c e l l s m us t be t r a ns pl a nt e d s o t ha t t he t he r a py w oul d not f a i l a nd t he di s e a s e or p a t ho l ogi c c ondi t i on r e e m e r ge S e c ondl y, t he bone m a r r ow c ont a i ns a m yr i a d of c e l l t ype s r a ngi ng f r om t hos e a l ong a ny poi nt of he m a t opoi e t i c de ve l opm e nt t o t he s uppor t i ng c e l l s of t he s t r om a D ur i ng a bone m a r r ow t r a ns pl a nt a num be r of t he s e c e l l s c oul d b e t r a ns pl a nt e d w i t h t he bol us c ont a i ni ng t he e nr i c he d H S C no m a t t e r s t r i nge nt t h e pur i f i c a t i on pa r a m e t e r s T he s e c ont a m i na t i ng c e l l s c oul d c ont r i but e t o t he t i s s ue t ype w he r e t he donor de r i ve d t a gge d c e l l s a r e f ound c onf oundi ng r e s ul t s I n or de r t o c o nc l u s i ve l y de m ons t r a t e t he pl a s t i c i t y of t he H S C c l ona l s t udi e s m us t be done T hr ough c l ona l t r a ns pl a nt s a s i ngl e c e l l m us t be s how n t o be a bl e t o pr oduc e t he bl ood a l ong w i t h t he non he m a t opoi e t i c t i s s ue T he s e e xpe r i m e nt s e xc l ude t he pos s i bi l i t y of s e ve r a l di f f e r e nt c e l l s a c c om pl i s hi ng di f f e r e nt r ol e s a nd t i s s ue w hi c h a r i s e s f r o m t he donor m us t ne c e s s a r i l y be f r o m t he s i ngl e c e l l F i na l l y, f or t he s e c e l l ba s e d t he r a pi e s t o be p r a c t i c a l i t m us t be de m ons t r a t e d t ha t t he pl a s t i c i t y m e a s ur e d i s r obus t a nd f unc t i ona l t r a ns di f f e r e nt i a t i on i nt o t he non he m a t opoi e t i c t i s s ue M a ny c e l l s e s pe c i a l l y t hos e of t he i m m une s ys t e m a r e c a pa bl e of a s s um i ng t he ge ne r a l m or phol ogy or e ve n s ur f a c e m a r ke r e xpr e s s i on of c e l l s t he y a r e ne a r by e i t he r due t o s t i m ul a t i on or m a c r opha ge e n gul f m e nt I t m us t be de m ons t r a t e d t ha t

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28 t he c e l l s a r e phys i ol ogi c a l l y pe r f or m i ng t he r o l e of t he t i s s ue t he y a r e r e pl a c i ng, i e c e l l s t ha t a r e r e s i di ng i n t he pa nc r e a s ha vi ng t he m or ph ol ogy a nd c ha r a c t e r i s t i c s of be t a c e l l s m us t a c t ua l l y pr oduc e i ns ul i n t o be t he r a pe ut i c I n a ddi t i on, a f e w i s ol a t e d c e l l s c a pa bl e of pr oduc i ng i ns ul i n w i l l not r e s c ue a pe r s on f r o m di a be t e s t he r e f or e t he t r a ns di f f e r e nt i a t i on o r pl a s t i c i t y m us t be r obus t pr o duc i ng a phys i ol ogi c a l l y r e l e va nt a m ount of t i s s ue O nl y w he n t he s e t hr e e s t r i nge nt c r i t e r i a ha ve be e n m e t c a n t he c e l l be c l a s s i f i e d a s pl a s t i c T o da t e t he r e ha s be e n r e l a t i v e l y f e w e xa m pl e s f ul f i l l i ng a l l t hr e e a l t hough t hos e t ha t ha ve p r e s e nt s om e e xc i t i ng pot e nt i a l O ne of t he i ni t i a l s t udi e s ha ve s how n t ha t a f t e r l on g t e r m s t a bl e he m a t opoi e t i c r e c ons t i t ut i on by a s i ngl e bone m a r r ow H S C dono r de r i ve d c e l l s c oul d be f ound i n m ul t i pl e t i s s ue s i nc l udi ng t he br a i n s ke l e t a l a nd c a r di a c m us c l e l i ve r a nd e ndot he l i a l c e l l s 8 2 T hi s e l e ga nt w o r k us e d a hom i ng a s s a y t o i s ol a t e H S C s w hi c h pr e s e nt e d s t e m c e l l s pe c i f i c s ur f a c e m a r ke r s a nd t he n w e r e a bl e t o s uc c e s s f ul l y hom e t o t he bone m a r r ow ni c he T he s e hom e d c e l l s w e r e t he n i s ol a t e d a nd s i ngl e c e l l s w e r e t r a ns pl a nt e d i nt o l e t ha l l y i r r a di a t e d r e c i pi e nt s W hi l e t hi s w o r k w a s of not e t he r e w a s a s s i gni f i c a nt l y l ow l e ve l of c ont r i but i on t o t he va r i ous t i s s ue s a nd t he r e w a s no f unc t i ona l a s s a y of t he donor de r i ve d c e l l s I t doe s how e ve r s ugge s t t he e xc i t i n g p os s i bi l i t y of r e ge ne r a t i on o f va r i ous da m a ge d t i s s ue s by H S C de r i ve d pr oge ni t or s T w o not a bl e s t udi e s a l s o de m ons t r a t e d t he pl a s t i c i t y of t he H S C i n l i ve r t o r e pl a c e he pa t oc yt e s i nj ur e d c he m i c a l l y. 8 3 8 4 E xc i t i ng l y, t he s e c e l l s w e r e a bl e t o r e s t or e l i ve r f unc t i on, how e ve r c l ona l a s s a ys w e r e not done i n t he s e t r a ns pl a nt s t udi e s I n a ddi t i on, O r l i c e t al de m ons t r a t e d t he f unc t i ona l r e c ove r y o f c a r di a c m us c l e t hr ough H S C t r a ns pl a nt a t i on. 5 0 A f t e r t he s e i ni t i a l pi one e r i ng pa pe r s a f l ood of w or k w a s e m ba r ke d upon, h ow e ve r s i nc e t he n t he t i de w a s s t e m m e d due t o t he

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29 di f f i c ul t y o f m e e t i ng a l l t h r e e c r i t e r i a 8 5 G r a nt e t a l ha s de ve l ope d a m ode l m i m i c ki ng di a be t i c r e t i nopa t hy, a nd us i ng t hi s m ode l w e ha ve be e n a bl e t o e xpa nd t he unde r s t a ndi ng of H S C w hi l e f ul f i l l i ng t he t hr e e pl a s t i c i t y c r i t e r i a 3 0 D i ab e t i c R e t i n op a t h y D i a be t i c r e t i nopa t hy i s t he l e a di ng s our c e of l e ga l bl i ndne s s a m ong w or ki ng a ge A m e r i c a ns I t i s c a u s e d by da m a ge t o t he s m a l l bl ood ve s s e l s i n t he r e t i na a s a r e s ul t of di a be t e s m e l l i t us I t i s e s t i m a t e d t ha t ove r f our t e e n m i l l i on pe opl e i n t he U ni t e d S t a t e s ha ve di a be t e s w i t h a ppr oxi m a t e l y ha l f of t he s e i ndi vi dua l s not ye t di a gnos e d a nd una w a r e of t he c ondi t i on N i ne t y pe r c e nt of pa t i e nt s w i t h d i a be t e s ha ve noni ns ul i n de pe nde nt di a be t e s m e l l i t us ( N I D D M ) a nd c ont r ol t he i r bl ood s uga r w i t h o r a l m e di c a t i ons or di e t a l one T he ot he r t e n pe r c e nt ha ve i ns ul i n de pe nde nt di a be t e s m e l l i t us ( I D D M ) a nd m us t us e i ns ul i n i nj e c t i ons da i l y t o r e gul a t e t he i r bl ood s uga r l e ve l s A l t hough di a be t i c r e t i nopa t hy i s f r e que nt l y s e e n i n bot h t ype s of di a b e t e s pa t i e nt s w i t h I D D M a r e a t gr e a t e r r i s k f or D i a be t i c R e t i nopa t hy c om pl i c a t i ons T he r i s k i nc r e a s e s ove r t i m e f or a l l pa t i e nt s w i t h di a be t e s A f t e r f i ve ye a r s a ppr oxi m a t e l y one qua r t e r o f pa t i e nt s w i t h I D D M ha ve r e t i nopa t hy a nd by f i f t e e n ye a r s ne a r l y e ve r yone w i t h I D D M e xpe r i e nc e s r e t i na l da m a ge D i a be t i c s a s a gr oup ha ve t w e nt y f i ve t i m e s t he us ua l r i s k of bl i ndne s s T he e nt i r e va s c ul a t ur e o f a di a be t i c i ndi vi dua l e xpe r i e nc e s t he pa t hol ogi c c ha nge s i nc l udi ng pl a que f o r m a t i on a nd s w e l l i ng of t he e nd ot he l i a l c e l l s T he s e ve s s e l s ha ve a di m i ni s he d c a pa c i t y t o c a r r y bl ood, a nd c ons e que nt l y a l l dow ns t r e a m t i s s ue be c o m e s i s c he m i c T hi s i s c he m i a c a us e s c ha nge s i n e xi s t i ng va s c ul a t ur e by s t i m ul a t i ng c om pe ns a t or y gr ow t h. T h i s pa t hol ogi c gr ow t h i s u ns t a bl e a nd t he ve s s e l s a r e f r a gi l e A s a r e s ul t t he i r r upt ur e c a n c a us e l e a ka ge of bl ood i nt o t he vi t r e ous a nd c ons e que nt l y vi s i on l os s

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30 O nc e pa t hol ogi c r e t i nopa t hy ha s de ve l ope d, l a s e r p hot oc oa gul a t i on i s c ur r e nt l y t he m a i ns t a y of t r e a t m e nt L a s e r s ur ge r y ha s be e n us e d i n t he t r e a t m e nt o f di a be t i c r e t i nopa t hy f o r m or e t ha n t w e nt y ye a r s a nd i t s be n e f i t ha s be e n c l e a r l y e s t a b l i s he d. T he a bnor m a l ne ova s c ul a r ve s s e l s of pr ol i f e r a t i ve di a b e t i c r e t i nopa t hy a r e t r e a t e d w i t h pa nr e t i na l l a s e r phot oc oa gul a t i on ( P R P ) T h i s t ype of l a s e r i nvol ve s t r e a t m e nt t o t he pe r i phe r a l r e t i na w hi c h i s not r e c e i vi ng a de qua t e b l ood f l ow due t o t he v e s s e l pa t hol ogy. B y phot oc oa gul a t i ng t he i s c he m i c r e gi ons t he s t i m ul us t ha t dr i ve s t he ne ova s c ul a r pr oc e s s m a y be ha l t e d. T hi s t ype of l a s e r t r e a t m e n t i s f r e que nt l y s uc c e s s f ul i n s t oppi ng t he gr ow t h o f t he a bnor m a l ve s s e l s but i n s om e c a s e s t he y m a y r e g r e s s I t i s not w i t hout s i de e f f e c t s a s s om e l os s of pe r i phe r a l a nd c ol or vi s i on i s nor m a l f ol l ow i ng t h i s t ype of t r e a t m e nt I r on i c a l l y i t i s t he e xi s t i ng P R P l a s e r t r e a t m e nt i n hum a ns f r om w hi c h w e de ve l ope d our m ous e ne ova s c ul a r i z a t i on m ode l de s c r i be d i n c ha pt e r 2 a nd us e d t hr oughout t hi s body of w or k. A n gi oge n e s i s vs N e ovas c u l ar i z at i on O ur di a be t i c m ode l i s a n e xa m pl e o f ne ova s c ul a r i z a t i on. D ur i ng ne ova s c ul a r i z a t i on, de nov o bl ood ve s s e l s a r e f or m e d w hi c h a r e not de r i ve d f r om pr e e xi s t i ng va s c ul a t ur e T he c e l l s w hi c h c ont r i but e t o ne ova s c ul a r i z a t i on a r e de r i ve d f r om a di s t a nt s our c e na m e l y t he H S C r e s i di ng i n t he bone m a r r ow C ont r a s t i ngl y, a ngi oge ne s i s i s t he pr oc e s s of e ndot he l i a l c e l l s pr o ut i ng f r o m pr e e xi s t i ng va s c ul a t ur e 1 5 L oc a l e ndot he l i a l c e l l s e ve n w i t h t he i r di m i n i s he d c a pa c i t y t o di vi de a r e a bl e t o p r oduc e e nough da ught e r c e l l s t o s uppl y bl ood ve s s e l l i ni n g i e no r m a l e ndot he l i a l c e l l s t ur nove r i s r e pl a c e d by ne i ghbor i ng c e l l s U nde r c ondi t i ons of s e ve r e i nj u r y o r i n s om e pa t hol ogi c c ondi t i on s uc h a s di a be t i c r e t i nopa t hy, t he s e ve s s e l s a r e de r i ve d f r om t he E P C I n v i t r o s t udi e s ha ve s how n t ha t E P C a r e c a pa bl e of p r odu c i ng t ube l i ke s t r uc t u r e s unde r c ul t ur e

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31 c ondi t i ons a nd c a n be de r i ve d f r om bone m a r r ow c e l l s 1 8 8 6 8 7 P r o a ngi oge ni c f a c t or s s uc h a s V E G F a nd G M C S F i nc r e a s e t he num be r of c i r c ul a t i ng E P C i n t he a dul t a nd ha ve be e n s how n t o pr om ot e bl ood ve s s e l gr ow t h 8 8 8 9 I n a ddi t i on hydr oxym e t hl ygl ut a r yl C oA r e duc t a s e i nhi bi t or s a r e e f f i c i e nt s t i m ul a t or s of E P C t r a ns di f f e r e nt i a t i on a nd f or m a t i on of e ndot he l i a l c e l l s i nvol vi ng t he A kt pr o t e i n ki na s e pa t hw a y. 9 0 I n v i v o s e ve r a l g r oups ha ve s how n t ha t E P C c ont r i but e t o bl ood ve s s e l s i n a dul t or ga ni s m s t o r e l i e ve c a r di a c i s c he m i a how e v e r t he s e m ode l s us e d s hor t t e r m pr oge ni t or c e l l s i n a n a c ut e i nj u r y m ode l 2 9 9 1 9 2 W hi l e c l e a r l y t he E P C c a n f unc t i ona l l y pr ovi de t he r a py f or i s c he m i c i nj ur y, t he s e s t udi e s di d not de m ons t r a t e w he t he r t he s e E P C w e r e de r i ve d f r om t he H S C or f r o m s om e ot he r c e l l s uc h a s t he m e s e nc hym a l s t e m c e l l D ur i ng de ve l opm e nt t he pl u r i pot e nt p r oge ni t or s w hi c h c ont r i but e t o t he f or m a t i on of bot h b l ood a nd b l ood ve s s e l s a r e t he he m a ngi ob l a s t s 9 3 9 6 T he he m a ngi obl a s t phe not ype c a n a l s o be de r i ve d i n v i t r o f r om e m b r y oni c s t e m c e l l s w he n c ul t ur e d w i t h V E G F 9 3 T he p r e s e nc e of a n a dul t he m a ngi obl a s t i n v i v o a nd t he r ol e bone m a r r ow de r i ve d c e l l s pl a y i n ne ova s c ul a r i z a t i on, how e ve r i s i nc om pl e t e T he w o r k de s c r i be d he r e w i l l e l uc i da t e t he r ol e H S C de r i ve d c e l l s ha ve i n pr o m ot i ng o r c ont r i but i ng t o ne ova s c ul a r i z a t i on a nd de s c r i be t he pl a s t i c na t ur e of t he s e c e l l s i n i s c he m i c t i s s ue R e s u l t s T he m e t hods us e d t o obt a i n t he f ol l ow i ng r e s ul t s a r e de s c r i be d i n de t a i n i n c ha pt e r t w o. A ny a l t e r a t i ons o r a ddi t i ons o f t he m ode l de s c r i be d w i l l be not e d. T h e C 57B L 6. G F P C h i m e r a A s de s c r i be d a bove t he r e a r e t hr e e s t r i nge nt c r i t e r i a f or t he de m ons t r a t i on o f H S C pl a s t i c i t y. B r i e f l y, t he c r i t e r i a a r e 1 ) t he c e l l m us t be s e l f r e ne w i ng a nd a bl e t o p r ovi de a l l of t he bl ood a nd bl ood p r oduc t s f or t he e nt i r e l i f e or t he or ga ni s m 2) t he c e l l m us t be

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32 a bl e t o do s o c l ona l l y a nd 3) t he c e l l m us t pr oduc t f unc t i ona l non he m a t opoi e t i c t i s s ue i n a r obus t m a nne r T he C 57B L 6 G F P c hi m e r a s t udi e s w i l l di r e c t l y a dd r e s s t he s e t hr e e c r i t e r i a T o a dd r e s s que s t i on one H S C w e r e i s ol a t e d f r om a donor G F P a ni m a l a s de s c r i be d. F i gu r e 3 1 i s a n e xa m pl e o f t he e nr i c he d H S C T he r ow o f pa ne l s w a s obt a i ne d f r om a w hol e bone m a r r ow pr e pa r a t i on p ur i f i e d w i t h a F I C O L L gr a di e nt A va s t m a j or i t y of c e l l s a r e l i ne a ge po s i t i ve ( > 80% ) a nd S c a 1 ne ga t i ve ( > 93% ) i ndi c a t i ng t ha t t he bul k of t he c e l l ul a r m a s s i n t he m a r r ow i s pr oge ni t or c e l l s O nc e t he c e l l s ha ve be e n e nr i c he d f or H S C w i t h M A C S a nd F A C S a h i gh pr opor t i on of c e l l s ha ve t he e xpe c t e d s ur f a c e m a r ke r phe not ype o f t he H S C ( > 98% S c a 1 pos i t i ve a nd > 99% l i ne a ge ne ga t i ve ) F i gur e 3 1 R e a na l ys i s of H S C pos t e nr i c hm e nt us e d f or t r a ns pl a nt a t i on. H S C w e r e f l us he d f r om t he bone m a r r ow e nr i c he d by M A C S s t a i ne d f o r t he S K L s ur f a c e m a r ke r s a nd e n r i c he d by F A C S P a ne l 1: S c a 1 e xpr e s s i on of e nr i c he d H S C a c hi e vi ng 98% pur i t y P a ne l 2: C e l l s e xpr e s s i ng a ny of t he l i ne a ge m a r ke r s w e r e de pl e t e d t o a 99% pur i t y. P a ne l 3: 99% of t he e nr i c he d c e l l s e xpr e s s t he pa n he m a t opoi e t i c m a r k e r C D 45. T he s e c e l l s w e r e t he n t r a ns pl a nt e d i nt o a l e t ha l l y i r r a di a t e d r e c i pi e nt a nd a l l ow e d t o l ong t e r m e ngr a f t f or t hr e e m ont hs O nc e l ong t e r m m ul t i l i ne a ge e ngr a f t m e nt w a s de m ons t r a t e d i n t he pe r i phe r a l bl ood of t he pr i m a r y r e c i pi e nt t he a ni m a l w a s s a c r i f i c e d a nd t he G F P H S C i s ol a t e d f r o m t he m a r r ow T he s e c e l l s w e r e onc e a ga i n t r a ns pl a nt e d i nt o s e c onda r y l e t ha l l y i r r a di a t e d r e c i pi e nt s a nd a l l ow e d t o e ngr a f t f or f ou r m ont hs T hi s

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33 c om bi ne d t ot a l r e p r e s e nt s m uc h l onge r t ha n a ny s hor t t e r m pr oge ni t or w oul d be a bl e t o pr ovi de he m a t opoi e s i s F i gur e 3 2 de pi c t s a r e pr e s e nt a t i ve F A C S a na l ys i s of t he pe r i phe r a l bl ood of a s e r i a l l y t r a ns pl a nt e d m ous e w i t h donor G F P H S C S i gni f i c a nt pr opor t i ons of t he T c e l l ( C D 4) B c e l l ( B 220) a nd m yl om onoc yt i c ( C D 11b) l i ne a ge s a r e donor de r i ve d ( s e e m e t hods c ha p t e r f o r de s c r i pt i on of G F P s t a nda r di z a t i on) T hi s c ont r i but i on c oul d onl y be f r om a l ong t e r m r e pop ul a t i ng, a nd t hus s e l f r e ne w i ng H S C F i gur e 3 2 H S C c a n e ngr a f t m u l t i pl e l i ne a ge s l ong t e r m a nd s e l f r e ne w E n r i c he d H S C w e r e t r a ns pl a nt e d i nt o a pr i m a r y r e c i pi e nt a n d he m a t opoi e t i c r e c ons t i t ut i on w a s de m ons t r a t e d l ong t e r m H S C w e r e t he n i s ol a t e d f r o m t he pr i m a r y r e c i pi e nt s a nd t r a ns pl a nt e d i nt o l e t ha l l y i r r a di a t e d s e c onda r y r e c i pi e nt s P e r i phe r a l bl ood w a s i s ol a t e d f r om s e c onda r y r e c i pi e nt s a nd s t a i n e d f or va r i ous he m a t opoi e t i c l i ne a ge s P a ne l 1: C D 4 ( T c e l l ) l i ne a ge s w e r e donor de r i ve d P a ne l 2: B 220 ( B c e l l ) l i ne a g e s w e r e donor de r i ve d P a ne l 3: C D 11b ( M yl om onoc yt i c ) l i ne a ge s w e r e d onor de r i ve d. T he s e c ond c r i t e r i a a dd r e s s e s t he c l ona l i t y of t he H S C i n i t s a bi l i t y t o pr oduc e a l l t he bl ood l i ne a ge s f r o m onc e s i ngl e c e l l T he s e e x pe r i m e nt s w i l l a l s o be c r uc i a l t o de m ons t r a t e t he a bi l i t y o f t he H S C t o pr oduc e a n a l t e r na t i ve non he m a t opoi e t i c t i s s ue t ype I n t he s e e xpe r i m e nt s H S C w e r e pu r i f i e d a s a bove e xc e pt t ha t du r i ng t he f i na l t r a ns pl a nt i ng i nt o t he l e t ha l l y i r r a di a t e d r e c i pi e nt s one s i ngl e c e l l w a s i s ol a t e d a nd t r a ns pl a nt e d a l ong w i t h non G F P r e s c ue pr oge ni t o r c e l l s F i gur e 3 3 i s t he pe r i phe r a l bl ood m ononuc l e a r c e l l s s t a i ne d w i t h t he s a m e l i ne a ge m a r ke r s T c e l l ( C D 4) B c e l l ( B 220) a nd m yl o m onoc yt e ( C D 11b) T hi s f i gur e de m ons t r a t e s t he c l ona l a bi l i t y o f t he

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34 H S C i n he m a t opoi e s i s or t he c a pa bi l i t y of a s i ngl e c e l l t o p r ovi de a l l of t he bl ood l i ne a ge s E a c h o f t he s e c ohor t s w a s t he n p l a c e d i n t o t he ne ova s c ul a r i z a t i on m ode l F i gur e 3 3 H S C c a n pr oduc e a l l he m a t opoi e t i c l i ne a ge s c l ona l l y. S i ngl e e n r i c he d H S C w e r e t r a ns pl a nt e d i nt o l e t ha l l y i r r a di a t e d r e c i pi e nt s P e r i phe r a l bl ood w a s i s ol a t e d a nd s t a i ne d f or va r i ous he m a t opoi e t i c l i ne a ge s P a ne l 1: C D 4 ( T c e l l ) l i ne a ge s w e r e donor de r i ve d. P a ne l 2: B 2 20 ( B c e l l ) l i ne a ge s w e r e donor de r i ve d P a ne l 3: C D 11b ( M yl om onoc yt i c ) l i ne a ge s w e r e donor de r i ve d. A s s e s s m e n t of G F P R e t i n al B l ood V e s s e l E n d ot h e l i al C e l l s O nc e l ong t e r m m ul t i l i ne a ge e ngr a f t m e nt ha s be e n de m ons t r a t e d i n t he s e a ni m a l s e xoge nous gr ow t h f a c t or ( V E G F ) w a s a dm i ni s t e r e d t o pr i m e t he s ys t e m f o r bl ood ve s s e l gr ow t h. A s not e d, V E G F i s a pot e nt s t i m ul a t or of e ndot he l i a l r e c r ui t m e nt a nd bl ood ve s s e l f or m a t i on. T he V E G F i s pa c ka ge d i nt o A A V w hi c h i nf e c t s t he c e l l s o f t he r e t i na a nd c a us e s ove r e xpr e s s i on a nd a c c um ul a t i on of t h e pr ot e i n. I nde e d, t he vi t r e ous of t he e ye i s a l m os t c om pl e t e l y l a c ki ng p r ot e a s e s s o t he r e i s a m pl e s i gna l f o r t he e ndot he l i a l c e l l f or m a t i on of bl ood ve s s e l s A f t e r one m on t h t o a l l ow f o r pe a k V E G F e xpr e s s i on, t he m a j or bl ood ve s s e l s of t he e ye a r e phot oc oa gul a t e d w i t h a l a s e r T hi s i s c he m i c i nj u r y, c om bi ne d w i t h t he V E G F e l i c i t s a d r a m a t i c ne ova s c ul a r r e s pons e i n t he r e t i na O ne m ont h a f t e r phot oc oa gul a t i on t he a ni m a l s w e r e s a c r i f i c e d t o m e a s ur e t he a m ount of H S C c ont r i but i on t o t he ne w va s c ul a t ur e T he m i c e w e r e pe r f us e d w i t h H oe c hs t s t a i n t o m a r k t he nuc l e i of c e l l s a nd de l i ne a t e ve s s e l l um e ns E y e s w e r e r e m ove d f o r s e c t i oni ng a nd i m m unohi s t oc he m i c a l a na l ys i s of t he donor c e l l s f or bot h b l ood a nd e ndot he l i a l c e l l

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35 s ur f a c e phe not ype s T h i s w a s done t o de t e r m i ne w he t he r t he c e l l s w e r e t r ul y t r a ns di f f e r e nt i a t e d i nt o e ndot he l i a l c e l l s or i f t he y w e r e i nva di ng l e ukoc yt e s or m a c r opha ge s E ye s w e r e s e c t i o ne d a l ong bot h s i d e s of t he opt i c ne r ve a nd m or e t ha n 30 s e c t i ons w e r e obt a i ne d f r om e a c h e ye T he s e c t i on s w e r e s t a i ne d w i t h he m a t oxyl i n F a c t or V I I I pl a t e l e t e ndot he l i a l c e l l a dhe s i on m ol e c ul e or m ous e e ndot he l i a l c e l l a nt i ge n 32. F i gur e 3 4 s how s t he G F P c e l l s w hi c h s ur r ound t h e l um e n of t he ne w l y f or m e d ve s s e l s T he s e s a m e s e c t i ons w he n c ount e r s t a i ne d w i t h t he e ndot he l i a l s pe c i f i c m a r ke r s de m ons t r a t e t ha t t he c e l l s l i ni ng t he l um e n of t he ve s s e l s a r e e ndot he l i a l i n na t ur e E a c h r ow i s of a di f f e r e nt c a pi l l a r y t uf t a nd e a c h i s s t a i ne d w i t h a d i f f e r e nt e ndot he l i a l m a r ke r T he t op r ow i s s t a i ne d w i t h F a c t or V I I I c onj uga t e d t o P E P a ne l C s how s t ha t t he ve s s e l l um e n i s e ndot he l i a l a s e xpe c t e d, a nd t he G F P c e l l s s e e n i n pa ne l B c ol oc a l i z e d w i t h F a c t or V I I I w hi c h m e r ge ye l l ow i n D P a ne l F s how s a not he r ve s s e l w i t h donor de r i ve d c e l l s ( G F P i n P a ne l F ) w hi c h c os t a i n w i t h P E C A M i n pa ne l G A not he r ve s s e l ha s t he s a m e donor de r i ve d e ndot he l i a l phe not ype e xpr e s s i ng M E C A 32 ( P a ne l J a nd K ) F i na l l y i n t he s e ve s s e l s w he n s t a i ne d w i t h C D 45, a he m a t opoi e t i c s pe c i f i c m a r ke r t he G F P c e l l s di d not e xpr e s s C D 45 a nd ha d e nt i r e l y a dopt e d t he e ndot he l i a l phe not ype T hi s r e s ul t w a s a l s o r e a di l y o bs e r ve d on a w hol e m ount e d r e t i na F i gu r e 3 5 s how s a n e nt i r e r e t i na f r om a n a ni m a l pe r f us e d w i t h t he r e d f l uor e s c e nt dye a s de s c r i be d i n t he m e t hods c ha pt e r U nde r l ow pow e r ( P a ne l A ) a r e a s of donor de r i ve d G F P c e l l s a r e vi s i bl e c ont r i but i ng t o t he va s c ul a t ur e i n t he t r e a t e d e ye s T he c ont r a l a t e r a l unt r e a t e d e ye ha s no s uc h e ndot he l i a l c ont r i but i on a l t hough a r e a s w he r e t he bl ood w a s not

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36 r e m ove d by t he pe r f us i on c a n be s e e n c ont a i ni ng t he G F P he m a t opoi e t i c c e l l s ( P a ne l B ) U nde r hi ghe r pow e r m a gni f i c a t i on G F P c e l l s c a n be s e e n w r a ppi ng a r ound t he ve s s e l s c ont a i ni ng t he r e d dye i n va r i ous s t a ge s of bl ood v e s s e l f or m a t i on ( P a ne l s C F ) O f not e t hi s H S C c ont r i but i on t o e ndot he l i a l c e l l s di d not o c c ur w he r e no i s c he m i c i nj u r y w a s i nduc e d. F i gur e 3 4 D onor de r i ve d H S C c ont r i but e t o e ndot he l i a l c e l l s of bl ood v e s s e l s i n t he e ye N e ova s c ul a r i z a t i on w a s i nduc e d i n H S C e ngr a f t e d a ni m a l s R e t i na s w e r e s e c t i one d a nd s t a i ne d w i t h e ndot he l i a l s pe c i f i c m a r ke r s P a ne l A ; A t r e a t e d a ni m a l w a s pe r f us e d w i t h a buf f e r c ont a i ni ng H oe s c s t dye t o de l i ne a t e ve s s e l l um e n a nd a t r e a t e d c ont r o l r e t i na w a s c r os s s e c t i one d. P a ne l B : T he s a m e c r os s s e c t i on ha d G F P dono r d e r i ve d c e l l s l i ni ng t he bl ood ve s s e l l um e n. P a ne l C : T he s a m e c r os s s e c t i on w a s s t a i ne d w i t h a n a nt i body t o F a c t or V I I I c onj uga t e d t o P E t o s t a i n e ndot he l i a l c e l l s P a ne l D : M e r ge d i m a ge s of B a nd C de m ons t r a t i ng e ndo t he l i a l c e l l s w hi c h w e r e donor de r i ve d. P a ne l s E H : A not he r c r os s s e c t i on w a s s t a i ne d w i t h P l a t e l e t E ndot he l i a l C e l l A dhe s i on M ol e c ul e 1 a nd i l l us t r a t e d i n t he s a m e m a nne r a s A D P a ne l s I L : A not he r c r os s s e c t i on w a s s t a i ne d w i t h M ous e E ndot he l i a l C e l l A dhe s i on 32 a nd i l l us t r a t e d i n t he s a m e m a nne r a s A D M a gni f i c a t i on i s x60

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37 F i gur e 3 5 D onor de r i ve d H S C p r oduc e f unc t i ona l e ndot he l i a l c e l l s s ur r oundi ng bl ood ve s s e l l um e ns M i c e w e r e l on g t e r m he m a t opoi e t i c e ngr a f t e d w i t h G F P H S C a nd pl a c e d i nt o t he ne ova s c ul a r i z a t i on m ode l T he a ni m a l s w e r e pe r f us e d w i t h T R I T C l a be l e d de xt r a n, s a c r i f i c e d, a nd r e t i na s w e r e i m a ge d by c onf oc a l m i c r os c opy. P a ne l A : A w ho l e m ount e d r e t i na i s de m ons t r a t e d u nde r l ow m a gni f i c a t i on ( x4) T he r e d f l uor e s c e nc e f i l l s t he pe r f us e d, f unc t i ona l bl ood ve s s e l s S m a l l c a pi l l a r y t uf t s of donor de r i ve d G F P c e l l s w hi c h a r e m a gni f i e d i n C D E & F c a n be obs e r ve d a r ound a r e a s of phot oc oa gul a t i on. P a ne l B : F r om a c on t r a l a t e r a l unt r e a t e d e ye c i r c ul a t i ng donor de r i ve d G F P he m a t opoi e t i c c e l l s a r e pr e s e nt i n t he l um e n of a bl ood ve s s e l M a gni f i c a t i on x40 P a ne l C : D onor de r i ve d G F P c e l l s a r e a s s oc i a t i ng w i t h a pe r f us e d bl ood ve s s e l ( l a r ge a r r ow he a d) O t he r donor de r i v e d c e l l s ha ve e i t he r not di r e c t l y a s s oc i a t e d w i t h ve s s e l l um e ns or ha ve e xt r a va s a t e d a nd not f or m e d e ndot he l i a l t ube s M a gni f i c a t i on x40. P a ne l s D F : H i gh m a gni f i c a t i on i m a ge s s how G F P c e l l s s ur r oundi ng ve s s e l l um e ns ( D & E ) a nd f or m i n g e a r l y ne ova s c ul a r i z a t i on ( F ) M a gni f i c a t i on x60

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38 T h e H S C h as H e m a n gi ob l as t F u n c t i on T he r e c r ui t m e nt of H S C de r i ve d c e l l s t o r e gi ons o f i nj ur y a gr e e s w i t h s t udi e s done w hi c h ha ve f ound l i m i t e d c ont r i but i on t o non i nj ur e d t i s s ue s 8 5 I n t he s e e xpe r i m e nt s t he donor de r i ve d G F P c e l l s w e r e a bl e t o c ont r i but e t o bot h t he bl ood pr oduc t s a nd e ndot he l i a l c e l l s of t he va s c ul a t ur e i n t he s a m e m o us e how e ve r t he e xa c t c e l l w hi c h c oul d a c c om pl i s h t he s e f e a t s c a nnot be e s t a bl i s he d by t he s e e xpe r i m e nt s T he c l a s s i c de f i ni t i on of a H S C i s a c e l l c a pa bl e o f l ong t e r m he m a t opoi e t i c r e c ons t i t ut i on a f t e r i r r a di a t i on o r s e l f r e ne w i ng. W e ha ve f ul f i l l e d t hi s de f i ni t i on t hr ough t he s e r i e s of t r a ns pl a n t a t i on s t udi e s de s c r i be d a bove how e ve r t he a bi l i t y of a s i ngl e H S C t o do s o c l ona l i t y, a nd t hus r u l i ng out a ny c ont r i but i on by ot he r c ont a m i na t i ng c e l l s w a s ne c e s s a r y t o pr ove H S C pl a s t i c i t y. A s de s c r i be d i n t he m e t hods c ha pt e r a s i ngl e H S C w a s e nr i c he d a nd i s ol a t e d t hough m i c r om a ni pu l a t i on. T he H S C w a s t r a ns pl a nt e d a l ong w i t h S c a 1 ne ga t i ve non G F P bone m a r r ow c e l l s ( s hor t t e r m pr oge ni t o r s ) a nd t r a ns pl a nt e d i nt o a l e t ha l l y i r r a di a t e d r e c i pi e nt O f t he 80 m i c e t r a ns pl a nt e d, pe r i phe r a l bl ood l ong t e r m m ul t i l i ne a ge e ngr a f t m e nt w a s de m ons t r a t e d i n 3 a ni m a l s w hi c h w e r e t he n s ubj e c t e d t o t he i s c he m i c ne ova s c ul a r i z a t i on m ode l E a c h o f t he t hr e e a ni m a l s e xhi bi t e d t he c a pi l l a r y t uf t gr ow t hs s e e n i n t he pr e vi ous e xpe r i m e nt s t ha t w e r e e nt i r e l y donor de r i ve d a s de m ons t r a t e d b y G F P e xpr e s s i on. I n a ddi t i on t he s e ve s s e l s w e r e f unc t i ona l i n t he i r a bi l i t y t o hol d t he r e d f l uor e s c e nt dye pe r f us e d i nt o t he va s c ul a t ur e S i nc e t he s e a ni m a l s ha d bo t h bl ood a nd bl ood ve s s e l s w hi c h w e r e de r i ve d f r om a s i ngl e t r a ns pl a nt e d H S C t he r e c a n be no c ont r i but i on f r om a not he r s our c e a nd a ny G F P c e l l s m us t ne c e s s a r i l y be de r i ve d f r om t he H S C A s s how n i n f i gur e 3 6 t he H S C de m ons t r a t e d he m a ngi obl a s t a c t i vi t y i n t he i r a bi l i t y t o pr oduc e bot h bl ood a nd bl ood ve s s e l s i n a c l ona l m a nn e r P a ne l s A C a r e f r om a s e r i a l l y t r a ns pl a nt e d m ous e T he r e d

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39 pe r f us e d bl ood ve s s e l ( P a ne l A ) c ol oc a l i z e s w i t h t he G F P donor de r i ve d e ndot he l i a l c e l l s ( P a ne l B ) t o s how donor de r i ve d ne ova s c ul a r i z a t i o n ( P a ne l C ) T he s e ve s s e l s w e r e de r i ve d f r om a s e l f r e ne w i ng H S C P a ne l s D F a r e f r om a s i ngl e c e l l t r a ns pl a nt e d a ni m a l T he r e d pe r f us e d bl ood ve s s e l ( P a ne l D ) c ol oc a l i z e s w i t h t he G F P dono r de r i ve d e ndot he l i a l c e l l s ( P a ne l E ) t o s how donor de r i ve d ne ova s c ul a r i z a t i on ( P a ne l F ) T he s e ve s s e l s a r os e f r om t he H S C i n a c l ona l m a nne r t he r e f or e t he H S C c a n gi ve r i s e t o bot h bl ood a nd bl ood ve s s e l s a nd f unc t i on a s a he m a ngi obl a s t D i s c u s s i on T he pr e vi ous w or k de m ons t r a t e s t he t r ue pl a s t i c i t y of t he H S C a nd f ul f i l l s t he t hr e e c r i t e r i a e s t a bl i s he d t o pr ove t hi s c a pa c i t y T he s e l f r e ne w i ng c a pa bi l i t y w a s de m ons t r a t e d t hr ough s e r i a l t r a ns pl a nt a t i ons a nd l o ng t e r m he m a t opoi e t i c r e c ons t i t ut i on. T he a bi l i t y of t he H S C t o pr ovi de he m a t opoi e s i s a l ong w i t h non he m a t opoi e t i c t i s s ue i n a c l ona l m a nne r w a s s how n t hr ough s i ngl e c e l l t r a ns pl a nt s B ot h e xpe r i m e nt s de m ons t r a t e d t he a bi l i t y o f t he H S C t o pr oduc e f u nc t i ona l ve s s e l s i n a r obus t m a nne r T a ke n t oge t he r t he s e e xpe r i m e nt s out l i ne a n a l t e r n a t i ve de ve l opm e nt a l f a t e of t he H S C na m e l y t he E P C a nd de s c r i be how t hi s out c om e c a n be i nduc e d t hr ough gr ow t h f a c t or a dm i ni s t r a t i on a nd i s c he m i c i nj ur y T he E P C w a s s how n t o be de r i ve d f r om t he H S C a nd not t he M S C a s pr e vi ous l y pos i t e d. 9 7 T hi s und e r s t a ndi ng i s e s pe c i a l l y va l ua bl e i n c ur r e nt t r e a t m e nt s w he r e t he E P C ha s be e n s how n t o ha ve t he a bi l i t y t o c ont r i but e t o t he r a pe ut i c ne ova s c ul a r i z a t i on i n s e ve r a l s t udi e s of i s c he m i c i nj ur y, s om e i n hu m a n c l i ni c a l t r i a l s 5 1 8 8 T hi s w or k de m ons t r a t e s t ha t ve s s e l gr ow t h i s not onl y c a r r i e d out by l oc a l or c i r c ul a t i ng e ndot he l i a l c e l l a ngi oge ne s i s but unde r c ondi t i ons of i n j ur y t he H S C c a n pr ovi de ne ova s c ul a r i z a t i on. N e w bl ood ve s s e l s f or m e d w e r e l a r ge l y de r i ve d f r om t he r e c r u i t m e nt of undi f f e r e nt i a t e d pr e c ur s or s c e l l s f r o m t he bone m a r r ow

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40 F i gur e 3 6 T he H S C i s s e l f r e ne w i ng a nd c a n c l ona l l y f or m e ndot he l i a l c e l l s B ot h s e r i a l l y t r a ns pl a nt e d l ong t e r m e ngr a f t e d a nd s i ngl e c e l l t r a ns pl a nt e d a ni m a l s w e r e pl a c e d i n t he ne ova s c ul a r i z a t i on m od e l A ni m a l s w e r e pe r f us e d w i t h t he T R I T C l a be l e d de xt r a n a nd r e t i n a s w e r e i m a ge d by c onf oc a l m i c r os c opy. P a ne l A C : A l ong t e r m e n gr a f t e d s e r i a l l y t r a ns pl a nt e d m ous e r e t i na w a s i m a ge d. P a ne l A s how s t he r e d c ha nne l onl y i ndi c a t i ng p e r f us e d, a nd t he r e f or e f unc t i ona l bl ood ve s s e l s P a ne l B i s t he donor G F P H S C c ont r i but i on t o t he ne ova s c ul a r i z a t i on. A a nd B w e r e m e r ge d i n C a nd ye l l ow a r e a s a r e donor de r i v e d c e l l s c ol oc a l i z i ng w i t h t he pe r f us e d ve s s e l T he H S C i s s e l f r e ne w i n g a nd c a n pr oduc e a l l bl ood l i ne a ge s a nd e ndot he l i a l c e l l s of t he va s c ul a t ur e P a ne l D F : A s i ngl e c e l l t r a ns pl a nt e d m ous e r e t i na w a s i m a ge d. P a ne l D s how s t he r e d c ha nne l onl y i ndi c a t i ng pe r f us e d, a nd t he r e f or e f un c t i ona l bl ood ve s s e l s P a ne l E i s t he donor G F P H S C c ont r i but i on t o t he ne ova s c ul a r i z a t i on. D a nd E w e r e m e r ge d i n F a nd ye l l ow a r e a s a r e don or de r i ve d c e l l s c ol oc a l i z i ng w i t h t he pe r f us e d ve s s e l T he H S C c a n c l ona l l y pr oduc e a l l he m a t opoi e t i c l i ne a ge s a nd e ndot he l i a l c e l l s l i ni ng bl ood ve s s e l w a l l s M a gni f i c a t i on x60. T he ne xt s e r i e s of e xpe r i m e nt s w i l l a s c e r t a i n t he p ot e nt i a l t o m odul a t e he m a ngi obl a s t f unc t i on. U nde r s t a ndi ng t he g r ow t h f a c t or s a nd bi o l ogi c a l c ondi t i ons dur i ng i s c he m i a a nd how t he y r e gul a t e c ont r i but i o n t o ne ova s c ul a r i z a t i on by t he H S C

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41 a nd H S C pr oge ni t or s m a y pr ovi de m e t hods t o m a n i pul a t e bl ood ve s s e l f or m a t i on M odul a t i on of t he H S C / E P C pa t hw a y m a y a l l ow f or t a i l or i ng of t he r a pi e s t o i nc r e a s e ne ova s c ul a r i z a t i on i n i s c he m i c c ondi t i ons s uc h a s s t r oke w ound he a l i ng or c a r di a c m us c l e da m a ge C onve r s e l y, t he a bi l i t y t o de c r e a s e pa t hol ogi c or unde s i r a bl e ne ova s c ul a r i z a t i on a s s e e n i n t um or ne ova s c ul a r i z a t i on or di a be t i c r e t i nopa t hy c oul d s t e m f r om a gr e a t e r unde r s t a ndi ng of t he H S C t o E P C d e ve l opm e nt a l f a t e

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42 C H A P T E R 4 M O D U L A T O R S O F H S C / H E M A N G I O B L A S T A C T I V I T Y F e w t opi c s ha ve s t i r r e d m or e r e c e nt de ba t e t ha n t h e pr om i s e of he m a t opoi e t i c s t e m c e l l s ( H S C ) e xhi bi t i ng f unc t i ona l p l a s t i c i t y. I nde e d, t he c a ndi da c y o f H S C f or t he r a pe ut i c t r e a t m e nt o f di s e a s e i s c ont i ng e nt upon de m ons t r a t i ng t he i r a bi l i t y t o f ul f i l l s t r i nge nt pl a s t i c i t y c r i t e r i a I ni t i a l pa pe r s de s c r i be d H S C t r a ns di f f e r e nt i a t i on i nt o a va r i e t y of non he m a t opoi e t i c t i s s ue s i n va r i ous o r g a ns s uc h a s t he l i ve r b r a i n, c a r di a c m us c l e bl ood va s c ul a t ur e i nt e s t i ne a nd pa nc r e a s 3 0 5 0 8 2 8 4 9 8 U s i ng va r i ous t i s s ue s pe c i f i c m a r ke r s a nd phe not ypi c c ha r a c t e r i s t i c s t he s e a ut hor s ha ve de s c r i be d t i s s ue s t o w hi c h H S C a r e a bl e t o c ont r i but e de m ons t r a t i ng pl a s t i c i t y of t he H S C i n t he i r e xpe r i m e nt a l s e t t i ngs H ow e ve r a t t e m pt s t o r e c a pi t ul a t e t he s e s t udi e s ha ve f ound l i m i t e d H S C pl a s t i c i t y. 8 5 9 9 A pos s i bl e r a t i ona l e f o r t he s e di c hot i c a c c ount s i s t ha t t h e s e s t udi e s e m pl oye d di f f e r i ng m e t hods t o i s ol a t e H S C a nd e xa m i ne t he t a r ge t or ga n of pot e nt i a l t r a ns di f f e r e nt i a t i on S pe c i f i c a l l y a va r i e t y of H S C i s ol a t i on a nd pu r i f i c a t i on s c he m a ha ve be e n e m pl oye d i nc l udi ng: e l ut r i a t i on, or s e pa r a t i on ba s e d on r e l a t i ve de ns i t y, s e r i a l t r a ns pl a nt a t i on w he r e onl y t hos e c e l l s w i t h t he c a pa bi l i t y t o hom e a nd l ong t e r m r e popul a t e t he bone m a r r ow ni c he r e s c ue a m ous e a nd c e l l s i s ol a t e d t hr ough f l uor e s c e nc e a c t i va t e d c e l l s or t i ng br oke n dow n i nt o s i de popul a t i on s t udi e s o f dye e xc l us i on, a nd s i ngl e K T L S ( c ki t + T hy 1 l o l i n S c a 1 + ) c e l l t r a ns pl a nt a t i ons 3 0 8 2 1 0 0 1 0 1 E a c h m e t hod i s ol a t e s f unc t i ona l H S C a s de f i ne d b y l ong t e r m m ul t i l i ne a ge he m a t opoi e t i c r e c ons t i t ut i on i n v i v o H ow e ve r e a c h t e c hni que m a y i s ol a t e f unc t i ona l H S C a t di f f e r e nt de ve l opm e nt a l s t a ge s w i t h r e s pe c t t o p l a s t i c i t y. I t m a y be i m pr ope r t o c om pa r e

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43 H S C i s ol a t e d by phys i c a l m e a ns i e e l ut r i a t i on a n d s i de popul a t i on, w i t h t hos e i s ol a t e d by bi ndi ng o f a nt i bodi e s t o c e l l s ur f a c e r e c e pt or s T he di f f e r i ng popul a t i ons i s ol a t e d a nd t he m a ni pul a t i ons w hi c h t he c e l l s unde r g o m a y i m pa c t t he i r be ha vi or i n t he e xpe r i m e nt a l s e t t i ngs T he s e m e t hods m us t be r e c onc i l e d be f or e a de f i ni t i ve a ns w e r c a n be r e a c he d. S ha r i ng a m e s ode r m a l k i ns hi p t o t he H S C a r e t he e ndot he l i a l c e l l s ( E C ) of t he va s c ul a t ur e D ur i ng e m br yoge ne s i s he m a t opoi e t i c a nd e ndot he l i a l p r e c ur s or s de ve l op i n bot h s pa t i a l a nd t e m por a l i m m e di a c y. I n t he a dul t E C c i r c ul a t e i n t he pe r i phe r a l b l ood w hi c h a r e phe not ypi c a l l y s i m i l a r i t y t o m a t ur e E C 1 6 T he s e c e l l s ha ve t he a bi l i t y t o c ont r i but e t o ne w ve s s e l f or m a t i on e i t he r i n pl a c e of or i n a ddi t i on t o r e s i de nt E C pr ol i f e r a t i on I n a ddi t i on, i t w a s s how n t ha t t he s e c i r c ul a t i ng c e l l s c ont a i ne d a popul a t i on w hi c h w e r e de r i ve d f r om t he bone m a r r ow c a l l e d e ndot he l i a l pr oge ni t or c e l l s ( E P C ) 1 7 I t i s now a c c e pt e d t ha t t he s e bone m a r r ow E P C e xi s t a nd c ont r i but e s i gni f i c a nt l y t o a dul t bl ood ve s s e l f or m a t i on, a nd t ha t t he s e E P C a r e H S C de r i ve d. 3 0 S e ve r a l e xpe r i m e nt a l s ys t e m s w hi c h da m a ge bl ood ve s s e l s ha ve be e n a bl e t o i nduc e r obus t H S C t r a ns di f f e r e nt i a t i on. 3 0 1 0 2 T he pr e c e di ng c ha pt e r s de s c r i be d how a dul t H S C e xhi bi t he m a ngi obl a s t f unc t i on by p r oduc i ng bot h bl ood a nd bl ood ve s s e l s i n a nove l m ode l of r e t i na l ne ova s c ul a r i z a t i on. T he m o de l us e s l ong t e r m bone m a r r ow c hi m e r i c m i c e t ha t ha ve be e n s t a bl y r e c ons t i t ut e d w i t h he m a t opoi e t i c s t e m c e l l s f r om G F P donor m i c e ( f ul f i l l i ng t he f i r s t p l a s t i c i t y r e qui r e m e nt ) T he s e c e l l s a r e pos i t i ve f or t he s ur f a c e m a r ke r s S c a 1 a nd c ki t a nd de m ons t r a t e r obus t G F P e xpr e s s i on i n bl ood pr oduc t s a f t e r a f our m ont h pe r i od T hi s t i m e i s s uf f i c i e nt t o e l i m i na t e a ny c ont a m i na t i ng pr oge ni t or c e l l w hi c h w oul d ha ve s i nc e di e d of f a n d c r e a t e d a de f i c i e nc y l e uke m i a i n m i c e e ngr a f t e d w i t h t he s e s hor t t e r m pr oge ni t o r c e l l s T he l ong t e r m e ng r a f t e d c hi m e r a s

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44 t he n r e c e i ve a c om bi na t i on of g r ow t h f a c t or a dm i n i s t r a t i on a nd l a s e r i nduc e d i s c he m i c i nj ur y t o pr om ot e ne w bl ood ve s s e l f or m a t i on i n a dul t m ur i ne r e t i na s B r i e f l y A de no A s s oc i a t e d V i r us ( A A V ) e xpr e s s i ng V a s c ul a r E ndot he l i a l G r ow t h f a c t or ( V E G F ) i s a dm i ni s t e r e d i nt r a vi t r e a l l y a nd a l l ow e d one m ont h t o r e a c h pe a k e xpr e s s i on. T he r e t i na i s t he n phot oc oa gul a t e d a nd ne w ve s s e l s a t t e m pt t o gr ow i nt o t he i s c he m i c r e gi on. S i nc e H S C ha ve t he a bi l i t y t o l ong t e r m r e popul a t e he m a t opoi e s i s l e t ha l l y i r r a di a t e d m i c e w e r e t he n t r a ns pl a nt e d w i t h a s i ngl e H S C T he s e a ni m a l s e xhi bi t e d s i gni f i c a nt G F P i n pe r i phe r a l bl ood a nd bone m a r r ow a l l of w hi c h w a s de r i ve d f r om t he s i ngl e t r a ns pl a nt e d H S C pr ovi ng c l ona l i t y i n t r a ns pl a nt e d H S C he m a t opoi e s i s C hi m e r a s de r i ve d f r om bot h s e r i a l l y t r a ns pl a nt e d a nd s i ngl e G F P + H S C pr oduc e d w hol e G F P va s c ul a r be ds a f t e r a c ut e i nj ur y a nd V E G F i nduc t i on. 3 0 T he ve s s e l s pr oduc e d w e r e not onl y r obus t but w e r e f unc t i ona l a s de t e r m i ne d by pe r f us i on a f t e r c a r di a c a dm i ni s t r a t i on of a f l uor e s c e nt dye T he pr e vi ous c ha pt e r ha s i l l us t r a t e d a ne w de ve l op m e nt a l out c om e of t he H S C : t he pr oduc t i on of E P C i n r e s pons e t o va s c ul a r i nj ur y. T he w or k de m ons t r a t e d t ha t bot h bl ood a nd bl ood ve s s e l s c a n be c l ona l l y de r i ve d f r om a dul t H S C v i a a c om bi na t i on of gr ow t h f a c t or a dm i ni s t r a t i on a nd i s c he m i c i nj ur y T hus a dul t H S C m e e t t he de f i ni t i on of a pl a s t i c s t e m c e l l i n t ha t t he y ha ve t he a bi l i t y t o a c t a s a he m a ngi obl a s t i n v i v o W he t he r t he H S C pa r t i c i pa t e s i n e ve r yda y m a i nt e na nc e va s c ul oge ne s i s or pa r t a ke s onl y i n r e s pons e t o c hr oni c ve s s e l i nj ur y r e m a i ne d t o be d e t e r m i ne d a nd w a s one of t he f oc us e s of t hi s w or k I n a ddi t i on, i t w a s de t e r m i ne d t ha t s om e f or m of s i gni f i c a nt i nj u r y i s ne e de d f or i nduc t i on of t he H S C t o E P C pa t hw a y, pr e s um a bl y s i nc e r e s i de nt E C a r e i na de qua t e t o s e e d a nd pr ol i f e r a t e t he da m a ge d a r e a s W hi l e t he H S C i s now know n t o

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45 pr oduc e E P C unde r i nj u r y c ondi t i ons t he pot e nt i a l r ol e o f phys i ol ogi c m e di a t o r s w hi c h i m pa c t va s c ul oge ne s i s i n r e l a t i on t o he m a ngi obl a s t H S C a c t i vi t y m e r i t e x a m i na t i on S i nc e t he i r f i r s t de s c r i pt i on i n 1989 t he ni t r i c oxi d e s ynt ha s e s ( N O S ) ha ve be e n s how n t o pl a y a r ol e i n a m yr i a d of bi ol ogi c a l f unc t i ons T he f r e e r a di c a l N O p r oduc e d f r om t he c onve r s i on of L a r gi ni ne t o c i t r ul l i ne i n t he pr e s e nc e of oxyge n, ha s be e n s how n t o f unc t i on i n di s t i nc t p r oc e s s e s s uc h a s i nf l a m m a t i on, hos t de f e ns e ne u r ot r a ns m i s s i on, a nd s m oot h m us c l e c ont r a c t i l i t y. T hr e e d i s t i nc t i s of or m s of t he e nz ym e ha ve be e n c ha r a c t e r i z e d i nc l udi ng nN O S w hi c h i s e xpr e s s e d i n ne ur ona l t i s s ue s i N O S e xpr e s s e d i n a w i de va r i e t y o f t i s s ue s a nd e N O S w hi c h i s pr e do m i na t e l y e xpr e s s e d i n t he e ndot he l i a l c e l l s of t he va s c ul a t ur e T he nN O S a nd e N O S i s of or m s a r e c ons t i t ut i ve l y a c t i ve i n t he i r e xpr e s s i ng t i s s ue s w hi c h t he i N O S i s of or m i s i ndu c e d i n r e s pons e t o pr oi n f l a m m a t or y c yt oki ne s or e ndot oxi ns f r om f o r e i gn ba c t e r i a T hi s i nduc t i on of i N O S p r oduc e s a 100 f ol d i nc r e a s e i n N O a s pa r t o f a n i m m une r e s pons e a nd N O pr oduc t i on i s m uc h hi ghe r t ha n i s s e e n c om pa r e d t o t he ba s a l l e ve l s of t he c ons t i t ut i ve l y a c t i ve i s of or m s 1 0 3 N O pr oduc e d by i N O S a c t s a s a n a nt i m i c r obi a l a nd a nt i vi r a l a ge nt by de c r e a s i ng D N A r e pl i c a t i on. N i t r i c O xi de ( N O ) a l s o m e di a t e s e ndot he l i a l c e l l f unc t i on a nd he nc e bl ood ve s s e l f or m a t i on. I t ha s be e n s how n t o i nf l ue nc e ne ova s c ul a r i z a t i on i n s e ve r a l m ode l s o f a ngi oge ne s i s 1 0 4 1 0 6 T he r ol e o f N O i n pr om o t i ng a ngi oge ne s i s ha s be e n c ont r ove r s i a l i n pa r t be c a us e of t he c om pl e x r e g ul a t i on of N O ge ne r a t i on a nd i na c t i va t i on I n a ddi t i on t o va s odi l a t a t i on, i nc r e a s e d l oc a l c onc e nt r a t i ons of N O s t i m ul a t e pr ol i f e r a t i on a nd m i gr a t i on of e ndot he l i a l c e l l s bot h o f w hi c h a r e e s s e nt i a l f or a ngi oge ne s i s 1 0 7 1 0 9 T he N O pr oduc e d by t he t hr e e s e pa r a t e i s of or m s a r e a c t i va t e d unde r di s t i nc t a c t i vi t i e s a nd ha ve uni que

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46 r e gul a t or y c ont r ol s 1 0 9 S i nc e i N O S i s a c t i va t e d un de r c e r t a i n pa t hol ogi c a l c ondi t i ons s uc h our i nj ur y m ode l a nd e N O S i s c ons t i t ut i ve l y a c t i va t e d i n e ndot he l i a l t i s s ue s t he s e i s of or m s m a y i nf l ue nc e t he pr oc e s s of ne ova s c ul a r i z a t i on. T he a l t e r e d a m ount o f N O due t o l a c k of t he s e e nz ym e s i n t he c e l l w i l l a f f e c t he m a ngi obl a s t r e c r ui t m e nt a nd f or m a t i on o f bl ood ve s s e l s A ngi oge ne s i s i s i ni t i a t e d by va s odi l a t i on i n o r de r t o ope n up ve s s e l s f a c i l i t a t i ng i nt r oduc t i on of c e l l s i n c i r c ul a t i on t o t he s i t e o f bl o od ve s s e l gr ow t h. N O i s know n t o ha ve s e ve r a l a ngi oge ne i c a f f e c t s i nc l udi ng i nc r e a s i ng m a t r i x m e t a l l opr ot e a s e e xpr e s s i on a l ong w i t h t y r os i ne phos phor yl a t i on of pr ot e i ns i n c e l l s popul a t i ng t he s pr out i ng c a pi l l a r y r e gi on. 7 4 I nt e r e s t i ngl y, i n va r i ous ne ova s c ul a r i z a t i on m ode l s N O ha s be e n s how n t o be bot h pr oa ngi oge ni c a nd a nt i a ngi oge ni c 7 4 1 0 4 T he t he or y i s t ha t t he t w o i s of o r m s a r e a c t i va t e d unde r di f f e r i ng c i r c um s t a nc e s a nd he nc e a r e t hought t o a f f e c t bl ood ve s s e l f or m a t i on di f f e r e nt l y I nde e d, i n v i v o t hi s i s t he c a s e B l ood ve s s e l f or m a t i on due t o H S C c ont r i but i on unde r c ondi t i ons o f i s c he m i c i nj ur y a r e i n f l ue nc e d by N O a s pr oduc e d by t he i N O S a nd pa r t i c ul a r l y t he e N O S i s of or m s 1 1 0 1 1 2 T he e ndot he l i a l N O S ( e N O S ) i s of or m i s c ons t i t ut i ve l y e xpr e s s e d a t ba s a l l e ve l s by e ndot he l i a l c e l l s a nd i s t hought t o pr o m ot e br a nc hi ng, or ga ni z a t i on a nd m a t ur a t i on o r e ndot he l i a l c e l l s dur i ng ve s s e l de ve l opm e nt e N O S de f i c i e nt ( e N O S / ) a ni m a l s e xhi bi t f e t a l gr ow t h r e s t r i c t i ons r e duc e d s ur vi va l a nd a n i nc r e a s e d r a t e of l i m b a bnor m a l i t i e s 1 1 3 T he y a l s o de m ons t r a t e m a r ke d va s c ul a r pa t hol ogy s uc h a s i nc r e a s e d c a r di om yoc yt e a popt os i s c onge ni t a l s e pt a l de f e c t s pos t na t a l he a r t f a i l ur e de c r e a s e d c a pi l l a r y de ns i t y a nd va s c ul a r pe r m e a bi l i t y. 1 1 4 E ndot he l i a l c e l l s f r o m e N O S / a ni m a l s de m ons t r a t e de c r e a s e d r a t e s of a ngi oge ne s i s w i t h r e duc e d br a n c hi ng i n v i t r o 1 1 5 T he s e a ni m a l s a l s o

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47 e xhi bi t a n i m pa i r m e nt of pos t na t a l a ngi oge ne s i s i n r e s pons e t o gr ow t h f a c t or s a nd i s c he m i a 1 1 6 C or r e s pondi ngl y, e N O S ha s be e n s ho w n t o m e di a t e t he m i t oge ni c e f f e c t of V E G F on c ul t ur e d m i c r ova s c ul a r e ndot he l i um 1 0 6 T he s e f i ndi ngs l e d t o t he i n v i v o w or k de m ons t r a t i ng t ha t N O p r oduc t i on i s e s s e nt i a l f or a ngi oge ne s i s i n hi ndl i m b i s c he m i a f or w ound he a l i ng, a nd c or ona r y c ol l a t e r a l gr ow t h a f t e r m yoc a r di a l i s c he m i a 1 1 5 1 1 7 V E G F ha s be e n s how n t o be a pot e nt va s c ul a r pe r m e a bi l i t y f a c t or a nd pl a ys a l e a di ng r ol e i n a ngi oge ne s i s a nd o ur m ode l t a ke s a dva nt a ge of t hi s a bi l i t y t o pr om ot e bl ood ve s s e l s ynt he s i s 7 3 T he a ngi oge ni c e f f e c t o f V E G F unde r bot h pa t hol ogi c a l a nd phys i ol ogi c a l c ondi t i ons ha s be e n r e ve a l e d t o be p r e dom i na nt l y m e di a t e d by e N O S 1 1 8 V E G F pr om o t e s N O pr oduc t i on f r om e N O S i n E C c e l l s a nd i nhi b i t i on o f e N O S by pha r m a c ol ogi c a l a ge nt s i n v i v o ha ve de c r e a s e d a ngi oge ne s i s a nd va s c ul a r pe r m e a bi l i t y i nduc e d by V E G F 1 0 5 T hi s de m ons t r a t e s t ha t e N O S i s bot h a dow ns t r e a m m e di a t or of V E G F i nduc e d a ngi oge ne s i s a nd a n ups t r e a m p r o m ot e r of V E G F e xpr e s s i on. T hi s r e s ul t s i n put a t i ve pos i t i ve f e e dba c k l oop be t w e e n N O a nd V E G F w hi c h pr om ot e s a ngi oge ne s i s 1 1 9 T he i nduc i bl e N O S ( i N O S ) i s of or m i s e xpr e s s e d b y e ndot he l i a l c e l l s i n r e s po ns e t o e xt e r na l s t i m ul i s uc h a s V E G F p r oi nf l a m m a t o r y c yt oki ne s or l i popol ys a c c ha r i de i N O S a c t i va t i on r e s ul t s i n a 1000 f ol d gr e a t e r ge ne r a t i on of N O t he n e N O S a c t i vi t y a l one 1 2 0 I t s i nduc t i on i s t hough t t o pr om ot e t ube e l onga t i on dur i ng ve s s e l de ve l opm e nt a l t hough e vi de nc e s uppor t s t ha t i t m a y ha ve a c ont r a s t i ng a n t i a ngi oge ni c e f f e c t 7 9 i N O S de f i c i e nt a ni m a l s ( i N O S / ) a r e r e l a t i ve l y he a l t hy but do ha v e a s l i ght de c r e a s e i n N O p r oduc t i on a nd va s c ul a r pe r m e a bi l i t y dur i ng a ngi oge ne s i s i n c ol l a ge n ge l s pl a c e d i n a m ous e c r a ni a l w i ndow 1 0 6 D u r i ng no r m a l bl ood ve s s e l f or m a t i on t he i nt e r pl a y be t w e e n e N O S a nd

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48 i N O S a c t i vi t y ha s be e n pos t ul a t e d t o di c t a t e ve s s e l s i z e a nd de gr e e of br a nc hi ng. I n t hi s c ha pt e r I w i l l de s c r i be e xpe r i m e nt s w he r e w i l d t yp e G F P + H S C a r e t r a ns pl a nt e d i nt o e N O S / a nd i N O S / r e c i pi e nt s t o a s s e s s t he e f f e c t of N O S dys f unc t i on i n t i s s ue on he m a ngi obl a s t a c t i vi t y. R e s u l t s i N O S an d e N O S G F P c h i m e r as d e m on s t r a t e d r ob u s t H S C e n gr af t m e n t T o di r e c t l y a s s e s s t he r ol e of N O S a c t i vi t y i n t he p r om o t i on o f H S C t r a ns di f f e r e nt i a t i on i nt o bl ood ve s s e l s c ohor t s of w i l d t ype ( W T ) C 57B L 6, i N O S / a nd e N O S / a ni m a l s w e r e ge ne r a t e d. A ni m a l s w e r e t r a ns pl a nt e d w i t h 2, 500 hi gh l y e nr i c he d G F P + H S C I t w a s ne c e s s a r y t o us e a hi ghl y e nr i c he d H S C popul a t i on r a t he r t he n a s i ngl e H S C due t o t he poor s ur vi va l of e N O S / a n i m a l s dur i ng t r a ns pl a nt a nd t he di f f i c ul t y i n pr oduc i ng s i ngl e c e l l t r a ns pl a nt e d a ni m a l s i n ge ne r a l T he e nr i c he d H S C popul a t i ons us e d w e r e i s ol a t e d us i ng t he s a m e pr ot oc ol pr e vi ous l y e m pl oye d f or s i ngl e c e l l t r a ns pl a nt s i n W T a ni m a l s 3 0 B r i e f l y, w hol e b one m a r r ow w a s obt a i ne d f r om t he bone m a r r ow o f G F P a ni m a l s C e l l s w e r e pl a t e d o n t i s s ue c ul t ur e t r e a t e d pl a t e s f or 2 hour s dur i ng w hi c h t i m e t he a dhe r e nt c e l l pop ul a t i on, w hi c h c ont a i ns p r oge ni t or c e l l s s uc h a s t he m e s e nc hym a l s t e m c e l l s t i c k t o t he pl a t e N on a dhe r e nt c e l l s a r e c ol l e c t e d a nd s t a i ne d w i t h S c a 1 c ki t a nd t he l i ne a ge m a r k e r s C e l l s w hi c h w e r e s or t e d by F A C S f or t he s t e m c e l l m a r ke r s of S c a 1 a n d c ki t a nd w e r e l i ne a ge ne ga t i ve w e r e i n j e c t e d i nt r a ve nous l y t hr ough t he r e t r o or bi t a l s i nus L ong t e r m m ul t i l i ne a ge he m a t opoi e t i c e ngr a f t m e nt w a s c onf i r m e d > 3 m ont hs pos t t r a ns pl a nt by f l ow c yt om e t r y a na l ys i s of pe r i phe r a l bl ood a nd i s s how n i n f i gur e 4 1 T he f i r s t c ol um n i n e a c h c ohor t r e pr e s e nt e d i s pe r i phe r a l bl ood s t a i ne d f or B c e l l s e xpr e s s i ng B 220, w i t h t he s e c ond c ol um n s t a i ne d f or m a c r opha ge s e xpr e s s i ng C D 11b, a nd t he t hi r d c ol um n T c e l l s e xpr e s s i ng C D 4. T he

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49 t op r ow ( C 57B L / 6) a nd t he s e c ond r o w ( G F P don or s t r a i n) a r e s uppl i e d f or r e f e r e nc e c ont r ol s t o f a c i l i t a t e c om pa r i s on be t w e e n r e c i pi e nt a nd donor ba c kgr ound f l uor e s c e nc e T he t hi r d r ow i s a t ypi c a l C 57B L / 6. G F P c hi m e r i c m ous e de m ons t r a t i ng r obus t he m a t opoi e t i c e ngr a f t m e nt T he f o r t h r ow ( i N O S G F P ) a nd f i f t h r ow ( e N O S G F P ) a r e r e pr e s e nt a t i ve of e ngr a f t m e nt l e ve l s of t r a ns pl a nt e d knoc kout a ni m a l s bl ood l i ne a ge pr of i l e s T he bot t om r ow i s pe r i phe r a l bl ood s t a i ne d f or V E G F R 2 de m ons t r a t i ng t ha t G F P E P C a r e i n t he c i r c ul a t i on of C 57B L / 6. G F P i N O S G F P a nd e N O S G F P a ni m a l s E ngr a f t e d r e c i pi e nt s w e r e s ubs e que nt l y t e r m e d C 5 7B L 6. G F P i N O S G F P or e N O S G F P c hi m e r a s R e c i pi e nt s t ha t w e r e r obus t l y r e c ons t i t u t e d by donor H S C ( > 75 % donor de r i ve d m ye l oi d c e l l s ) t he n unde r w e nt ou r m ode l o f i s c he m i c i nj ur y t o i nduc e a dul t r e t i na l ne ova s c ul a r i z a t i on ( n > 10 f or a l l c ohor t s ) B y w a i t i ng > 3 m ont hs pos t t r a ns pl a nt be f or e i nduc i ng r e t i na l i s c he m i a i t i s a s s ur e d t ha t t he a bi l i t y of H S C e xc l us i ve l y t o r e ge ne r a t e bl ood ve s s e l s i s be i ng a s s e s s e d. N o ot h e r c e l l t h a t c a n be di r e c t l y i s ol a t e d f r om t he m a r r ow ha s be e n s how n t o be c a pa bl e of l ong t e r m r e c ons t i t ut i on i n a t r a ns pl a nt s e t t i ng. A ny c ont a m i na t i ng pr e c ur s or c e l l s w oul d not ha ve ha d t he a bi l i t y t o r e popul a t e t he bone m a r r ow f or t hi s e xt e nde d pe r i od of t i m e a nd w oul d ha ve l ong s i nc e di s a ppe a r e d f r om t he c i r c ul a t i on. F ur t he r pr oof of t he pl a s t i c a bi l i t y of t he H S C i s de m ons t r a t e d i n pr e vi ous w or k w he r e w e i l l us t r a t e how a s i ngl e a d ul t H S C i s c a pa bl e of m a ki ng bo t h bl ood a nd bl ood ve s s e l s i n a t r a ns pl a nt r e c i pi e nt e l i m i na t i ng t he pos s i bi l i t y o f a ny ot he r c ont a m i na t i ng c e l l A l s o, t hi s a c t i vi t y i s s e r i a l l y t r a ns pl a nt a bl e pr oduc i ng f unc t i ona l ve s s e l s i n a r obus t m a nne r 3 0

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50 F i gur e 4 1 N O S knoc kout a ni m a l s e xhi bi t l ong t e r m m ul t i l i ne a ge donor G F P pe r i phe r a l bl ood e ngr a f t m e nt P e r i phe r a l bl ood m ononuc l e a r c e l l s w e r e a na l yz e d by f l ow c yt om e t r y 3 m ont hs pos t t r a ns pl a nt T he f i r s t c ol um n i s B c e l l s e xpr e s s i ng B 220, t he s e c ond c ol um n i s m a c r opha ge s e xpr e s s i n g C D 11b, a nd t he t h i r d c ol um n i s T c e l l s e xpr e s s i ng C D 4. T he t op r ow ( C 57B L / 6) a nd t he s e c ond r ow ( G F P donor s t r a i n) r e f e r e nc e c ont r ol s s how r e c i pi e nt a nd donor f l uo r e s c e nc e T he t hi r d r ow i s a r e pr e s e nt a t i ve C 57B L / 6. G F P c hi m e r i c m ous e de m ons t r a t i ng r ob u s t he m a t opoi e t i c e ngr a f t m e nt T he f or t h r ow ( i N O S G F P ) a nd f i f t h r ow ( e N O S G F P ) a r e r e pr e s e nt a t i ve of e ngr a f t m e nt l e ve l s of t r a ns pl a nt e d knoc kout a ni m a l s T he bot t om r ow i s pe r i phe r a l b l ood s t a i ne d f o r V E G F R 2. C i r c ul a t i ng V E G F R 2 pos i t i ve c e l l s a r e i n C 57B L / 6. G F P i N O S G F P a nd e N O S G F P a ni m a l s N um be r s i n t he t op r i ght c or ne r a r e pe r c e nt a ge s of doubl y l i ne a ge s t a i ne d a nd G F P pos i t i ve c e l l s i N O S = I n duc i bl e N i t r i c O xi de S ynt ha s e e N O S = E ndot he l i a l N i t r i c O xi de S ynt ha s e

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51 T h e N O p at h w ay af f e c t s b l ood ve s s e l f or m at i on A f t e r i nduc t i on of r e t i na l i s c he m i a by l a s e r a bl a t i o n i nj ur y C 57B L 6. G F P c hi m e r a s pr oduc e d a va r i e t y of G F P + bl ood ve s s e l s a t t he s i t e s of i nj ur y r a ngi ng f r om s m a l l c a pi l l a r i e s t o l a r ge r ve s s e l s S i z e w a s m os t l i ke l y d i c t a t e d by t he de gr e e of t he l a s e r i nj ur y a s s e e n i n t he or i gi na l he m a ngi obl a s t c ha r a c t e r i z a t i on ( F i g 3 5 C a nd F i g. 3 6 C & F ) S t r i ki ngl y t he N O S G F P c hi m e r a s pr oduc e d a m a r ke r phe not ype c ha nge f r om t he w i l d t ype m i c e i ndi c a t i ng a r ol e f or t he N O S pa t hw a y i n he m a ngi obl a s t f u nc t i on. i N O S G F P c hi m e r a s pr oduc e d pr i m a r i l y s m a l l hi ghl y b r a nc he d bl ood ve s s e l s t ha t pe r f us e d r e a di l y ( F i g 4 2 E a nd G ) w he n i nj u r e d. T he s e ve s s e l s w e r e l a r ge l y dono r de r i ve d a s s how n i n t he r e d gr e e n m e r ge d i m a ge s de m ons t r a t i ng t he c o l oc a l i z a t i on of t he pe r f us e d f l uo r e s c e nt dye a nd t he G F P c e l l s T he c ont r a l a t e r a l e ye s ha d l i t t l e t o no donor c ont r i but i on a s i s s e e n i n F i gur e 4 2 D a nd F T hi s i ndi c a t e d t ha t t he e N O S i s of or m w hi c h i s s t i l l p r e s e nt i s s uf f i c i e nt f o r m a i nt e na nc e of va s c ul a r he a l t h, a n d t ha t i N O S pl a ys a r ol e i n bl ood ve s s e l br a nc hi ng. I n c ont r a s t e N O S / m i c e r e t i na s e xhi bi t e d a m a r k e d phe not ype w he n c om pa r e d t o bot h c ont r ol a nd i N O S / a ni m a l s done i n pa r a l l e l S t r i ki ngl y, e N O S G F P c hi m e r a s onl y pr oduc e d r e l a t i ve l y l a r ge a nd unb r a nc he d ve s s e l s o f donor or i gi ns r e ga r dl e s s of i s c he m i c i ns ul t ( F i g 4 3 D F ) T he s e ve s s e l s t e nde d t o pe r f us e poor l y i n s pi t e of t he i r l a r ge s i z e O f not e t hi s phe not ype w a s not due t o t he i na bi l i t y t o vi s ua l i z e t he r e d dye i n l a r ge ve s s e l s due t o t he f a c t t ha t t he f l uo r e s c e nt pe r f us a nt c a n be e a s i l y vi s ua l i z e d i n B 6 c ont r ol ve s s e l s of s i m i l a r s i z e I n a ddi t i on a f e w s m a l l ve s s e l s w e r e r e a di l y pe r f us e d a nd t he a ni m a l de m ons t r a t e d t he gr os s m us c l e c ont r a c t i on a nd l i ve r c ol or c ha nge i ndi c a t i ve o f s uf f i c i e nt pe r f us i ng T hi s i s c ons i s t e nt w i t h t he kn ow n va s c ul a r de f e c t s of e N O S /

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52 a ni m a l s W he t he r t hi s l a c k o f ve s s e l f unc t i ona l i t y i s due t o s om e va s c ul a r bl oc ka ge of s om e a l t e r na t i ve de f e c t i s not know n. F i gur e 4 2 T he i N O S pa t hw a y m odul a t e s he m a ngi obl a s t ne ova s c ul a r i z a t i on. i N O S G F P c hi m e r i c m i c e unde r w e nt t he r e t i na l i s c he m i a m ode l f ol l ow e d by pe r f us i on w i t h T R I T C l a be l e d d e xt r a n be f or e e ye e nuc l e a t i on a nd c onf oc a l i m a gi ng of t he r e t i na s A l l pa ne l s a r e r e d a nd gr e e n m e r ge d c onf oc a l i m a ge s P a ne l s D a nd F a r e r e t i na s f r om c ont r ol un t r e a t e d e ye s T he r e i s l i t t l e G F P c ont r i but i on obs e r ve d ( ye l l ow ) P a ne l s E a nd G a r e f r om t r e a t e d e ye s w he r e r obus t G F P c ont r i but i on c a n be s e e n t o va s c ul a t ur e M a gni f i c a t i on i s 60X a nd s i z e ba r i s ~ 10 M

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53 F i gur e 4 3 d e m ons t r a t e s t he l a r ge a nd unbr a nc hi ng c ha r a c t e r i s t i c s of t he dono r de r i ve d ve s s e l s T he s e pi c t ur e s a r e r e d gr e e n m e r ge d c onf oc a l i m a ge s a nd t he l a c k of r e d pe r f ua s nt i ndi c a t e s how poor l y t he s e ve s s e l s f unc t i on. P a ne l s E a nd G a r e f r om t r e a t e d e ye s w he r e r obus t G F P c ont r i but i on c a n be s e e n t o va s c ul a t ur e f or m i ng l a r ge unbr a nc he d ve s s e l s t ha t do not c ont a i n t he T R I T C de xt r a n. P a ne l s D a nd F a r e r e t i na s f r om c ont r ol unt r e a t e d e ye s T he r e i s s i gni f i c a nt G F P c ont r i but i on obs e r ve d w i t h or w i t hout i s c he m i c t r e a t m e nt i nd i c a t i ng t ha t t he e N O S pa t hw a y pl a ys a s i gni f i c a nt r ol e i n e ndot he l i a l c e l l m a i nt e na nc e T he pr of ound c ont r i but i on of H S C de r i ve d G F P + c e l l s t o t he unt r e a t e d r e t i na s of e N O S / r e c i pi e nt s s t r ongl y s ugge s t e d t ha t de l e t i on of t h i s ge ne i nduc e s c h r oni c va s c ul a r i nj ur y. W hi l e i nj ur y w a s know n t o be ne c e s s a r y f or H S C he m a ngi obl a s t a c t i vi t y t hi s w or k de m ons t r a t e s t ha t a c h r oni c l a c k o f e N O S c a n a l s o i nduc e ne ova s c ul a r i z a t i on. I f t hi s pos t ul a t e i s t r ue t he t r a ns pl a nt e d G F P + H S C s houl d c ont r i but e t o va s c ul a r r e ge ne r a t i on t hr oughout t he e N O S / r e c i pi e nt T h e e va l ua t i on of ne ova s c ul a r i z a t i on i n c ont r a l a t e r a l e ye s de m ons t r a t e s a nd a gr e e s w i t h c u r r e nt s t udi e s t ha t de t e r m i ne d s om e t ype of i nj u r y i s r e qui r e d f or f unc t i ona l p l a s t i c i t y o f H S C I n t ypi c a l p hys i ol ogi c c ondi t i ons l i t t l e or no H S C c ont r i but i on t o no r m a l t i s s ue oc c ur s but w he n a c ut e ( i s c he m i c i nj ur y) or c h r oni c ( e N O S knoc kout ) pa t hol ogi c c ondi t i ons a r i s e H S C r e a di l y c ont r i but e t o va s c ul a r t i s s ue T he s e e xpe r i m e nt s f or m a l l y de m ons t r a t e t ha t i N O S a c t i vi t y a t t he s i t e o f va s c ul a r i nj ur y di c t a t e s t he s i z e a nd b r a nc h c ha r a c t e r i s t i c s o f ne w ve s s e l s f or m e d i n a dul t a ni m a l s F ur t he r m or e t he ne w ve s s e l s a r e f or m e d i n a l l or l a r ge pa r t f r o m c i r c ul a t i ng e ndot he l i a l pr oge ni t or s of H S C or i gi n

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54 F i gu r e 4 3 T he e N O S pa t hw a y m odul a t e s he m a ngi obl a s t ne o va s c ul a r i z a t i on. e N O S G F P c hi m e r i c m i c e unde r w e nt t he r e t i na l i s c he m i a m ode l f ol l ow e d by pe r f us i on w i t h T R I T C l a be l e d de xt r a n be f or e e ye e nuc l e a t i on a nd c onf oc a l i m a gi ng of t he r e t i na s A l l pa ne l s a r e r e d a nd gr e e n m e r ge d c onf oc a l i m a ge s P a ne l s D a nd F a r e r e t i na s f r om c ont r ol un t r e a t e d e ye s T he r e i s s i gni f i c a nt G F P c ont r i but i on obs e r ve d ho w e ve r t he ve s s e l s f or m e d do not c ont a i n t he T R I T C de xt r a n t he r e f o r e a r e poor l y f unc t i ona l P a ne l s E a nd G a r e f r om t r e a t e d e ye s w he r e r obus t G F P c ont r i but i on c a n be s e e n t o va s c ul a t ur e f or m i ng l a r ge unbr a nc he d v e s s e l s w hi c h do not c ont a i n t he T R I T C de xt r a n, t he r e f o r e a r e poo r l y f u nc t i ona l P a ne l D i s 60X P a ne l E i s 4X m a gni f i c a t i on. P a ne l F i s 10X m a gni f i c a t i on. P a ne l G i s a c om pos i t e of 60X i m a ge s S i z e ba r i s ~ 10 M unl e s s not e d ~ 100 M

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55 T h e N O S p at h w ay af f e c t s b l ood ve s s e l b r an c h i n g c h ar ac t e r i s t i c s T o f u r t he r e xa m i ne t he r ol e of N O S i n ne ova s c ul a r i z a t i on, r e t i na s f r om t he non t r e a t e d c ont r a l a t e r a l e ye s w e r e c om pa r e d t o t he i nj ur e d r e t i na s of W T i N O S / a nd e N O S / r e c i pi e nt s T hi s w a s done i n or de r t o e l uc i da t e w he t he r N O S c oul d dr i ve H S C f or m a t i on o f va s c ul a t ur e w i t hout i s c he m i c i nj ur y a nd gr ow t h f a c t or a dm i ni s t r a t i on. i N O S / a ni m a l s r e s ponde d i n a s i m i l a r f a s hi on t o W T a ni m a l s w i t h p r oduc t i on o f G F P + H S C de r i ve d ve s s e l s i n t he i nj ur e d r e t i na ( F i g 4 2 E & G ) but l i t t l e o r no c ont r i but i on c oul d be f ound i n t he r e t i na s f r om t he c ont r a l a t e r a l unt r e a t e d e ye ( F i g. 4 2 D & F ) U ne xpe c t e dl y, r e t i na s f r om e N O S / r e c i pi e nt s w h i c h a s de s c r i be d i n ot he r s t udi e s ha ve s ys t e m i c va s c ul a r dys f unc t i on, de m ons t r a t e d r obus t G F P + H S C de r i ve d c ont r i bu t i on t o t he pr e e xi s t i ng va s c ul a r e ndot he l i um o f bot h t e s t ( F i g. 4 3 E & G ) a nd c ont r ol e ye s ( F i g 4 3 D & F ) A f t e r i nduc t i on of r e t i na l i s c he m i a by l a s e r a bl a t i o n i nj ur y C 57B L 6. G F P c hi m e r a s pr oduc e d a va r i e t y of G F P + bl ood ve s s e l s a t t he s i t e s of i nj ur y r a ngi ng f r om s m a l l c a pi l l a r i e s t o l a r ge r ve s s e l s I n C 57B L 6. G F P c hi m e r a s s i z e w a s m os t l i ke l y di c t a t e d by t he d e gr e e of t he l a s e r i nj u r y ( F i g. 3 5 D F ) a nd no G F P + c ont r i but i on t o va s c ul a t ur e w a s obs e r ve d i n c ont r ol e ye s ( F i g. 3 5 B ) i N O S G F P c hi m e r a s pr oduc e d pr i m a r i l y s m a l l hi ghl y br a nc he d bl ood ve s s e l s t ha t pe r f us e d r e a di l y i n t r e a t e d e ye s ( F i g. 4 2 D & F ) T he s e a ni m a l s ha d l i m i t e d dono r E P C c ont r i bu t i on i n c ont r a l a t e r a l unt r e a t e d e ye s ( F i g. 4 2 E & G ) S t r i ki ngl y, e N O S G F P c hi m e r a s onl y p r oduc e d r e l a t i ve l y l a r ge a nd unbr a nc he d ve s s e l s r e ga r dl e s s of i s c he m i c i ns ul t ( F i g. 4 3 D t hr ough G ) T he br a nc hi ng c ha r a c t e r i s t i c s of t he t hr e e s t r a i ns w e r e m a r ke dl y d i f f e r e nt s ugge s t i ng t ha t t he N O pa t hw a y f unc t i ons i n ve s s e l or ga ni z a t i on. T ot a l b r a nc h poi nt s of G F P ve s s e l s pe r 60X f i e l d of vi e w w e r e c ount e d f or e a c h ge not ype ( F i g ur e 4 4) C 57B L 6 m ode l c ont r ol

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56 c ohor t s a ve r a ge d a bout 18 b r a nc h poi nt s pe r vi s ua l f i e l d. i N O S / r e c i pi e nt s ha d ne a r l y 3 f ol d m or e br a nc h poi nt s pe r f i e l d, w hi l e e N O S / r e c i pi e nt s a ve r a ge d 61 t i m e s l e s s F i gur e 4 4 T he ni t r i c oxi de pa t hw a y a l t e r s he m a ngi obl a s t bl o od ve s s e l f or m e d br a nc hi ng c ha r a c t e r i s t i c s C onf oc a l Z s e r i e s i m a g e s w e r e c om pr e s s e d a nd c ount e d bl i ndl y f or num be r of ve s s e l br a nc h poi nt s pe r i m a ge C 57B L / 6. G F P r e t i na s a ve r a ge d 17 8 br a nc he s pe r i m a ge ( n= 5) i N O S G F P r e t i na s a ve r a ge d 48 br a nc h poi n t s pe r i m a ge ( n= 4) e N O S G F P r e t i na s a ve r a ge d 0. 29 br a nc h po i nt s pe r i m a ge ( n= 38) T he bl ood ve s s e l s of i N O S / r e t i na s w e r e 2 7 t i m e s m o r e br a nc he d t ha n W T a ni m a l s ( p< 0. 0001 ) w hi l e e N O S / w e r e 61. 5 t i m e s l e s s br a nc he d t ha n W T ( p< 0. 0002 ) T he s e e xpe r i m e nt s f or m a l l y de m ons t r a t e t ha t N O S a c t i vi t y a t t he s i t e of va s c ul a r i nj ur y di c t a t e s t he s i z e a nd b r a nc h c ha r a c t e r i s t i c s o f ne w ve s s e l s f or m e d i n a dul t a ni m a l s F ur t he r m or e t he ne w ve s s e l s a r e f or m e d i n a l l or l a r ge pa r t f r o m c i r c ul a t i ng e ndot he l i a l pr oge ni t or s of H S C or i gi n I n a ddi t i on, a c hr on i c l a c k of e N O S a c t i vi t y ove r t i m e c om bi ne d w i t h our i s c he m i c i nj ur y m ode l r e s ul t s i n a pr ol i f e r a t i ve r e t i nopa t hy i nt o t he pr e r e t i na l s pa c e t he ha l l m a r k o f pr o l i f e r a t i ve r e t i n opa t hy de ve l ope d i n di a be t i c pa t i e nt s

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57 N O p r od u c t i on af f e c t on vas c u l at u r e i n n on oc u l ar t i s s u e T he f i ndi ng t ha t H S C ha ve t he a bi l i t y t o c ont r i but e t o va s c ul a r t i s s ue i n non t r e a t e d e ye s dur i ng a di s e a s e s t a t e l e nds t o t he e xa m i na t i o n of t i s s ue s f a r r e m ove d a nd un r e l a t e d t o t he e ye T o de t e r m i ne t he e xt e nt of dono r G F P + H S C c ont r i but i on t o t he ove r a l l va s c ul a r s ys t e m m ul t i pl e t i s s ue s ( s pl e e n, t hym us b r a i n, ki dne y l i ve r m us c l e s ki n, a nd gut ) f r om t he C 57B L 6. G F P i N O S G F P a nd e N O S G F P c hi m e r a s ( n= 10 pe r c ohor t ) w e r e ha r ve s t e d. E a c h of t he s e a ni m a l s ha d de m ons t r a t e d l ong t e r m m u l t i l i ne a ge he m a t opoi e t i c e ngr a f t m e nt a nd ha d unde r gone t he r e t i na l i s c he m i a m ode l A t one m ont h a f t e r t he i nduc t i on of r e t i na l i s c he m i a t he a ni m a l s w e r e e ut ha ni z e d a nd pe r f us e d w i t h t e t r a m e t hyl r hoda m i ne i s ot hi oc ya na t e c onj uga t e d d e xt r a n ( T R I T C a r e d f l uor e s c e nt dye ) t hr ough t he l e f t ve nt r i c l e T i s s ue s w e r e ha r ve s t e d a nd i m m e di a t e l y pl a c e d i n opt i m um c ut t i ng t e m pe r a t ur e m e di um a nd f r oz e n t o pr e s e r v e G F P 10 m i c r on t hi c k s e c t i ons w e r e t he n c ut a nd m ount e d w i t h D A P I t o s t a i n t he nuc l e i S e c t i ons w e r e e xa m i ne d by f l uor e s c e nt m i c r os c opy f or G F P + c ont r i but i ons t o t he va s c ul a t ur e R e s ul t s f or t he s pl e e n, t hym us a nd br a i n a r e s how n ( F i g 4 5) I n a l l c a s e s t he C 57B L 6. G F P a nd i N O S G F P yi e l de d s i m i l a r r e s ul t s : l i m i t e d e vi de nc e f o r G F P + c e l l s be i ng i nc or por a t e d i nt o bl ood ve s s e l s i n a ny t i s s ue out s i de of t he t r e a t e d r e t i na ( F i g. 4 5 A F a nd da t a not s how n) T hi s i ndi c a t e s t ha t w hol e body i r r a di a t i on a l one i s not s uf f i c i e nt f or i nduc t i on of H S C c ont r i but i on t o va s c ul a t ur e i n t i s s ue s w hi c h a r e not t r e a t e d f u r t he r I n c ont r a s t e N O S G F P c hi m e r a s e xhi bi t e d r obus t G F P + c ont r i but i ons t o t he va s c ul a t ur e ( a s de t e r m i ne d by c o l oc a l i z a t i on w i t h t he pe r f us e d r e d f l uor e s c e nt dye ) i n a l l t i s s ue s e xa m i ne d ( F i g. 4 5 G L a nd da t a not s how n) L a c k of e N O S c r e a t e s a pa t hol ogi c va s c ul a r c ondi t i on w he r e H S C a r e i nduc e d t o c ont r i but e t o va s c ul a r r e pa i r t h r oughout a n or ga ni s m

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58 F i gur e 4 5 C hr oni c va s c ul a r i nj ur y i n e N O S G F P c hi m e r a s i n duc e s w i de s pr e a d he m a ngi obl a s t a c t i vi t y f r om a dul t H S C N O S kno c kout a ni m a l s w hi c h unde r w e nt t he ne ova s c ul a r i z a t i on m ode l a nd s pl e e n ( A G B & H ) t hym us ( C I D & J ) a nd br a i n ( E K F & L ) w e r e ha r ve s t e d f r om T R I T C pe r f us e d a ni m a l s 10 M c r yos e c t i ons w e r e pr e pa r e d a nd m ount e d w i t h V e c t a s hi e l d pl us D A P I i N O S G F P ( A F ) a nd e N O S G F P ( G L ) c hi m e r a s w e r e e xa m i ne d by f l uor e s c e nc e m i c r os c opy. T he dono r G F P H S C de r i ve d c e l l s a r e g r e e n, a nd t he T R I T C l a be l e d de xt r a n pe r f us a nt i s r e d. P a ne l s A & G a r e m a gni f i c a t i on X 40 A l l r e m a i ni ng pa ne l s a r e a l l m a gni f i c a t i on X 64.

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59 T o a s c e r t a i n t he e ndot he l i a l c e l l na t ur e of t he G F P + c e l l s s ur r oundi ng t he ve s s e l l um e ns t i s s ue s e c t i ons w e r e s t a i ne d f or t he pa n e ndot he l i a l c e l l m a r ke r M E C A 32. T e n m i c r on f r oz e n s e c t i ons w e r e s t a i ne d w i t h a p r i m a r y a nt i bo dy t o M E C A 32 f ol l ow e d by a T e xa s R e d c onj uga t e d s e c onda r y a nt i body a nd D A P I E n dot he l i a l c e l l s w e r e t he n s c or e d f or t he pr e s e nc e of bot h M E C A 32+ a nd G F P + c e l l s vi a f l uor e s c e nt m i c r os c opy. S p l e ni c s e c t i ons de m ons t r a t e t he c ha r a c t e r i s t i c r e s ul t s obs e r ve d i n a l l t i s s ue s s t udi e d ( F i g. 4 6) D o nor de r i ve d he m a t opoi e t i c c e l l s i n t he s pl e e n s e r ve a s i nt e r na l ne ga t i ve s t a i ni ng c ont r ol s f or M E C A 32 i n e a c h s e c t i on. W T a ni m a l s s how e d oc c a s i ona l G F P + M E C A 32+ e ndot he l i a l c e l l s i n t he br a i n ( c l os e s t or ga n t o t he s i t e of V E G F a dm i ni s t r a t i on ) w i t h t he m a j or i t y of t i s s ue s s uc h a s t he s pl e e n ( F i g. 4 6 A D ) ki dne y, l i ve r a nd m us c l e s how i ng no donor de r i ve d e ndot he l i a l c e l l s i N O S / a ni m a l s w hi c h e xhi bi t m i nor s ys t e m i c va s c ul a r de f e c t s ha d oc c a s i ona l G F P + M E C A 32+ e ndot he l i a l c e l l s i n t he s pl e e n ( F i g. 4 6 E H ) a nd ot he r t i s s ue s O ve r a l l G F P + H S C de r i ve d c ont r i bu t i on t o t he va s c ul a t ur e of i N O S / a ni m a l s out s i de t he a r e a of r e t i na l i s c he m i a w a s a t m os t 1 % i n > 150 s e c t i ons e xa m i ne d f or M E C A 32+ ve s s e l s R obus t G F P + donor de r i ve d e ndot he l i a l c e l l pr odu c t i on w a s obs e r ve d i n e N O S / r e c i pi e nt s w hi c h ha ve be e n d e m ons t r a t e d t o ha ve c hr oni c a nd s e ve r e va s c ul a r pa t hol ogy. M os t ve s s e l s w e r e qui t e l a r ge a nd m os t s how e d e xt e ns i ve G F P + H S C de r i ve d M E C A 32+ e ndot he l i a l c e l l c ont r i bu t i ons i n t he s pl e e n ( F i g. 4 6 I P ) a nd ot he r t i s s ue s e xa m i ne d. T he H S C c ont r i but i on t o va s c ul a t ur e de t e c t e d i n unt r e a t e d t i s s ue w a s a na l ogous t o t ha t obs e r ve d i n t he t r e a t e d r e t i n a s de m ons t r a t i ng t ha t c hr oni c va s c ul a r i nj ur y a ppe a r s t o be s uf f i c i e nt t o i nduc e t he he m a n gi obl a s t a c t i vi t y of a dul t H S C

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60 F i gur e 4 6 D onor de r i ve d c e l l s l i ni ng va s c ul a r l um e ns i n e N O S G F P a ni m a l s a r e M E C A 32 pos i t i ve S pl e ni c c r yos e c t i ons w e r e pr e pa r e d f r om C 57B L / 6. G F P ( A D ) i N O S G F P ( E H ) a nd e N O S G F P ( I P ) c hi m e r a s S e c t i ons w e r e s t a i ne d w i t h a nt i M E C A 32 a nt i bod y a nd a T e xa s R e d c onj uga t e d s e c onda r y a nt i body t o de l i ne a t e va s c ul a r e ndot he l i um S e c t i ons w e r e m ount e d w i t h D A P I ( A E I M ) t o de l i ne a t e nuc l e i w i t h bl ue f l uor e s c e nc e e xa m i ne d f or G F P e xpr e s s i on ( B F J N ) vi a gr e e n f l uor e s c e nc e or M E C A 32 s t a i ni ng ( C G K O ) vi a r e d f l uor e s c e nc e M e r ge d i m a ge s of t he D A P I G F P a nd M E C A 32 T e xa s R e d s t a i ns a r e s how n i n D H L a nd P ( A L ) M a gni f i c a t i on X 64. ( M P ) M a gni f i c a t i on X 32. Q u an t i t a t i on an d l oc at i on o f N O S p r od u c e d i n k n oc k ou t a n i m al s T o a s c e r t a i n t he i n f l ue nc e a nd de t e r m i ne t he e xpr e s s i on of N O S i n r e t i na s l a c ki ng one s pe c i f i c N O S i s of or m r e t i na s w e r e di s s e c t e d a nd s t a i ne d w i t h i s of or m s pe c i f i c a nt i bodi e s i N O S / ( F i g 4 7 A C t op ) a nd e N O S / ( F i g. 4 7 A C bot t om ) a ni m a l s w e r e

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61 qua nt i t a t e d f or N O S e xpr e s s i on i n pa r a l l e l A ni m a l s w e r e s a c r i f i c e d a nd t he e ye s e nuc l e a t e d a s de s c r i be d. T he d i s s e c t e d r e t i na s w e r e t he n i m a ge d t h r ough c onf oc a l m i c r os c opy. F i gur e 4 7 ( A t op a nd bot t om ) de m o ns t r a t e s t ha t i n e a c h knoc kout s t r a i n t he i s of or m w hi c h i s de l e t e d i s not e xp r e s s e d i n v i v o a t de t e c t a bl e l e ve l s T he i N O S / ha s r e l a t i ve a m ount s of N O S e xpr e s s e d ( B a nd C t op) w hi l e t he e N O S / r e t i na s de m ons t r a t e a n i nc r e a s e i n i N O S e xp r e s s i on a s s e e n t hr oughout t he l a r ge a nd pa r t i c ul a r l y t he s m a l l e r ve s s e l s ( B a nd C bot t om ) T hi s c on f i r m s t ha t t he r e i s a n upr e gul a t i on of i N O S e xpr e s s i on i n e N O S knoc kout r e t i na s i ndi c a t i ng a dys r e gul a t i on i n a m ount o f N O pr oduc e d r e s ul t i ng i n t he pa t hol ogi c bl ood ve s s e l f or m a t i on obs e r ve d i n t he s e a ni m a l s D i s c u s s i on T hr ough g r ow t h f a c t or a dm i ni s t r a t i on a nd i s c he m i c i nj ur y t o t he r e t i na H S C c a n be i nduc e d t o t r a ns di f f e r e nt i a t e i nt o va s c ul a r e ndot he l i um F ur t he r m or e t r a ns di f f e r e nt i a t i on c a n a l s o be obs e r ve d dur i ng a pa t hol ogi c di s e a s e s t a t e of c hr oni c va s c ul a r i nj ur y. T he s ubs t a nt i a l r ol e N O pl a ys i n v a s c ul a r t one a nd t he pr e s e nc e of a N O S i s of or m s pe c i f i c a l l y f ound i n e ndot he l i a l c e l l s hi nt e d a t a r ol e of N O i n bl ood ve s s e l f or m a t i on a nd r e m ode l i ng. N O S a c t i vi t y c a n a l s o di c t a t e t he ge ne r a l s i z e a nd b r a nc h c ha r a c t e r i s t i c s of ne w bl ood ve s s e l s f or m e d i n r e s p ons e t o i s c he m i c i nj ur y a nd gr ow t h f a c t or a dm i ni s t r a t i on U s i ng t he ne ova s c ul a r m ode l of i nduc i ng H S C he m a ngi obl a s t a c t i vi t y t o p r om ot e bl ood ve s s e l f or m a t i on i n t he a dul t r e t i na donor W T H S C t r a ns pl a nt e d i nt o i N O S / r e c i pi e nt s pr oduc e d hi gh l y br a nc he d ve s s e l s t ha t a r e ge ne r a l l y s m a l l e r i n s i z e T he s e H S C a r e f unc t i oni ng i n a n e nvi r onm e nt w he r e l oc a l N O pr oduc t i on i s s i m i l a r t o w ha t i s s e e n i n w i l d t ype n on i nf e c t i on c ondi t i ons due t o t he e N O S i s of or m w hi c h i s c o ns t i t ut i ve l y a c t i ve i n e nd ot he l i a l c e l l s A w i de va r i e t y of ve s s e l s i z e s a r e f or m e d w hi c h a r e f unc t i ona l a s m e a s ur e d by pe r f us i on o f m a r ke r dye

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62 F i gur e 4 7 N i t r i c oxi de p r oduc t i on i s dys r e gul a t e d i n e N O S k noc kout a ni m a l s i N O S / a nd e N O S / r e t i na s w e r e s t a i ne d w i t h N O S i s of or m s pe c i f i c a nt i bodi e s V e s s e l s w e r e i l l um i na t e d by a ggl u t i n s t a i ni ng ( r e d ) a nd r e gi ons w hi c h w e r e pos i t i ve f or t he N O S a nt i body a r e gr e e n. I n t he t op r ow pa ne l A de pi c t s a n i N O S / r e t i na s t a i ne d w i t h i N O S s pe c i f i c a nt i b ody. P a ne l s B a nd C a r e i N O S / s t a i ne d w i t h e N O S i s of or m s pe c i f i c a nt i body. I n t he bot t om r ow P a ne l A de pi c t s a n e N O S / r e t i na s s t a i ne d w i t h e N O S i s of or m s pe c i f i c a nt i body. P a ne l B a n d C a r e e N O S / s t a i ne d w i t h i N O S i s of o r m s pe c i f i c a nt i body T he br a nc h pa t t e r n i ng i s s i m i l a r t o nor m a l m ous e va s c ul a t ur e a l t hough s l i ght l y i nc r e a s e d. C ont r a s t i ngl y, e N O S / r e c i pi e nt s pr od uc e d pr i m a r i l y unbr a nc he d ve s s e l s of l a r ge s i z e T hi s i ndi c a t e s t ha t t he l oc a l N O p r oduc t i on due t o t he e N O S i s of or m a c t i vi t y i s ne c e s s a r y f or nor m a l he m a ngi obl a s t de r i ve d bl o od ve s s e l f or m a t i on. M odi f i c a t i on of N O pr oduc t i on vi a t he e N O S i s of o r m w hi c h c a n n ow be s pe c i f i c a l l y t a r ge t e d w i t h pha r m a c ol ogi c a l a ge nt s c oul d pr ov i de a m e a ns t o i nf l ue nc e ne ova s c ul a r i z a t i on a nd

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63 a ngi oge ne s i s i n pa t hol ogi c di s e a s e s s u c h a s D i a be t i c R e t i nopa t hy, a nd R e t i nopa t hy of P r e m a t ur i t y. T hi s a l t e r e d H S C r e s pons e w a s i n a d di t i on t o t he w i de s pr e a d va s c ul a r r e m ode l i ng by dono r H S C s e e n t h r oughout t he e N O S / r e c i pi e nt s e ve n i n non i nj ur e d or ga ns a nd t i s s ue s T he ve s s e l s of e N O S / r e c i pi e nt s w e r e di f f i c ul t t o pe r f us e i ndi c a t i ng t he i r ge ne r a l va s c ul a r dys f unc t i on T hi s f ur t he r e m pha s i z e s t he c r uc i a l na t ur e of t he e N O S i s of or m s N O p r oduc t i on t o e ns ur e pr ope r v e s s e l f or m a t i on a nd f unc t i ona l i t y f or e s ha dow i ng s t udi e s oc c ur r i ng out s i de t he r e a l m of t he e ye on w hi c h ou r m ode l f oc us e s T hi s c ha pt e r r e i t e r a t e s w or k de m ons t r a t i ng t ha t a n i nj ur y s t a t e w he t he r i t be a c ut e a s s e e n i n t he phot oc oa gul a t i on of bl ood ve s s e l s o r c hr oni c a s s e e n i n t he va s c ul a r pa t hol og y obs e r ve d i n t he e N O S knoc kout a ni m a l s i s r e qui r e d f o r H S C pl a s t i c i t y. 8 5 F ur t he r m or e l oc a l m e di a t or s i nc l udi ng V E G F a nd N O c a n g r e a t l y i n f l ue nc e not onl y t he s i z e a nd a m ount of ne w bl ood ve s s e l s f or m e d b ut a l s o t he i r f unc t i ona l i t y T he r e gul a t i on of N O S a c t i vi t y a s a m e a ns t o i n f l ue nc e t he r e m ode l i ng o f va s c ul a r be ds m a y pr ovi de s pe c i f i c t r e a t m e nt r e gi m e s I t m a y p r ove b e ne f i c i a l t o s e l e c t i ve l y i nhi bi t a p a r t i c ul a r N O S i s of or m t o c or r e c t a n i m ba l a nc e t h us a l t e r i ng t he de ve l opm e nt of ne w bl ood ve s s e l s W he t he r a de r e gul a t i on of N O S o r N O a c t i vi t y i s a c a us a l e f f e c t i n hum a n pr ol i f e r a t i ve r e t i nopa t hy r e m a i ns t o be de t e r m i ne d. I f t hi s i s t he c a s e pha r m a c e ut i c a l s t ha t a f f e c t t he s e a c t i vi t i e s s om e a l r e a dy i n us e f or non r e l a t e d di s e a s e t r e a t m e nt m a y pr ovi de a n e f f e c t i ve t he r a py o r p r e ve nt a t i ve f or hu m a n di s e a s e s i n r e l a t i on t o p r ol i f e r a t i ve or pa t hol ogi c a l bl ood ve s s e l f or m a t i on due t o he m a ngi obl a s t a c t i vi t y

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64 C H A P T E R 5 L I M I T A T I O N S O F S T E M C E L L R E S E A R C H A N D E T H I C A L C O N S I D E R A T I O N S W hi l e t he pr om i s e o f s t e m c e l l s a s t he r a py i s c ons i de r a bl e t he r e a r e l i m i t a t i ons t o t he i r us a ge i nc l udi ng t he i r bi ol ogi c a l a c t i vi t y a nd e t hi c a l i m pl i c a t i ons T he bi ol ogi c a l l i m i t a t i on s of s t e m c e l l pl a s t i c i t y a r e r e l a t e d t o t he i r a bi l i t y of t o t r a ns di f f e r e nt i a t e a nd t he i r pot e nt i a l t o f o r m t i s s ue s T he e t hi c a l i m pl i c a t i ons a r e m a ny, a nd I w i l l a dd r e s s s om e of t he m i n t hi s c ha pt e r B i ol ogi c al l i m i t a t i on s I n t he c ur r e nt r e s e a r c h e nvi r on m e nt t he r e i s c ur r e nt l y he a t e d c ont r ove r s y ove r t he r e por t e d pl a s t i c i t y o f s t e m c e l l s pa r t i c ul a r l y t he H S C i n r e l a t i on t o c a r di a c m us c l e l i ve r a nd t he ne r vous s ys t e m 1 2 1 T he l i m i t a t i ons of a dul t s t e m c e l l s i nc l ude : t he i r di m i ni s he d c a pa c i t y t o p r ol i f e r a t e w he n c om pa r e d t o e m br yon i c s t e m c e l l s A ut o l ogous t r a ns pl a nt a t i on of c e l l s ba c k i nt o a pa t i e nt w i l l s t i l l r e t a i n ge ne t i c a bno r m a l i t i e s C or r e s pondi ngl y, e x vi vo e pa nde d or ge ne t i c a l l y m odi f i e d c e l l s us e d f or t he r a py m a y pr oduc e unf or e s e e n c ons e que nc e s S om e t i s s ue s a r i s e t hr ough a c om pl e x de ve l opm e nt a l f a t e s uc h a s t he pa nc r e a s w hi c h i s de r i ve d f r om t h e i nf ol di ng o f s e ve r a l t i s s ue l a ye r s w hi c h w i l l not be e a s i l y m i m i c ke d i n vi t r o. T he r e s i de nt s t e m c e l l s w i t hi n a t i s s ue t ype a r e e xt r e m e l y r a r e T h i s pa uc i t y of c e l l s m a ke s t he m e xt r e m e l y di f f i c ul t t o i de nt i f y, i s ol a t e a nd pur i f y. I n a ddi t i on, i t ha d pr ove n di f f i c ul t t o c ul t u r e a dul t s t e m c e l l s c om pa r e d t o e m br yoni c s t e c e l l s i n vi t r o l i m i t i ng t he i r us e a s pot e nt i a l t he r a py F i na l l y f us i on I t i s be l i e ve d t ha t c e l l f us i on m a y be a pot e n t i a l m e t hod f or i n t r oduc i ng ne w ge ne t i c m a t e r i a l t o c or r e c t m ut a t e d or m a l f unc t i oni ng ge ne s t ha t c a us e di s e a s e C e l l

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65 f us i on oc c ur s w he n t w o o r m o r e c e l l s c om bi ne t o f or m one c e l l w hi c h t he n c ont a i ns m o r e ge ne t i c m a t e r i a l t ha n no r m a l F us i on ha s be e n s how n t o oc c ur i n e m br yon i c a l ong w i t h a dul t s t e m c e l l s 1 2 2 1 2 3 I n a dul t m i c e f us e d l i ve r c e l l s m a y c ont a i n 80 c hr om os om e s doubl e t he a m ount f ound i n a no r m a l m ous e l i ve r c e l l s O t he r c e l l t ype s i nc l udi ng m e ga ka r yoc yt e s a nd m us c l e c e l l s f unc t i on w i t h a n i nc r e a s e d pl oi dy a s w e l l I n m os t c e l l t ype s how e ve r a ne upl oi dy w oul d be de t r i m e nt a l of t e n i nduc i ng a popt os i s or c e l l t r a ns f or m a t i on. T he r e s ul t i ng i m ba l a nc e i n ge ne d os a ge c oul d l e a d t o nonf unc t i ona l t i s s ue or c a nc e r I t i s not c l e a r l y unde r s t ood, nor h a s i t be e n de f i ni t i ve l y pr ove n w he t he r f us i on i s a pa t hw a y f o r s t e m c e l l pl a s t i c i t y w i t h r e s e a r c h i ndi c a t i ng t ha t f us i on c a n r e s ul t i n, but i s not ne c e s s a r y f or pl a s t i c i t y. 1 2 4 1 2 6 I f t he f or m e r i s t he c a s e t he n i nve s t i ga t i on i nt o t he e f f e c t of pr oduc i ng c e l l s w i t h a n i nc r e a s e d pl oi dy a t a ny t i m e m us t be done A f us i on e ve nt dur i ng c ont r i but i on t o t he r e ge ne r a t e d t i s s ue c oul d pr e c l ude s t e m c e l l ba s e d t he r a py. T he f i r s t w or k c ha r a c t e r i z i ng f us i on done by T e r a d a e t al f ound t ha t f us i on e ve nt s i n v i t r o w e r e e xt r e m e l y r a r e 1 2 2 C ons e que nt l y, t he r obus t a m ount of donor de r i ve d c ont r i but i on i s unl i ke l y t o a r i s e f r om s uc h r a r e e ve nt s R e c e nt w or k f r om our l a b de m ons t r a t e d t ha t t he c e l l s de r i ve d a r e di pl oi d a nd a ny unr e s ol ve d f us i on o f t he H S C w oul d ha ve r e s ul t e d i n a n i nc r e a s e i n pl oi dy s how n i n f i gur e 5 1. 1 2 7 T he c i r c ul a t i ng e ndot he l i a l pr e c ur s or c e l l s de r i ve d f r om t he dono r m a r r o w e xhi bi t nor m a l 2N pl oi dy w he n s t a i ne d w i t h t he D N A dye pr opi di um i odi de T h i s doe s not r u l e out t he pos s i bi l i t y of a f us i on e ve nt w hi c h w a s r e s ol ve d r e s ul t i ng i n n or m a l pl oi dy how e ve r a nd s pe c i a l s pe c i f i c e xpe r i m e nt s m uc h be done t o a s c e r t a i n i f t hi s f us i on r e s ol ut i on i s oc c ur r i ng.

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66 F i gur e 5 1 P r opi di um i odi de s t a i ni ng of c i r c ul a t i ng E P C doe s not i ndi c a t e a bnor m a l pl oi dy. L ong t e r m G F P e ngr a f t e d C 57B L / 6 r e c i pi e nt s unde r w e nt t he ne ova s c ul a r i z a t i on m ode l A ni m a l s w e r e bl e d, a nd F I C O L L e nr i c he d pe r i phe r a l bl ood w a s s t a i ne d a nd F A C S e nr i c he d f or V E G F R 2 e xp r e s s i on. C e l l s w e r e s t a i ne d w i t h pr opi di um i odi de a nd a na l yz e d by F A C S L e f t pa ne l de pi c t s E P C f r om a nont r a ns pl a nt e d C 57B L / 6 a ni m a l bl e d i n pa r a l l e l R i ght de pi c t s a t e s t a ni m a l B ot h e xhi bi t c l a s s i c a l di pl oi d s t a i ni ng pr of i l e s E t h i c s A ny c om pr e he ns i ve a na l ys i s of t he s t e m c e l l f i e l d w oul d be r e m i s s t o no t i nc l ude t he e t hi c a l i m pl i c a t i ons f o r t he i r us e i n t he r a py. T he f i e l d ha s pr ove n t o be a pol a r i z i ng i s s ue w hi c h i nf l ue nc e s m a ny r e l i gi ous a nd pol i t i c a l r e f e r e nda bo r n s i m ul t a ne ous l y w i t h D ol l y, t he f i r s t c l one d m a m m a l T he de ba t e a r i s e s f r om t he us e of e m b r yoni c s t e m c e l l s w hi c h a t t hi s t i m e c a n onl y be i s ol a t e d f r o m a n e m br yo r e s ul t i ng i n t he e m b r yo s de s t r uc t i on. T he s e c e l l s a r e a t t r a c t i ve how e ve r b e c a us e of t he i r a bi l i t y t o p r oduc e a l l o f t he c e l l t ype s i n a n a dul t a ni m a l or pr ovi de a n e nvi r onm e nt i n w hi c h D N A c a n be t r a ns f e r r e d i n nuc l e a r t r a ns f e r T o da t e no s i ngl e a dul t c e l l ha s be e n s how n t o ha ve t he pl ur i pot e nc y of e m br yoni c c e l l s a l ong w i t h p r ovi di ng t he i nt r a c e l l ul a r e nvi r onm e nt a l c ue s ne c e s s a r y t o r e pr og r a m t r a ns f e r r e d D N A A dul t s t e m c e l l s do not ha ve t he s a m e c a pa c i t y

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67 t o pr oduc e a ny t i s s ue or c e l l t ype s T hi s i nhe r e nt e xt r a or di na r y t he r a pe ut i c pot e nt i a l r e s ul t i ng f r om t he de s t r uc t i on of a n e m br yo l e a ds t o oppos i t i on ba s e d on t he i de a of w he n l i f e be gi ns I n t he U ni t e d S t a t e s m a ny F unda m e nt a l i s t C hr i s t i a n gr oups a r e s t r ong l y oppos e d t o e m br yoni c s t e m ( E S ) c e l l r e s e a r c h a s t he de s t r uc t i on of t he e m br yo i s c ons i de r e d a bor t i on, o r m u r de r T he y be l i e ve t ha t a ny a nd a l l r e s e a r c h us i ng hum a n s t e m c e l l s i s m or a l l y una c c e pt a bl e O t he r r e l i gi ons how e ve r a r e s uppor t i ve of e m br yoni c r e s e a r c h. M a ny J e w i s h gr oups of di f f e r i ng de nom i na t i ons d o not vi e w a n e a r l y s t a ge e m br yo a s a hum a n be i ng, t he r e f or e us a ge of e m br yoni c t i s s ue i s not de s t r uc t i on of a hum a n M a ny H um a ni s t s U ni t a r i a n U ni ve r a s l i s t s a nd M us l i m c l e r i c s ha ve a l s o c om e out i n f a vor of s t e m c e l l r e s e a r c h. I n a ddi t i on p r opone nt s poi nt o ut t ha t s t e m c e l l r e s e a r c h us e s di s c a r de d e m br yos f r om i n v i t r o f e r t i l i z a t i on a nd t ha t f e r t i l i t y c l i ni c s r out i ne l y de s t r oy t hous a nds of e m br yos T he s e unus e d e m br yos w o ul d nor m a l l y be di s c a r de d or ke pt f r oz e n i nde f i ni t e l y i f not us e d i n r e s e a r c h. T he r e i s no ge ne r a l c ons e ns us a m ong r e l i gi ou s gr oups w hi c h gi ve s r i s e t o m a ny c onc e r ns ove r t he us e of E S c e l l s C on c e r n s O ve r S t e m C e l l U s e C e r t a i nl y, s t e m c e l l s a r e no t t he f i r s t hu m a n di s c ove r y t o r e vol ut i oni z e s c i e nt i f i c know l e dge a nd c r e a t e w a ve s of e t hi c a l de ba t e S i n c e a nc i e nt t i m e s s oc i e t y ha s a dm oni s he d m a n f or a ppr oa c hi ng t he s e bounda r i e s a s e xe m pl i f i e d i n t he G r e e k m yt h o f I c a r us w ho di d no t he e d hi s f a t he r s c om m a nd; he r e ve l e d i n t he unna t u r a l s e ns a t i on of f l i ght a nd t he n pl um m e t e d t o hi s de a t h a f t e r t he s un m e l t e d hi s w i ngs T hi s G r e e k m yt h e m bodi e s our a ppr e he ns i ons a bout i nt e r f e r i ng w i t h na t ur e G a l i l e o G a l i l e i e xpa nde d t he f r ont i e r s of a s t r onom y a nd pos i t e d t ha t t he E a r t h r ot a t e s on i t s a xi s a nd r e vol ve s a r ound t he S un. T hi s l e d t o hi s e ve nt ua l l y c onde m na t i on f or he r e s y. I n V i c t or i a n t i m e s s oc i e t y

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68 gr a ppl e d w i t h t he ba l a nc e be t w e e n t he know l e dge ga i ne d f r om pe r f or m i ng a ut ops i e s f or c r uc i a l unde r s t a ndi ng of hum a n a na t om y ve r s us t he de s e c r a t i on of t hos e w ho w e r e de a d. E ve n r e c e nt l y, c om pl e t e c ons e nt t o p r oduc e r e c om bi na nt D N A f or l i f e s a vi ng m e di c a t i ons s uc h a s i ns ul i n ha s be e n gr a nt e d but onl y a f t e r ve h e m e nt pr ot e s t a t i ons ove r ge ne t i c e ngi ne e r i ng. T he r e a r e s ha r e d c onc e r ns a m ong a l l i ns t a nc e s of t e s t i ng m e di c a l bounda r i e s a nd t he c onc e r ns o f s t e m c e l l t e c hnol ogy i nc l ude i s s ue s of s a f e t y, e f f i c a c y, a nd r e s our c e a l l oc a t i on. F or de c a de s pa t i e nt s ha v e unde r gone a dul t H S C t r a ns pl a nt a t i on i n t he t r e a t m e nt of i m m une de f i c i e nc i e s a nd c a nc e r A l t hough g r a f t ve r s us hos t di s e a s e a nd pos t t r a ns pl a nt a t i on i nf e c t i ons a r e m a j o r r i s ks of a l l oge ne i c bone m a r r ow t r a ns pl a nt s i nve s t i ga t or s ha ve w or ke d t o m i ni m i z e t he s e c ons e que nc e s a nd m a ny pa t i e nt s a c c e pt t he s e r i s ks i n t he hope o f t he l i f e s a vi ng be ne f i t o f di s e a s e e r a di c a t i on. H ow e ve r t he f i e l d of s t e m c e l l t he r a py i s s t i l l i n i t s i nf a nc y a nd r e s e a r c he r s a r e i nc r e m e nt a l l y i m p r ovi ng s a f e t y, e f f i c a c y, a nd a ppl i c a bi l i t y t o a w i de r s pe c t r um of d i s e a s e I n a l l i ns t a nc e s of e xpa ndi ng t he hor i z ons of our know l e dge a s oc i e t a l c ons c i ous ne s s w a s a t pl a y, of t e n t i m e s e nc um be r i ng pr ogr e s s a nd que s t i oni ng t e c hn i que s of i nt e r ve nt i on S t e m c e l l t he r a py di f f e r s f r om pr e vi ous t e c hnol ogi e s i n how t he s e pot e nt i a l s our c e s of r e ge ne r a t i ng t i s s ue a r e t a ppe d. A dul t s t e m c e l l s a r e t ypi c a l l y a c qui r e d by ha r ve s t i ng a dul t t i s s ue s P a t i e nt s gi ve i n f or m e d c ons e nt a nd u s ua l l y unde r go l i t t l e r i s k a t dona t i on. I n c ont r a s t hum a n e m br yoni c s t e m c e l l s ( h E S ) a r e obt a i ne d by c ul t u r i ng c e l l s f r om t he i nne r c e l l m a s s of a bl a s t oc ys t T hi s bl a s t oc ys t i s u s ua l l y a c qui r e d f r om a n unus e d hum a n e m br yo pr oduc e d by i n v i t r o f e r t i l i z a t i on or f r om a n a bor t e d f e t us T he ha r ve s t i ng pr oc e s s r e qui r e s di s s ol vi ng t he bl a s t oc ys t br i ngi ng i nt o que s t i on t he m o r a l a nd l e ga l s t a t us of t he hum a n e m b r yo.

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69 M a ny r e l i gi ous pe r s pe c t i ve s c ons i de r t he hum a n f e t us a n i ndi vi dua l hum a n e nt i t y. H ow e ve r t he r e i s s ubs t a nt i a l de ba t e r e ga r di ng a t w hi c h s pe c i f i c s t a ge di gni t y i s c onf e r r e d i n de ve l opm e nt r a ngi ng f r om c onc e pt i on, t o p r i m i t i ve s t r e a k de ve l opm e nt i m pl a nt a t i on, or bi r t h T a ki ng i nt o a c c ount t he m a ny pe r s pe c t i ve s on t he m or a l s t a t us of t he hum a n e m br yo w e i ghe d a ga i ns t t he s c i e nt i f i c pr om i s e s of a he a l t hi e r t om o r r ow t h r ough s t e m c e l l t e c hnol ogy, our s oc i e t y ha s a t t e m pt e d t o de f i ne t he l e ga l s t a t us of t he hum a n e m br yo I n t he U ni t e d S t a t e s t he f i r s t m a nda t e w a s out l i ne d i n 1973 w he n t he U S S up r e m e C our t r ul e d t ha t a f e t us i s not a pe r s on i n t e r m s o f c ons t i t ut i ona l pr ot e c t i on ( R oe v W a de 410 U S 113 [ 1973 ] ) F or a be t t e r e xa m i na t i on of t he d e c i s i on s e f f e c t on r e s e a r c h, t he N a t i ona l I ns t i t ut e s of H e a l t h ( N I H ) i m pos e d a m or a t or i um on f e t a l r e s e a r c h, a nd C ongr e s s f ounde d t he N a t i ona l C om m i s s i on c ha r gi ng i t t o put t oge t he r pol i c y a nd gui de l i ne s on f e t a l r e s e a r c h. T he c om m i s s i on publ i s he d a r e por t e nc our a gi ng f e t a l r e s e a r c h due t o i t s pot e nt i a l p r ovi de d t ha t t he r e s e a r c h r i s ks t o t he f e t us w e r e m i n i m a l a nd w e r e onl y t hos e t ha t w oul d be a c c e pt e d f or a t e r m f e t us T hus de s pi t e R oe v W a de t he c om m i s s i on e xt e nde d pr ot e c t i on t o a f e t us e qu a l t o a dul t pa t i e nt s i n r e s e a r c h. T hi s i nc l ude d f e t us e s pl a nne d f or e l e c t i ve a bor t i on. T he N I H m or a t or i um w a s l i f t e d i n 1975 how e ve r d ur i ng P r e s i de nt R ona l d W R e a ga n s s e c ond t e r m C ongr e s s e na c t e d l e gi s l a t i o n t ha t f u r t he r pr ot e c t e d t he f e t us by e ndi ng f e de r a l s uppor t of f e t a l r e s e a r c h i nvol vi ng a ny l e ve l of r i s k. I n 1996 C ongr e s s e xt e nde d t hi s r e s t r i c t i on by ba nni ng f e de r a l f undi n g f or t he c r e a t i on o f a hum a n e m br yo or e m br yos f or r e s e a r c h pur pos e s T h i s l e d t he N I H t o di s t i ngui s h be t w e e n de r i vi ng a nd us i ng e xi s t i ng hum a n e m br yos t o s uppor t e m b r yon i c s t e m c e l l r e s e a r c h. U nde r t he s e gui de l i ne s r e s e a r c he r s us i ng a l r e a dy e s t a bl i s he d hE S c e l l l i ne s de r i ve d f r om p r i va t e

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70 s e c t or s uppor t c a n r e c e i ve publ i c s e c t or m oni e s pr ovi de d t ha t t he f e r t i l i z e d e m b r yos w oul d ot he r w i s e ha ve be e n di s c a r de d a f t e r I V F or w e r e f r om a l r e a dy a bor t e d f e t us e s donor s a r e a w a r e of t he r e s e a r c h us e a nd n o pa ym e nt w a s m a de t o t he dono r s P r e s i de nt W i l l i a m J C l i nt on c r e a t e d T he N a t i ona l B i oe t hi c s A dvi s or y C om m i s s i on ( N B A C ) t o t hor ough l y r e vi e w m or a l a nd l e ga l i s s ue s of s t e m c e l l r e s e a r c h. T hi s c om m i s s i on l a r ge l y f r a m e d i t s m or a l pos i t i on ba s e d on a ut i l i t a r i a ni s m a r gu m e nt t he good of m a ny out w e i gh t he s t a t us of one I n a ddi t i on, i t dr e w on m e di c i ne s a i m s t o he a l a nd pr e ve nt di s e a s e ur gi ng c ons i de r a t i on o f a l ong t e r m be ne f i t t o ha r m ba l a nc e T he N B A C c onc l us i on r e c om m e nde d a l l ow i ng f e de r a l f undi ng f o r h E S r e s e a r c h on e xc e s s I V F e m br yos R e a s ons s uppor t i ng t hi s pos i t i on i n c l ude t he pot e nt i a l o f E S c e l l s i n r e ge ne r a t i ve a nd r e pr oduc t i ve m e di c i ne a nd t he ne e d f or f e de r a l s uppor t t o a voi d pr i va t e s e c t or c onf l i c t s of i n t e r e s t w hi c h s om e t i m e s i nvok e s s e c r e c y l i m i t i ng t he s pr e a d of know l e dge a nd pl a c e s s ha r e hol de r s i nt e r e s t s a he a d of publ i c good. T a ki ng a l l t he s e gui de l i ne s a nd pe r s pe c t i ve s i nt o a c c ount P r e s i de nt G e or ge W B us h m a de a n e xe c ut i ve or de r on A ugus t 9, 2001 t o l i m i t f e de r a l f undi ng o f hE S r e s e a r c h t o c e l l l i ne s a l r e a dy de r i ve d f r om 64 e m b r yos T he c os t t o s oc i e t y o f f o r e goi ng us e of t hi s t e c hnol ogy, e i t he r by f a i l ur e t o c or r e c t ge ne t i c a bnor m a l i t i e s or by i m p r ovi ng t he s uc c e s s of l i f e s a vi ng o r ga n t r a ns pl a nt a t i ons m a y be e qua l t o or g r e a t e r t ha n t he pe r c e i ve d c os t s t o t he di gni t y o f l i f e due t o de s t r uc t i on of a hum a n e m br yo. T he r e m us t be a ba l a nc e be t w e e n t he pe r c e i ve d c os t s t o t he di gn i t y of l i f e he l d by t hos e w i t h t he m os t s a c r os a nc t c onc e pt of t he m or a l s t a t us of e m br yos a nd t hos e w ho w oul d di r e c t l y be ne f i t f r om s t e m c e l l ba s e d t he r a py. T he c l i m a t e i n w hi c h s t e m c e l l s a r e e xpl or e d c a n be nur t ur i ng or pr of ou ndl y l i m i t i ng A s m e di c a l s c i e nt i s t s

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71 w e m us t not m a ke j udg m e nt s or e t hi c a l de c i s i ons on our ow n; r a t he r w e m us t e ns ur e f ul l i nf or m e d c ons e nt of t he popul a t i on a s a w hol e T h i s a ppr oa c h m a y l i m i t qui c k p r ogr e s s a nd m a y di s qua l i f y a ve nue s of r e s e a r c h a nd t he r a p y, but a s r e s pons i bl e r e s e a r c he r s w e m us t us e t he r e s our c e s of s oc i e t y i n a w or t hy m a nn e r t o e xpl o r e f ul l y t he t r e m e ndous pot e nt i a l of e m b r yoni c s t e m c e l l s T he us e of a dul t s t e m c e l l s e l i m i na t e s a ny e t hi c a l c onc e r ns a s t he c e l l s us e d f or t he r a py c a n be obt a i ne d w i t h onl y s l i ght di s c om f or t a nd po t e nt i a l l y f r om t he i ndi vi dua l pa t i e nt t he m s e l ve s T hi s i s a t t r a c t i ve due t o t he f a c t t h a t t he r e w oul d be no hum a n l e ukoc yt e a nt i ge n m i s m a t c hi ng, a nd t he r e f or e no n e e d f or i m m une s uppr e s s i ng dr ugs or t he f e a r o f t i s s ue r e j e c t i on a nd gr a f t ve r s us hos t di s e a s e I f a dul t s t e m c e l l s p r ove t o ha ve t he s a m e pot e nt i a l i t y a s E S c e l l s e i t he r i ndi vi d ua l l y or a s a c ol l e c t i on, t he i r us e w oul d e nd t he ne e d f o r e m br yoni c t i s s ue a nd t he i r s ubs e que nt de s t r uc t i on e l i m i na t i ng a ny e t hi c a l c onc e r ns ove r t he e nds j us t i f yi ng t he m e a ns

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72 C H A P T E R 6 G E N E R A L C O N C L U S I O N S T he goa l o f t hi s w o r k w a s t o c ha r a c t e r i z e t he he m a t opoi e t i c s t e m c e l l s pl a s t i c a bi l i t y a nd de s c r i be bi ol ogi c a l pa t hw a ys w hi c h c a n m odul a t e t hi s a bi l i t y. I n a hi s t or i c c ont e xt t hi s w or k s pa ns s e ve r a l s t a ge s of t he he s t e m c e l l f i e l d a s i t m a t u r e d f r om t he be gi nni ng f l ur r y o f a c t i vi t y i nt o i t s c ur r e nt s t a ge of c a r e f ul e va l ua t i on T he pi one e r i ng w or k de m ons t r a t e d t he e xc i t i ng pot e nt i a l o f t he f i e l d a s a n a l t e r na t i ve a nd pow e r f ul t ool f or us e a s a c e l l ba s e d t he r a py f or m a ny di s e a s e s r a ngi ng f r om c a nc e r t o d i a be t e s he a r t a t t a c k, a nd P a r ki ns on s d i s e a s e T hos e f ounde r s d e m ons t r a t e d t ha t t he s e c e l l s s pe c i f i c a l l y t he he m a t opoi e t i c s t e m c e l l w a s c a pa bl e of p r oduc i ng m a ny d i f f e r e nt t i s s ue t ype s I t w a s f ound t ha t t he H S C i s c a pa bl e of pr o duc i ng not onl y a l l o f t he b l ood l i ne a ge s but a l s o m us c l e pa nc r e a t i c c e l l s he a r t i n t e s t i na l e pi t he l i um b r a i n, a nd bl ood ve s s e l e ndot he l i um T he s e e xc i t i ng r e s ul t s s pa r ke d a r e vol ut i on i n t he s c i e nt i f i c f i e l d w i t h ne w f i ndi ngs c ons t a nt l y be i ng publ i s he d i n pe e r r e vi e w e d s c i e nt i f i c j ou r na l s a l ong w i t h t he f r ont pa ge s of popul a r ne w s pa pe r s a nd m a ga z i ne s T hi s i ni t i a l i m pe t us s oon pl a ye d out how e ve r a s t he he r a l de d pl a s t i c i t y o f s t e m c e l l s c a m e unde r t ho r ough a nd c r i t i c a l s c r ut i ny, a nd j us t i f i a bl y s o. I t be c a m e a ppa r e nt t ha t t he c e l l t ype s pr oduc e d w e r e not ne c e s s a r i l y f unc t i ona l no r c oul d t he r e be c e r t a i nt y of t he s our c e of t he dono r c e l l s s e ve r a l di f f e r e nt c e l l t ype s w i t hi n t he t r a ns pl a nt c o ul d be i ndi vi dua l l y c ont r i but i ng t o t he obs e r ve d t i s s ue t he r e f or e no one c e l l w oul d be c a p a bl e of f o r m i ng s e ve r a l t i s s ue t ype s T hi s c r i t i que w a s a ddr e s s e d i n s e ve r a l s t udi e s w hi c h be ga n t he s e c ond s t a ge of s t e m c e l l r e s e a r c h. K r a us e e t al d i d a s i ngl e c e l l t r a ns pl a nt hom i ng a s s a y t o pr ove s e ve r a l t i s s ue

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73 t ype s c oul d a r i s e f r om one c e l l 8 2 G r a nt e t al a l s o di d s i ngl e c e l l t r a ns pl a nt s a nd de m ons t r a t e d t he f unc t i ona l a bi l i t y o f e ndot he l i a l c e l l s w hi c h c l ona l l y a r os e f r om t he H S C t o c a r r y r e d f l uor e s c e nt dye pe r f us e d i nt o t he c i r c ul a t or s ys t e m 3 0 I n a ddi t i on L a ga s s e e t al r e s c ue d l i ve r f unc t i on i n a m e t a bol i c l i ve r d i s e a s e w i t h H S C 8 4 C l e a r l y, s t e m c e l l s pa r t i c ul a r l y t he H S C a r e c a pa bl e of pr o vi di ng f unc t i ona l r e s c ue of di s e a s e c ondi t i ons i n a c l ona l m a nne r W i t h t hi s unde r s t a n di ng, w e now e m ba r k on t he t h i r d s t a ge of s t e m c e l l pl a s t i c i t y r e s e a r c h de f i ni ng t he ge ne s a nd bi ol ogi c a l pa t hw a ys i nvol ve d i n r e gul a t i ng or c ont r ol l i ng s t e m c e l l a c t i vi t y. M a ny ge ne s ha ve be e n s how n t o m a i nt a i n t he s t e m ne s s of s t e m c e l l s by c ont r ol l i ng t he i r s e l f r e ne w i ng a nd pr ol i f e r a t i ng c a pa c i t y. I n a ddi t i on w e a r e s t a r t i ng t o unde r s t a nd f a c t or s s uc h a s t he ni t r i c oxi de pa t hw a y, w hi c h pl a y a r o l e i n s t e m c e l l hom i ng a nd f unc t i ona l be ha vi or 1 2 8 T hi s w or k a l o ng w i t h ot he r e xc i t i ng w o r k done i n our l a b on c he m oki ne s s uc h a s s t r om a l de r i ve d f a c t or 1 s i nvol ve m e nt i n ho m i ng, hi ghl i ght s t he t he r a pe ut i c be ne f i t of not onl y s t e m c e l l r e s e a r c h, but a l s o f or t i f i e s t he f ounda t i on f o r c e l l ba s e d t he r a py 1 2 9 T hi s t hi r d a g e of di r e c t e d s t e m c e l l r e pa i r of da m a ge d or non f unc t i ona l t i s s ue ha s t he pot e nt i a l f or di r e c t t r a ns l a t i on i n t o di s e a s e t he r a py a l ong w i t h ope ni ng e xc i t i ng ne w a ve nue s of or i gi na l r e s e a r c h. T hi s body of w or k c hr oni c l e s t he r e l a t i ve l y ne w f i e l d of s t e m c e l l pl a s t i c i t y r e s e a r c h f r om t he i ni t i a l c ha r a c t e r i z i ng of H S C pl a s t i c i t y up t o de s c r i bi ng b i ol ogi c a l pa t hw a ys w hi c h c a n or c he s t r a t e H S C a c t i vi t y. T he f oundi ng w or k a nd i ni t i a l a ppl i c a t i on t o H S C pl a s t i c i t y de s c r i be d he r e c a n l e a d t o m a ny nove l s t e m c e l l t h e r a py s t r a t e gi e s f or de bi l i t a t i ng c ondi t i ons a nd di s e a s e s A ddi t i ona l e f f o r t w i l l f oc us on di r e c t t r a ns l a t i on of t hi s know l e dge i nt o hum a n di s e a s e t he r a py.

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74 L I S T O F R E F E R E N C E S 1 P a l i s J R obe r t s on, S K e nne dy, M W a l l C & K e l l e r G D e ve l opm e nt of e r yt hr oi d a nd m ye l oi d pr oge ni t or s i n t he yol k s a c a nd e m br yo pr ope r of t he m ous e D e ve l opm e nt 122, 1363 1371 ( 1999) 2. W ong, P C hung, S C hui D & E a ve s C P r ope r t i e s of t he e a r l i e s t c l onoge ni c he m opoi e t i c pr e c ur s or t o a ppe a r i n t he de ve l opi ng m ur i ne yol k s a c P r oc N a t l A c a d S c i 83, 3851 3854 ( 1986) 3. D r a ke C & F l e m i ng, P V a s c ul oge ne s i s i n t he da y 6. 5 t o 9 5 m ous e e m br yo B l ood 95, 1671 1679 ( 2000) 4. H a a r J & A c ke r m a n, G A pha s e a nd e l e c t r on m i c r os c opi c s t udy of va s c ul oge ne s i s a nd e r yt hr opoi e s i s i n t he yol k s a c o f t he m ous e A na t R e c 170, 199 223 ( 1971 ) 5. R i s a u, W & F l a m m e I V a s c ul oge ne s i s A nnua l R e vi e w of C e l l & D e v e l opm e nt a l B i ol ogy 11, 73 91 ( 1995) 6. F l a m m e I & R i s a u, W I nduc t i on of va s c ul oge ne s i s a nd he m a t opoi e s i s i n v i t r o D e ve l opm e nt 116, 435 9 ( 1992 ) 7. G odi n, I G a r c i a P o r e r o, J C out i nho, A D i e t e r l e n L i e vr e F & M a r c os M P a r a a or t i c s pl a nc hnopl e ur a f r om e a r l y m ous e e m b r yos c ont a i ns B 1a c e l l pr oge ni t or s N a t ur e 364, 67 70 ( 1993) 8. G odi n, I G a r c i a P o r r e r o J D i e t e r l e n L i e vr e F & C um a no, A S t e m c e l l e m e r ge nc e a nd he m a t opoi e t i c a c t i vi t y a r e i nc om pa t i bl e i n m ous e i nt r a e m br yoni c s i t e s J E xp M e d 190 43 52 ( 1999 ) 9. G odi n, I D i e t e r l e n L i e vr e F & C um a no, A E m e r ge nc e of m ul t i pot e nt he m opoi e t i c c e l l s i n t he yol k s a c a nd pa r a a or t i c s pl a nc hnopl e ur a i n m ous e e m br yos be gi nni ng a t 8. 5 da ys pos t c oi t us P r oc e e di ngs of t he N a t i ona l A c a de m y of S c i e nc e s of t he U ni t e d S t a t e s o f A m e r i c a 92 77 3 7 ( 1995 ) 10. M e dvi ns ky, A S a m oyl i na N M ul l e r A & D z i e r z a k, E A n e a r l y pr e l i ve r i nt r a e m br yoni c s our c e of C F U S i n t he de ve l opi ng m o us e N a t ur e 364, 64 70 ( 1993) 11. T a ka ha s hi T K a l ka C M a s uda H C he n D S i l ve r M K e a r ne y M M a gne r M I s ne r J M & A s a ha r a T I s c he m i a a nd c yt oki ne i nduc e d m obi l i z a t i on of bone m a r r ow de r i ve d e ndot he l i a l pr oge ni t o r c e l l s f o r ne ova s c ul a r i z a t i on. N a t M e d 5, 434 8 ( 1999 )

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86 B I O G R A P H I C A L S K E T C H S t e ve G ut hr i e w a s bor n a nd r a i s e d i n L a nc a s t e r P e nns yl va ni a H e a t t e nde d A l br i ght C ol l e ge i n R e a di ng, P e nns yl va ni a w he r e he gr a dua t e d i n 1998 w i t h t w o m a j or s ( bi ol ogy a nd ph i l os ophy) r e c e i vi ng t he G a r y K e nni s P hi l os ophy A w a r d a nd E r n e s t J P a s t or e l l o B i ol ogy P r i z e H e t he n m o ve d t o G a i ne s vi l l e F l or i da w he r e he w or ke d a s a l a b t e c hni c i a n f or D r A l f r e d L e w i n a nd t he n a s a bi ol ogi c a l s c i e nt i s t f or D r E dw a r d S c ot t f or 2 ye a r s H e j oi ne d t he I nt e r di s c i pl i na r y P r ogr a m i n B i om e di c a l S c i e nc e s a t t he U ni ve r s i t y of F l or i da C ol l e ge o f M e di c i ne i n 2000 w he r e he be ga n hi s doc t or a l s t udy unde r t he gui da nc e of D r E dw a r d S c ot t i n t he D e p a r t m e nt of M ol e c ul a r C e l l B i ol ogy. H e w a s a w a r de d a G r i nt e r F e l l ow s hi p, a nd r e c e i ve d f i r s t p l a c e i n hi s de p a r t m e nt a nd f i f t h pl a c e ove r a l l a t t he 2003 M e di c a l G ui l d R e s e a r c h D a y s pons or e d by C ol l e ge of M e di c i ne S t e ve w i l l be doi ng pos t doc t or a l r e s e a r c h a t t he U ni ve r s i t y of A l a ba m a a t B i r m i ngha m be gi nni ng S um m e r o f 2005


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Title: Hemangioblasts: From Hematopoietic Stem Cells to Endothelial Progenitor Cells and Their Effector Molecules
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Title: Hemangioblasts: From Hematopoietic Stem Cells to Endothelial Progenitor Cells and Their Effector Molecules
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
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HEMANGIOBLASTS: FROM HEMATOPOIETIC STEM CELLS TO
ENDOTHELIAL PROGENITOR CELLS AND THEIR EFFECTOR MOLECULES















By

STEVEN MITCHELL GUTHRIE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Steven Mitchell Guthrie
































This work is dedicated to my mother, Bernadette Guthrie, and my father, Edwin Guthrie.















ACKNOWLEDGMENTS

I would first like to thank my mentor, Dr. Edward Scott, for the excellent training

and opportunities I received during my stay in his lab. I would also like to thank all of

my committee members-- Dr. Maria Grant, Dr. Jorg Bungert, Dr. Bryon Petersen, and Dr.

Naohiro Terada-- for their time, energy, and guidance. I thank Dr. Chris Cogle, also my

good friend, for his constant sharing of ideas and discussions including but not limited to

science. My deepest thanks also are extended to current and past members of the "Scott

lab," especially Gary Brown for has vast animal knowledge and expertise; Doug Smith

for his very capable FACS analysis and confocal imaging; Chris Culler and Dustin Hart

for maintaining an organized and efficient lab; Jen Targac for always cleaning up after

me; and Jason Butler with whom I worked side by side on many experiments. In

addition, I would like to thank Jeff Harris, Dr. Robert Fisher, Dr. Ron Sanders and

especially Dr. Bill Slayton, Chris Bray, and Greg Marshall for constant and engaging

discussion.

Outside the lab, and most importantly, I would like to thank my parents and my

sister, Alisa, in Pennsylvania. Although we were separated by a thousand miles, I could

always hear their encouragement and feel their caring. Finally I would like to thank my

fiance, Dr. Christina Covelli, who has been through thick and thin during my science

career and has provided strength, wisdom, motivation, and love.
















TABLE OF CONTENTS


page

A C K N O W L E D G M E N T S ............................................................................ ...... ........ iv

LIST O F FIG U RE S .............................................................................................. .......vii

ABSTRACT ............. ........................... .................... viii

CHAPTER

1 INTRODUCTION AND BACKGROUND INFORMATION ............................. 1

Hematopoiesis and Vasculogenesis during Embryonic Development...................... 2
Form ation of Blood Vessels in Adults .............................................. .............. 3
Regulation of Neovascularization.......... ... ................... ....... ....... 5
Stem Cell Transplantation ...................... ..... ..................... .. ...... .............. 8
Endothelail Progenitor Cells for Neovascularization ........................................ 9
Nitric Oxide as Potential Regulator of Vascular Formation.................................. 11
Role of Nitric Oxide Synthase in Vessel Formation............................. ............. 13

2 GENERAL METHODS AND MATERIALS...................... ..... ............ 16

G enerating the G FP/BL6 Chim era................................. .............. ... ................ 16
Harvesting Bone M arrow .......................................................................... 16
Initial Purification of Hematopoietic Stem Cells by Magnetic Activated Cell
S o rtin g .............. ..... ..... ...... .............................................. .......... 17
Final Purification of Hematopoietic Stem Cells by Flourescence Activated
Cell Sorting .................. ..... ..................... ......... .... .... ... .......... 18
Harvesting of BL6 Rescue Marrow with Hematopoietic Stem Cells
Depletion, and Irradiation of Recipient Animals...................... ......... 19
Purified Green Fluorescence Protein Hematopoietic Stem Cells and Depleted
Rescue Marrow Transplantation and Ensuing Animal Husbandry Concerns. 20
Verification of M ultilineage Reconstitution.................. ...................... .............. 21
Induction of Retinal Ischem ia.................................................. 21
Eye Removal ................................... .......... .......... ........ 23

3 THE HEMATOPOIETIC STEM CELL HAS HEMANGIOBLAST ACTIVITY ... 25

A dult H em atopoietic Stem Cells............................. .............. .............. 26
D iabetic R etinopathy ............................................................. ................. 29









Angiogenesis vs. Neovascularization................... ...... .......................... 30
R e su lts .................................................................................................... 3 1
The C57BL6.GFP Chim era.......................................................... ............... 31
Assessment of Green Fluorescence Protein Retinal Blood Vessel Endothelial
Cells .......................... .................. ...... .......... ... ............. 34
The Hematopoietic Stem Cells has Hemangioblast Function ........................... 38
D iscussion.............................. ........... .......... 39

4 MODULATORS OF HSC/HEMANGIOBLAST ACTIVITY ............................ 42

R e su lts ............... .............. ..... ..................... ..... ...... ....... .................... 4 8
Inducible Nitric Oxide Synthase and Endothelial Nitric Oxide Synthase
Green Fluorescence Protein Chimeras Demonstrated Robust Hematopoietic
Stem C ells E ngraftm ent ............................................................ ................ 48
The Nitric Oxide Pathway Affects Blood Vessel Formation ............................ 51
The Nitric Oxide Synthase Pathway Affects Blood Vessel Branching
Characteristics. ............................................ ... ..... ......... 55
Nitric Oxide Production Effect on Vasculature in Non-ocular Tissue............ 57
Quantitation and Location of Nitric Oxide Synthase Produced in Knockout
A nim als. ............. ......... ... ............ ............ ................ ......... 60
D discussion ................ ... .................. ........ .................... ......... 61

5 LIMITATIONS OF STEM CELL RESEARCH AND ETHICAL
C O N SID E R A T IO N S ......... ................. ............................................................ 64

Biological Limitations ..... ......... .. ........ ......... ........ 64
E th ic s ................. ............... .............................................................. ................. 6 6
Concerns Over Stem Cell Use ............. .................................. 67

6 GENERAL CONCLUSIONS .................. ................... ................... 72

L IST O F R E FE R E N C E S ......... ................. .............................................................74

BIOGRAPHICAL SKETCH ............. ....................................... 86
















LIST OF FIGURES


Figure page

2-1. Fluorescence activated cell sorting gates for isolating HSC ................. 19

3-1. Reanalysis of HSC post-enrichment used for transplantation ................. 32

3-2. HSC can engraft multiple lineages long-term and self-renew.................. 33

3-3. HSC can produce all hematopoietic lineages clonally ............................ 34

3-4. Donor-derived HSC contribute to endothelial cells of blood vessels in
the ey e.................................................. ....................... .......... 36

3-5. Donor-derived HSC produce functional endothelial cells surrounding
blood vessel lum ens.................................. ............... ............ 37

3-6. The HSC is self-renewing and can clonally form endothelial cells............ 40

4-1. NOS knockout animals exhibit long-term, multi-lineage, donor GFP
peripheral blood engraftm ent.......................................... ... ... .............. 50

4-2. The iNOS pathway modulates hemangioblast neovascularization............. 52

4-3. The eNOS pathway modulates hemangioblast neovascularization ........... 54

4-4. The nitric oxide pathway alters hemangioblast blood vessel formed
branching characteristics .................................................. ............ .. 56

4-5. Chronic vascular injury in eNOS.GFP chimeras induces widespread
hemangioblast activity from adult HSC .............. ........ ........ ......... 58

4-6. Donor-derived cells lining vascular lumens in eNOS.GFP animals are
MECA-32 positive ....................... ........... .... .......... 60

4-7. Nitric oxide production is dysregulated in eNOS knockout animals.......... 62

5-1. Propidium iodide staining of circulating EPC does not indicate abnormal
ploidy .................................................. ........................... ...... 66















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

HEMANGIOBLASTS: FROM HEMATOPOIETIC STEM CELLS TO
ENDOTHELIAL PROGENITOR CELLS AND THEIR EFFECTOR MOLECULES

By

Steven Mitchell Guthrie

May 2005

Chair: Edward Scott
Major Department: Molecular Genetics and Microbiology

Research in the field of stem cell has received much attention in the past few years.

Stem cells hold tremendous potential for treating many debilitating conditions and

diseases. My study describes how the hematopoietic stem cell is plastic, or capable of

producing non-hematopoietic tissue in addition to all of the expected blood lineages.

Specifically, the hematopoietic stem cell is capable of producing endothelial cells of

blood vessels. I describe this through a series of experiments where I transplanted a

single hematopoietic stem cell into a lethally irradiated recipient and reconstituted all of

the blood lineages. This single cell was then able to produce endothelial cells under

conditions of injury and ischemia in an attempt to relieve the ischemic pressure. I found

that the hematopoietic stem cell can function as a hemangioblast, capable of producing

not all of the blood lineages and also blood vessels. This activity suggests the possibility

of modulating this hemangioblast activity.









I determined that two genes play a role in blood-pressure maintenance and immune

responses in the Nitric Oxide Synthase pathway. These genes are also able to modulate

hemangioblast function in mice. This ability to alter blood vessel formation would be

extremely useful in conditions of pathologic blood vessel growth such as diabetic

retinopathy, the leading cause of blindness worldwide, or tumor blood vessel growth

where decreasing the blood supply could starve the cancer cells. Conversely, wound

healing, and therapy for conditions such as stroke or cardiac ischemia, would benefit

from increased blood vessel growth. This knowledge can be directly applied by using

pharmacological agents that either inhibit or upregulate the Nitric Oxide Synthase genes

to modulate blood vessel formation for therapies useful in human patients.














CHAPTER 1
INTRODUCTION AND BACKGROUND INFORMATION

The discovery of the ability of stem cells to differentiate along alternative

developmental fates heralded a new tool for the treatment of many debilitating diseases.

The ability of exogenous cells to home to areas of injury, take up residence, and

reprogram themselves to new tissue types allows for functional repair of dysfunctional

tissues. While in some tissue types this has been known to occur, such as vasculature

reperfusion in wound healing, the exact cells contributing to the endothelial tissue were

identified only recently. Elucidation of the contributing cell to certain types of vascular

repair, viz. hematopoietic stem cells, now allows exploration of the molecules that play

parallel roles in both hematopoiesis and blood vessel formation. Indeed, tailoring of the

hematopoietic stem cell's hemangioblast activity could improve currently limited

palliative care for conditions such as diabetic retinopathy or could provide an improved

targeted approach for tumor growth suppression and elimination. The potential for

clinical therapies is profound.

The unifying goal of my study was to further describe the characteristics of the

hematopoietic stem cell in relation to its plastic ability to produce the endothelial tissue

lining the blood vessel walls. I do this immersed in the current environment of sanguine

skepticism towards stem cell plasticity highlighting how this work addresses the

controversy. I begin by outlining the backdrop for current research and provide a

barometer to measure the current stem cell climate. I outline the limitations to stem cell

research in relation to the hematopoietic exploration along with the methods by which









they were addressed. Chapter 2 describes the development of a novel, robust, and

reproducible model for inducing hematopoietic stem cell hemangioblast activity thereby

promoting an alternative developmental fate along the endothelial lineage. Chapter 3

underscores how this model was applied to the critiques of stem cell plasticity and how

the hematopoietic stem cell functions in conditions of injury. There are many biological

molecules that can modulate hematopoiesis and neovascularization. In chapter 4 I

describe how nitric oxide has the ability to play a significant role in hematopoietic stem

cell derived hemangioblast activity. Finally, in chapter 5 I will identify some of the

limitations of stem cell based research and therapy including both biological and ethical

implications.

Hematopoiesis and Vasculogenesis During Embryonic Development

The rapid growth of the early embryo necessitates conversion from a mechanism

where simple diffusion provides the necessary nutrients and removes metabolic

byproducts for the ever-increasing cell number to a mechanism of circulated transport.

The developing blood and vasculature provide this circulation. During murine

development, hematopoiesis and vasculogenesis begin as early as Day 7 in the region of

the yolk sac.1, 2 Endothelial cells are derived from mesodermal precursors in the yolk sac

and begin to constitute the primary vascular system in parallel with initiation of

premature hematopoiesis.3-5 This vasculogenesis begins with a cluster of cells, called

blood islands, composed of a "nucleus" containing hematopoietic stem cells (HSC)

surrounded by more differentiated angioblasts, the cells which will form blood vessels,

on the periphery.6 The close proximity of the two precursor cells and the developmental

relationship between the formation of blood and blood vessels suggest a shared parent

cell from which both are derived: the hemangioblast.









Until the Day 10 of development, the yolk sac remains the primary site of

hematopoiesis. Around Day 12 the liver which then becomes the primary site of

hematopoiesis.7 However, there are other regions of potential hematopoiesis in the para-

aortic splanchnopoeura (PAS) from Day 8.5 to 10, and the aorta-gonad-mesonephrous

(AGM) region from Day 10.5 through Day 12.7-11 The potential of these areas was

determined through a series of transplantation studies where cells isolated from these

regions are able to rescue lethally irradiated recipients.12-14 This hematopoietic rescue

capability defines the first location from where functionally defined HSC arise.

Endothelial cells on the ventral surface of the aorta are derived from the PAS/AGM

regions, and HSC are also found nestled in the endothelial floor of the aorta, again

suggesting the that this area contains cells which are have the capabilities of the

hemangioblast.12

Formation of Blood Vessels in Adults

Vasculogenesis and angiogenesis are two distinct roles of the hemangioblast.

Vasculogenesis is defined as the de novo generation of blood vessels via the recruitment

of undifferentiated progenitor cells to the site of vessel formation where they differentiate

into vascular endothelium.11 During embryonic development, the vascular system is

formed through vasculogenesis. After development is complete, new blood vessel

formation is attributed to the process of angiogenesis where vessels are formed by

sprouting from the pre-existing vasculature.15 Until 1991, angiogenesis was thought to

occur by the proliferation of resident endothelial cells at the site where new vessels are

forming, but George et al.16 showed that endothelial cells circulate in the blood. They

found that peripheral blood contained endothelial cells by staining blood samples with the









endothelial cell specific antibody S-Endo 1 and analyzing these cells by Fluorescence

Activated Cell Sorting (FACS). The discovery of circulating endothelial cells

unvaryingly leads us to question where these cells are derived.

There are two possibilities of circulating endothelial cell parentage: the existing

vasculature where cells extrude themselves from blood vessel walls and enter the

circulation; the bone marrow itself, via an endothelial cell progenitor (EPC) intermediate.

Several studies describe endothelial cells which derived from the bone marrow.17-22 If

this is the case, the HSC and EPC populations could possible be distinguished through

their cell surface marker expression, or through "tagging" of the parent cell. No studies

have yet directly addressed this question; however there is significant indirect evidence

linking endothelial cells to the EPC and its involvement in adult neovascularization. One

such study described several cell surface antigens present on the EPC, such as CD133

and CD34, that are also present on the HSC.23 However, there are differences in the two

populations, namely that fetal liver kinase-2 (VEGFR-2) expression is only found on

committed progenitors.24 This is one of the first hints that EPC may be a more

differentiated or committed HSC daughter cell. CD 34 positive cells can phenotypically

function as endothelial cells after several days of culture on fibronectin. They are

capable of incorporating acetylated LDL, producing nitric oxide when stimulated with

VEGF, and express of PECAM-1 and Tie-2, both of which are specific to endothelial

cells.25 CD133 positive cells appear to be a more immature subgroup of the CD34

population. The CD133 positive cells are able to repopulate the bone marrow

compartment of radioablated sheep, and evidence shows that a subset of cells which are

CD34, CD133 and VEGFR-2 positive may be EPC.26-28 CD133 and CD34 positive cells









are believed to be more primitive EPC because they lack VE-cadherin or Von Willebrand

expression. Only 3% of these cells express VEGFR-2.27 CD34 negative, CD133

positive, and VEGFR-2 positive cells may represent a more mature or further

differentiated population of endothelial cells.

The exact markers and phenotype of EPC are not known, and the conditions under

which these cells are stimulated to proliferate, circulate, and home to sites of injury are

poorly understood. There is disparity in the amount of neovascularization occurring in

certain vascular beds with some tissue-types experiencing significantly more vessel

formation in relation to others. In addition, the wide range of ischemia models utilized

for study have been found to induce different levels of neovascularization. Crosby et

al.29 have shown that up to 11% of endothelial cells contributing to neovascularization

are EPC derived. This contribution occurred during injury and was not observed under

normal physiologic conditions. Grant et al.30 demonstrated that circulating endothelial

cells, specifically endothelial cells which contribute to the formation of blood vessels

during injury repair, arise from the HSC through an EPC intermediate. This finding lends

to the possibility of regulating vessel formation at a precursor level through a molecular

mediator. The ability to orchestrate formation of blood vessels is highly desired for

conditions in which pathological vascular growth, or lack of growth, and leads to

damaging conditions ultimately decreasing the quality of life.

Regulation of Neovascularization

Vascular endothelial cells maintain a tight border between the circulating blood and

the outside tissue. This monolayer of cells acts as a non-adherent surface where

circulating cells cannot interact and adhere without the presence of certain surface

markers, such as the integrins or selections of the cellular adhesion molecule family.









While this boundary must necessarily remain intact, mechanisms exist in which cells

within the blood can extravasate into the surrounding tissue in order to fight infection or

provide repair. Conversely, mechanisms exist by which cells in tissue can enter the

bloodstream illustrated by bone marrow cells ability to proliferate in the bone marrow

compartment, migrate to the inner marrow vessels, and enter the circulation. Endothelial

cells generally have a very low level of apoptosis and thus a low turnover rate. Cells in

certain organs, such as the eye, can live for years without being replaced.31 As a result,

there are infrequent endothelial cells circulating in healthy adults usually numbering 1-3

per milliliter of blood.32 This emphasizes how the steady state of endothelial cells is non-

dividing unless stimulated by injury when mechanisms to upregulate endothelial mitosis

stimulate proliferation.

Positive regulators are growth factors frequently detected in adult tissues in which

there is apparent angiogenesis and include Vascular Endothelial Growth Factor (VEGF)

and basic Fibroblast Growth Factor (bFGF).33 In vitro, it has been found that VEGF and

bFGF upregulate many endothelial cell functions, including proliferation, migration,

extracellular proteolytic activity, and tube formation.34 This has led to the notion that

these factors act directly on endothelial cells to upregulate their activity. Indeed, VEGF

is increased in tumors when the transformed cells begin to recruit blood vessels for

growth.34 Conversely, a method must exist that can limit the amount of

neovascularization occurring so as to not produce pathologic vasculature. Endothelial

quiescence is thought to be maintained by the presence of endogenous downregulators

such as Tumor Growth Factor-beta (TGF-B) and Tumor Necrosis Factor-alpha (TNF-

a).35 Unlike to VEGF and bFGF, angiogenic downregulators may act directly on









endothelial cells, or indirectly by inducing the production of inflammatory and other non-

endothelial cell regulators.36'37 TGF-B and TNF-A inhibit endothelial cell growth in vitro

and have therefore been considered as direct acting negative regulators.35 Unexpectedly,

TGF-B and TNF-a are angiogenic in vivo, and it has been demonstrated that these

cytokines induce angiogenesis indirectly by stimulating the production of stromal and

chemoattracted inflammatory cell positive regulators.38

Other cytokines that have been reported to regulate angiogenesis in vivo include

HGF, EGF/TGF-, PDGF-BB, interleukins (IL-1, IL-6, and IL-12), interferons, GM-CSF,

P1GF, proliferin, and proliferin-related protein.3941 Chemokines that regulate

angiogenesis in vitro have also been identified including IL-8, platelet factor IV, and

groB.41-43 Angiogenesis can also be regulated by a variety of noncytokine or

nonchemokine factors, including enzymes (angiogenin and PD-ECGF/TP), inhibitors of

matrix-degrading proteolytic enzymes (TIMPs), plasminogen activator inhibitor-1

(PAls), extracellular matrix components, coagulation factors or fragments

(thrombospondin, angiostatin, hyaluronan, and its oligosaccharides), soluble cytokine

receptors, prostaglandins, adipocyte lipids, and copper ions.39 42-45 This plethora of

cytokines demonstrates the complexity of regulating of the angiogenic process, and

justifies assessing their role in stem and progenitor cell governance of neovascularization.

These positive and negative regulators often coexist in tissues in which endothelial cell

turnover is increased. Although this has yet to be proven in vivo, the current working

hypothesis is that the angiogenic switch of tumors involves either the induction of a

positive regulator and/or the loss of a negative regulator.









Stem Cell Transplantation

The adult bone marrow (BM) is a rich reservoir of tissue specific stem and

progenitor cells. BM cells may be a source of EPC. Therefore tapping into BM in

combination with neovascularization regulators may provide significant and manageable

therapy. Stimulation of angiogenesis may be of benefit in wound healing and fracture

repair. Therapeutic growth will also be beneficial in the treatment of ischemia, and

substantiated by extensive experimental data.46-49 Pesce et al.49 demonstrated that under

ischemic conditions, transplanted umbilical cord cells gave rise to enhanced arteriole

length and density along with skeletal muscle fibers. Another group transplanted early

bone marrow cells into nonirradiated, aged mice and found a contribution to vasculature

from subsequently transplanted neonatal myocardium.48 In addition, Orlic et al.50

demonstrated that bone marrow cells can differentiate into myocytes and vascular

structures. They also mobilized bone marrow cells with stem cell factor and granulocyte-

colony stimulating factor and found that marrow cells could home to infarcted regions of

the heart, replicate, differentiate, and ultimately promote myocardial repair.51 This could

lead to significant alterations and improvements in treatment for cardiac ischemia.

Current therapy for myocardial ischemia relies on drugs that reduce myocardial

oxygen demand, mechanical endovascular revascularization procedures (angioplasty), or

bypass surgery.52 However, compensatory neovascularization is an important

physiological process that occurs in chronic myocardial ischemia.53 It has recently been

demonstrated in experimental models of myocardial ischemia and infarction in the pig

and rat that VEGF and VEGF receptors 1 and 2 are increased in chronically ischemic

myocardium and also in regions of ischemia surrounding an area of infarction.5456 Those

studies demonstrated that the VEGF ligand is upregulated in cardiomyocytes and its









cognate receptors exhibited increased expression in endothelial cells. Further studies

have revealed that hypoxia is a potent inducer of VEGF in cultured cardiac myocytes.7

Correspondingly, escalated bFGF activity has been shown in myocardium after coronary

artery ligation.58 This occurs in parallel with an increase in collateral blood flow in dogs,

and elevated levels of bFGF (but not VEGF) have been detected in the pericardial fluid of

patients with unstable angina.52 These observations on the molecular mechanisms of

physiological angiogenesis in ischemic myocardium led to the notion that cell based

therapy or pharmacological stimulation of angiogenesis may augment or even replace

more conventional forms of therapy. As will be described next, this notion has recently

received considerable experimental support in animal models.

Vascular healing may be mediated in part by the recruitment of EPC. In several

studies, genetically marked bone marrow-derived EPC were recruited to the ischemic

limbs of mice.11'17 In addition, transplantation of mature endothelial cells (EC) derived

from in vitro generated, human bone marrow-derived, multipotent adult progenitor cells

has facilitated revascularization of various tissues.59 The physiologic significance of

EPCs and EC in neovascularization was further underscored when thoracic aorta from

adult dogs previously transplanted with haploidentical bone marrow,were replaced with

Dacron grafts impervious to the ingrowth of established EC. In 3 month old grafts, the

newly established EC layer were determined to arise from donor derived cells from the

bone marrow.58 These findings indicate that EC derived from the EPC of bone marrow

origin can contribute to new blood vessel formation.

EPC for Neovascularization

This low number of EPC in the circulation increases dramatically under conditions

such as acute stress or injury to vasculature walls where there is a large apoptotic event of









EC. Normal replacement of the EC is usually accomplished by the surrounding local

endothelial cells which increase their proliferation and migrate to the areas of ischemia.

The terminally differentiated EC, however, are not able to proliferate considerably and

may not have the capacity to provide for the demand for new vessels. As described in

numerous studies, researchers have isolated circulating cells that are bone marrow

derived yet have endothelial potential-the EPC. These EPC are capable of lessening the

ischemic pressure of injured organs by revascularizing injured areas and restoring organ

function.

Our current understanding of the neovascularization process is founded on the

classical light-microscopy observations made by Clark and Clark in 1953.60 They were

among the first to reveal the sequence of events leading to the formation of new capillary

blood vessels in the translucent tails of amphibian larvae. These and later observations in

nondevelopmental settings provided a detailed histological account of new blood vessel

formation.61 62 On these pioneering results our current knowledge was founded. Clark

and Clark described a local angiogenic stimulus that causes endothelial cells of

preexisting capillaries or postcapillary venules to become activated. Although the precise

molecular consequences of this activation process remain to be clearly defined, activated

blood vessels are vasodilated, have increased vascular permeability, and experience

accumulation of extravascular fibrin as well as proteolytic degradation of the basement

membrane of the parent vessel.46-48 The endothelial cells then extend thin cytoplasmic

arms which direct migration into the surrounding matrix towards the angiogenic stimulus.

Migrating endothelial cells elongate and align with one another to form a capillary sprout,

and endothelial cell division, which occurs proximal to the migrating tip, further









increases the length of the sprout. The solid sprout gradually develops a lumen proximal

to the region of proliferation. Contiguous tubular sprouts fuse at their tips to form a

functional capillary loop in which blood flow is soon established. Vessel maturation is

accomplished by reconstitution of the basement membrane and recruitment of mural

cells.49 These cellular functions contribute to the formation of patent, endothelium-lined,

blood vessel structures.

Nitric Oxide as Potential Regulator of Vascular Formation

The process of angiogenesis in the adult is a complex sequence of growth factor

release, vasodilation, and recruitment or proliferation of endothelial cells to build the

vessels. These events are heralded by EC activation, most notably vasodilation, which

facilitates growth by granting access for cells to enter the area and remove any damaged

and dead cells/debris, increases nutrient depositing and breakdown of existing

extracellular matrix, and allows cells to establish permanent residence. One of the

molecules which has been shown to play an extensive role in vasodilation is Nitric Oxide

(NO).

NO has been used in nature for over 250 million years, longer than mammals have

existed. The horseshoe crab uses NO to prevent blood cell aggregation, and this function

is still retained in mammals. Other kingdom and phyla also utilize NO including fireflies

for their flashes, and plants that use NO's cytotoxic effects to fight infection. Victorian

physicians recognized its vasodilatory effect, even if they did not understand its

mechanism, and its medicinal value was written in a Sherlock Holmes story.130 The

medical uses for NO continued into World War I where doctors noticed that factory

workers in ammunition plants had lower blood pressures. This led directly to the

nitroglycerine tablet still used today to treat angina. The gas molecule itself, however,









was considered only a pollutant until recently. In the early 1990s the journal Science

named it molecule of the year. During this time over 250 articles per month were written

further characterizing NO and its effects. Robert F. Furchgott, Louis J. Ignarro, and

Ferid Murad received the Nobel Prize in Medicine in 1998 for their work on "nitric oxide

as a signaling molecule in the cardiovascular system." One historic irony is that Alfred

Nobel made his fortune by making dynamite from nitroglycerine, a known NO donor.

NO is unique among physiologic substances in the body as it is the only gas

produced in mammals that has a biological effect. This singular messenger molecule is

involved in the regulation of diverse physiologic functions including central and

peripheral nerve cell neurotransmission, promotion of the cytotoxic actions of immune

cells, and preventing/increasing leukocyte adhesion.63-67 It also has profound vasomotor

regulatory affect on vascular beds, specifically the regulation of smooth muscle

contractility and thus vasodilation.63'64

Three distinct isoforms of the enzyme that synthesizes NO (NOS) have been

identified, all of which share a 50-60% homology.67 Two isoforms are constitutively

active: the form expressed primarily in neuronal tissue (nNOS) and the form first found

in vascular endothelial tissue (eNOS). The third form's activity can be induced in a

variety of cell types usually in response to inflammatory signals and bacterial products,

and has been named inducible NOS (iNOS). Each of the three isoforms require

homodimerization for activity. The C-terminal portion of the NOS protein closely

resembles the cytochrome P-450 reductase possessing many of the same cofactor binding

sites.68 The extreme C-terminus contains an NAPDH binding region, conserved in all

three isoforms, that exactly aligns with the binding region of the cytochrome P-450.68









Following this is a flavin adenine dinucleotide and flavin mononucleotide consensus

sequence that is self-sufficient, unlike the P-450 enzyme, in that the oxygenation of its

substrate L-arginine occurs at the heme site in the N-terminal region.69 NO is generated

via a 5-electron oxidation of a terminal guanidinium nitrogen on L-arginine.68

Most of the physiologic actions of NO are brought about by the activation of

soluble guanylate cyclase. Binding of NO to the heme moiety of the enzyme causes a

conformational change that upregulates the activity over 400-fold resulting in the

formation of the intracellular second messenger cyclic GMP.70 NO has numerous

angiogenic effects, including (but not limited to) increasing matrix metalloprotinase

(MMP) expression and tyrosine phosphorylation of proteins in sprouting tips of

capillaries.65 Inhibiting NO production has been shown to decrease capillary formation

in rats with portal hypertension.66 In addition, DNA synthesis can be impaired by the

inhibitory effect of NO on ribonucleotide reductase which addresses the cytotoxic and

cytostatic effect of NO during an immune response. In the aqueous environment of the

cytosol, NO interacts with water to form the free radical peroxynitrate.67 Peroxynitrate

interacts with DNA leading to oxidation and initiation of a complex series of

transformations involving base damage or strand breaks as well as reactions with the

deoxyribose portion of the DNA.71 The DNA damage itself, along with the cell cycle

arrest as repeated and costly DNA repair occurs, ultimately leads to apoptosis.

Role of NOS in vessel formation

The process of angiogenesis can be divided into two components: endothelial cell

proliferation and blood vessel tube formation. The potent angiogenic agent VEGF

stimulates NO release from endothelial cells.72 VEGF-induced NO release has been

shown to modulate angiogenesis both in vitro and in vivo.73'74 The adult mouse model we









have developed utilizes the angiogenic influence of VEGF as we artificially increase

local expression of this growth factor in the retina mimicking the pathophysiology that

occurs in diseases associated with retinal neovascularization such as Diabetic

Retinopathy and Retinopathy of Prematurity. The established resident vascular

endothelial cells, the endothelial cells found in the circulation, and those derived from

HSC all respond to VEGF and influence local NO concentration. NO is crucial for the

myriad of physiological vascular functions, and its inappropriate production and release

has been linked to several pathologies.75 Consequently, agents which modulate NO

activity could find beneficial use in a therapeutic setting. As has been shown, NO plays

an integral role in blood vessel formation, and consequently makes a good starting

candidate for manipulating hemangioblast function.

The two isoforms which have a direct influence over endothelial cells are the iNOS

and eNOS isoforms as nNOS is found only in neuronal tissue.67 The role of eNOS in

angiogenesis is complex. Brooks et al. have demonstrated that eNOS deficiency, either

through gene disruption or through pharmacological inhibition, significantly protects the

developing retina from oxygen-induced retinopathy.76 The fact that nonspecific

inhibitors of NOS activity produced quantitatively similar levels of vaso-obliteration

compared to eNOS gene disruption also suggests that eNOS may be an isoform involved

in blood vessel regulation. Evidence suggests that NO and VEGF are reciprocally

regulated such that stimulation of VEGFR-2 activates eNOS leading to NO formation.76

NO inhibits VEGF production in adjacent cells by a paracrine feedback mechanism

involving inhibition of AP-1 binding to the VEGF promoter.7









iNOS has consensus sequences in its promoter for the transcription factors

hypoxia inducible factor (HIF) and NF-kappa B, both of which are activated under

conditions of ischemia.78 Consequently, iNOS is thought to be induced under conditions

of ischemia. Sennlaub et al. perfused retinas of wild type and iNOS knockout (iNOS -)

mice exposed to hypoxic conditions. They found that iNOS -/- animals had normal

intraretinal vasculature patterning whereas wild type animals had persistent avascular

areas.79 Interestingly, there was a reduction in preretinal neovascularization in iNOS --

mice indicating a dual role of iNOS in distinct retina layers. They corroborated these

observations with pharmacological inhibition of iNOS which increased retinal

neovascularization and decreased preretinal neovascularization. They found that

pathological intraretinal neovascularization was more severe in iNOS expressing

animals.80 These studies suggest that NO can be an important modulator of angiogenesis

in the retina, and that local levels of NO can influence the location and degree of

neovascularization. To our knowledge our model is the only one which allows for the

simultaneous examination of preretinal and intraretinal neovascularization at the same

time in an adult animal. We will use this model to understand the requirement of

beneficial intraretinal neovascularization compared to pathological preretinal

neovascularization allowing for the dissection of NO and other molecules which affect

vascular growth.














CHAPTER 2
GENERAL METHODS AND MATERIALS

The methods detailed below are used extensively in each chapter. Any

modifications made to this framework during an experiment are noted in the specific

chapter. Methods will be described in this basic outline: (1) the generation of the

GFP/BL6 chimera, (2) the induction of the retinal neovascularization, (3) the enucleation

of the eye for mounting, (4) examination of neovascularization via confocal microscopy

and (5) immunohistochemistry staining of serial sections.

Generating The GFP/BL6 Chimera

The generation of the chimeric GFP/BL6 animal will be described below. This

includes the harvesting of bone marrow from the GFP donor animal, the purification and

preparation of the marrow for FACS sorting of HSC, the preparation of the C57BL6

rescue marrow and recipient animals, and the HSC transplant and commensurate animal

husbandry concerns.

Harvesting Bone Marrow

The generation of the GFP/BL6 chimera animals requires extensive animal use and

cell manipulation. The transgenic mouse used as the donor strain was obtained from

Andras Nagy at mount Sanai in Toronto Canada.81 The strain carries green fluorescent

protein (GFP) driven by chicken beta-actin promoter and CMV intermediate early

enhancer and is ubiquitously expressed. The BL6 females were obtained from Jackson

Laboratories (Bar Harbor, Maine) and were at least 5 weeks old at the time of bone

marrow transplantation. Recent controversy concerning the events during stem cell









transdifferentiation for repair has led to the possibility that this may not be an inherent

ability stem cells, but rather a fusion event occurring between the stem cell and target

tissue. The transplantation of male HSC into female recipients directly addresses this

issue by allowing for fluorescent in situ hybridization of tissue samples looking for the Y

chromosome and determination if a fusion event has occurred. After fully-grown GFP

males are euthanized and sacrificed, the long bones in the legs were immediately

removed. All muscle, tendon, and ligature was dissected from the bone which was

immediately placed in ice-cold PBS. Each bone end was then pruned back about 1-2

millimeters to expose the hollow core of the marrow space. The bone marrow was

flushed out into a tissue culture treated plate by inserting a 26-gauge needle into one end

of the bone and washing 1-2 milliliters of Dulbecco's Modified Eagle's Medium (Gibco)

through the hollow bone core. The cells were kept on ice at all times. The liberated

marrow was then triturated with a 26-gauge needle to break up the cell clumps and

allowed to adhere to a tissue culture treated plate (Gibco) for 120 minutes. This step

allows for an initial enrichment of HSC from other adherent progenitor cells such as

mesenchymal stem cells (MSC) since hematopoietic progenitor and stromal cells adhere

to the tissue culture treated plastic, while HSC will remain suspended in the media. The

complete volume of media containing the nonadherent HSC was then gently drawn up,

washed in >10mL volume of cold media, and pelleted by centrifugation at 1000 x g

performed at 4 degrees Celsius. The cells were resuspended and stained as outlined by

the protocol of the Milteny MACS system in the following section.

Initial Purification of HSC by MACS

Initial HSC purification was done through sorting of the cells by magnetic beads

using the Milteny Magnetic Activated Cell Sorting (MACS) system. Briefly, cells were









stained with an antibody conjugated to a magnetic bead. The antibody, and subsequently

the bead, is bound to the cell. When these cells are then run over a column in the

presence of a magnetic field, those cells which have the specific surface antigens, and

thus the antibody-bead bound to them, will adhere to the column (termed positive

fraction). Cells which do not present that surface marker (negative fraction) will pass

directly through the magnetic field and be removed from the positive fraction of cells.

The magnetic field can then be removed and the positive fraction collected from the

column.

To begin the MACS enrichment, cell number and viability were determined from

the total marrow flushed from the long bones to ensure that the correct amount of

antibody, beads, and staining volume will be used. To determine the cell number, I

resuspended the washed cells in trypan blue and counted bright cells using a

hemacytometer under a phase-contrast microscope. The enumerated cells were then

washed in >10mL cold PBS and stained with Sca-1 microbeads (Milteny) in appropriate

volume. The cells were run over 2 separate columns to insure enrichment, and the flow-

through was discarded and the positive fraction retained. At this time a >90% Sca-1

positive purity typically has been achieved. After enrichment, cells were immediately

pelleted and placed back on ice for fluorescent antibody staining for FACS sorting.

Final Purification of HSC by FACS

Again all antibody concentrations and incubation times were followed according to

the parameters described by the manufacturer guidelines. For HSC purification I used

three different fluorochromes: C-KIT conjugated to APC, biotynylated Sca-1 (with

Streptavidin-PharRed secondary antibody), and the lineage markers B220, CD3, CD4,

CD8, CD11B, GR-1, and TER-119 all directly conjugated to PE (Pharmingen). The










FACSvantage SE is able to isolate single cells based on the surface antigen bound by

antibodies and hence the spectrum of absorbance and fluorescence emitted by that cell.

Two rounds of purification are needed to ensure complete removal of all non-HSC cells.

See Figure 2-1 for of an example of the gates used to enrich and isolate single HSC.

o K00301 31.004 KO 03 01 31.004 KO 03 01 31.004 KO 03 01 31.007
R3

a- C\j < c,. < c\



0 50 100 150 200 250 '-' n'1 '- n4 lO0 101 102 103 104 oO 101 12 103 4
Side Scatter GFP CKIT APC CKIT APC


Figure 2-1. Fluorescence activated cell sorting gates for isolating HSC. HSC were
removed from bone marrow, enriched by MACS, and stained for SKL
0 50 100 150 200 250 1, 1',2 ,14 -00 101 102 103 104 700 101 102 103 104
Side Scatter GFP CKIT APC CKIT APC


Figure 2-1. Fluorescence activated cell sorting gates for isolating HSC. HSC were
removed from bone marrow, enriched by MACS, and stained for SKL
surface expression. First panel: Forward and Side Scatter of MACS
enriched cells with gate R1 drawn. Second panel: Cells are enriched for
GFP and Lineage positive cells (B220, CD3, CD4, CD1 b, Gr-1, Ter-119)
are depleted excluding gate R2. Third panel: Sca-1 and c-kit positive cells
from gate R1 and R2 are enriched in gate R3. Cells are then further
enriched by gate R4 based on the same parameters. Panel 4: Reanalysis of
cells based on Sca-1 and c-kit expression. These doubly sorted enriched
cells were used for transplantation.

The flow rate is set at 10,000 events per second with no greater than a 10% abort

proportion. The cells were then collected in media immediately after completion of the

sort, isolated, and injected into the recipient animals following "rescue" marrow isolation

and recipient preparation kept on ice at all times.

Harvesting of BL6 Rescue Marrow with HSC Depletion, and Irradiation of
Recipient Animals.

The harvesting of non-GFP female BL6 marrow was performed in the same

manner as the HSC, except these cells were not given time to adhere to the tissue culture

treated plate. Once the marrow was flushed, washed, and counted, a Sca-1 depletion was

done to remove any HSC from the rescue marrow which would compete with the donor









GFP HSC. This rescue dose is administered for twofold reasons. The immune system of

the irradiated animal will experience an interruption and often the animal will become

anemic. Until the HSC can engraft and repopulate hematopoiesis, these short term rescue

progenitors will help the animal mount an immune response and provide the necessary

blood products as needed. Again cells were stained as described in the MACS magnetic

bead section, but this time the cells were Sca-1 depleted three times to ensure that the

rescue marrow was devoid of HSC. Recipient BL6 mice were finally irradiated with 950

RADS of gamma radiation to prepare the bone marrow for transplantation.

Purified GFP HSC and Depleted Rescue Marrow Transplantation and Ensuing
Animal Husbandry Concerns

The HSC depleted rescue marrow was count as above and 1 x 106 cells in a 100

microliter volume were aliquoted into a fresh Eppendorf tube. The highly enriched HSC

were then singly isolated in the following manner. A volume of the sorted sample was

placed on a glass drop slide and examined under a phase-contrast microscope. The cells

were diluted to a concentration where single cells can be visualized, isolated, and

captured one at a time with a micropipette. Under the scope a single, round, bright,

viable cell was isolated and drawn up into a pulled glass micropipette by mouth pipetting

with a suction tube. The needle was examined to visualize the cell to ensure that only

one cell was drawn. The cell was then place into the 100 microliter aliquot containing

the HSC depleted rescue dose. The rescue/single HSC mixture was drawn into a fresh

insulin needle and syringe to ensure no contamination of other samples. Finally, an

anaesthetized, irradiated BL6 animal was injected in the retro-orbital sinus cavity. The

animals were monitored until they overcome the effects of the anesthetic and then be









placed on a regime of antibiotics for the next month until multilineage engraftment had

been verified.

Verification of Multilineage Reconstitution

The recipient animals were given one month for the HSC to home to the bone

marrow niche and begin to divide to produce progenitor cells which will contribute to the

various hematopoietic cell lineages. Determination of engraftment was resolved by

peripheral blood sampling and FACS analysis to determine whether the marrow was

repopulated or if the animal's native marrow recovered. Each animal had a peripheral

blood sample drawn through a tail vein bleed and the blood was collect in a tube

containing PBS and 5mM EDTA to act as an anticoagulant. The erythrocytes were

removed with a FICOLL PLAQUE (Amersham Biosciences) purification. Briefly, the

blood/PBS sample was layered on top of two times greater volume of FICOLL. The

emulsion was centrifuged and the "buffy" layer containing the nucleated cells at the

interface was removed. The lymphocyte layer containing the nucleated cells was washed

in 5X volumes of PBS and stained with the various lineage marker antibodies conjugated

to PE. Samples were analyzed by FACS caliber, and animals exhibiting GFP positive

cells of the various lineages were scored positive for engraftment. The positive animals

were then monitored an additional three months where multi-lineage reconstitution is

reconfirmed to demonstrate long-term engraftment by HSC. Exogenous growth factor

was then administered as described below.

Induction of Retinal Ischemia

The next step involves administration of an endogenous growth factor and vessel

damage in order to promote blood vessel growth in the retina. Fully and robustly

engrafted animals were selected and anaesthetized. VEGF was administered directly into









the vitreous using a 36-gauge needle and Hamilton syringe. Either purified (40ug/kg)

VEGF protein (Sigma) or (2 x 108 particles) AAV-VEGF (VectorCore, UF), where CMV

promoter drives expression of VEGF in an Adeno Associated Vector, was used. VEGF is

an endothelial cell-specific mitogen which is transcriptionally regulated by the

cytomegalovirus promoter/enhancer when packaged in AAV. AAV mediates long-term

expression in nondividing cells, which allows for stable expression and constant amounts

of VEGF to reach the area of ischemia to promote neovascularization.30

The study of clinical diseases such as Diabetic Retinopathy and Retinopathy of

Prematurity has led to an understanding of the pathology which occurs in these diseases.

In these conditions the eye "detects" a lack of oxygen, either due to the diabetic condition

leading to leaky vessels, or the removal of a prematurely born baby from an incubator's

oxygen-rich environment. The model takes advantage of this neovascularization by

creating a local region of ischemia in the eye through cauterizing of large blood vessels

with a laser. As a result, the cells signal new blood vessel growth in the region in an

attempt to relieve the ischemic pressure.

Peak expression of VEGF by AAV has been determined to be at 3-6 weeks,

therefore the physical disruption of the blood vessels is done during this time

(unpublished data). First, mice were anaesthetized normally with a general anesthetic,

and concurrently a 10% sodium fluorescein (Akorn) solution was administered

intraperitineally. This dye labels blood vessels facilitating visualization during

photocoagulation. The eyes were dilated with 1% atropine (Akorn) for 5 minutes,

washed with PBS (Gibco), and subsequently dilated with 2.5% phenylephrin (Akorn) for

5 minutes. Immediately after the two 5 minute treatments the mice underwent laser









treatment. An Argon Green laser system (HGM Corporation) was used for retinal vessel

photocoagulation with the aid of a 78-diopter lens. The blue-green argon laser

(wavelength 488-514 nm) was applied to various venous sites juxtaposed the optic nerve.

The venous occlusion were accomplished with >60 burns of 1-sec duration, 50 millimeter

spot size, and 50-100 milliwatt intensity. Again the animals were allowed to recover for

30 days while the transplanted HSC, directed by the ischemia and induced by the VEGF,

contributed to the neovascularization in order to relieve the hypoxia produced by the

cauterizing of the existing vessels.

Eye Removal

One month after ischemic injury the eyes were ready to be enucleated and

neovascularization imaged by confocal microscopy. Mice were first anesthetized and

then perfused while sedated. Peripheral blood and bone marrow was collected to confirm

donor contribution analysis by FACS with lineage specific antibodies conjugated to PE

(BD BioSciences) similarly to the procedure outlined above. First, the chest cavity was

opened and the ribs cut away to expose the heart completely. The left atria was

punctured with a 26-gauge needle and injected with >3 mL of 50 mg/mL tetramethyl

rhodamine isothiocyanate (TRITC)-conjugated dextran (160,000 avg. MW, Sigma

Chemical) in phosphate-buffered formaldehyde, pH 7.4. The perfusion was performed

slowly into the left ventricle and is integral for the functional assay. Immediately

afterwards the eyes were removed by sliding a curved forceps underneath the eyeball and

pulling the globe out. The eye was punctured with a 26-gauge needle to allow complete

perfusion. The eye was placed in fresh 4% PFA and shaken at room temperature for 30

minutes. The globe was then transferred to 1X PBS and washed by shaking at room

temperature for 30 minutes to overnight. After washing with PBS the eyes were









dissected. To do this I placed the eye under a surgical microscope and made an initial

incision in the cornea. The opening was enlarged until it could accommodate the lens of

the eye. The lens was gently pushed forward until it exited through the hole cut in the

cornea. The remaining cornea was then trimmed to where the sclera and cornea meet.

The retina was dissected away from the retina pigment epithelial (RPE). To do this I

gently pushed down on the posterior portion of the RPE and rolled the forceps forward.

The retina then detached and was readily mounted. The thickness of the retina (>200um)

prevents adequate perfusion of antibody, therefore the retina was placed on a glass slide

and 5-6 cuts were made around the periphery so that the retina lies flat when mounted.

The tissue was placed in Vectashield mounting medium (Vector Laboratories) to inhibit

photo-bleaching. The retinas were immediately imaged. I used an Olympus IX-70, with

inverted stage, attached to the Bio-Rad Confocal 1024 ES system for fluorescence

microscopy. A Krypton-Argon laser with emission detector wavelengths of 598nm and

522nm differentiated the red and green fluorescence. The lenses used in our system were

the (Olympus) 10X/0.4 Uplan Apo, 20X/0.4 LC Plan Apo, 40X/0.85 Uplan Apo,

60X/1.40 oil Plan Apo and 100X/1.35 oil Uplan Apo. The software was OS/2 Laser

Sharp.














CHAPTER 3
THE HEMATOPOIETIC STEM CELL HAS HEMANGIOBLAST ACTIVITY

During development there are several types of stem cells broadly classified based

on their ability for form specific tissue types. After fertilization during the first few days

of division, the embryonic cells are described as totipotent. They have the capacity to

produce all the cells, tissues and organs that make up the body along with all of the

extraembryonic tissue of the trophectoderm. After the first four to five cell divisions, the

embryo forms a hollow sphere called the blostocyst. The blastocyst contains a population

of cells located in the inner wall which are capable of producing each of the over two

hundred different cell types of an organism. These differ from the totipotent cells in that

no one of them can produce an entire organism, nor can they produce the cells of the

trophectoderm. Finally, after birth and into adulthood, several types of tissues have cells

residing within them which are able to produce the tissue type where they reside. This

can occur constantly, such as the hematopoietic stem cell producing all of the blood cells,

or only in times of stress or injury such as the oval cells producing hepatocytes. These

stem cells are called multipotent, and in most cases under "normal" conditions these cells

are thought to produce only one cell type.

In the adult, stem cells are believed to define unspecialized cells that can self-renew

(or proliferate) for extended periods of time without differentiating. This process is not

well understood, but is believed to involve asymmetric cell division where a copy of

itself is produced along with a further differentiated daughter cell. These stem cells

exhibit a stable, normal chromosome complement and cannot perform any specialized









functions. However, they do have the potential to give rise to cells with specialized

functions-- a process known as differentiation. It is suggested that some of these cells

may be able to differentiate into multiple non-related cell types, a characteristic called

plasticity.

Adult Hematopoietic Stem Cells

Adult hematopoietic stem cells are defined by their ability to both self renew and

provide all of the hematopoietic cells necessary to replace those lost each day. The bone

marrow produces an estimated 2-3 million cells per second or over 200 billion per day.

The tremendous proliferative potential of these cells would quickly be exhausted

throughout a lifetime if there were not some self-renewing parent call to maintain

hematopoietic and lymph system progenitor cells. This proliferative and self-renewing

capacity make HSC excellent clinical tools for the treatment of hematological

malignancies such as leukemias and lymphomas. In these conditions, the bone marrow

population, most notably the HSC, is replaced by cells which are non malignant and

healthy to reconstitute normal hematopoiesis of an individual. In research, our ability to

enrich for HSC coupled with their easy transplantability opens up large realms of

exploration. Similarly to other multipotent stem cells, HSC and believed to retain a

significant ability to transdifferentiate. These two characteristics make the HSC ideal for

identifying the potential of HSC to regenerate or contribute to non-hematopoietic tissues

following injury or stress. This data has yielded a large amount of initial excitement,

however there has since been a cooling in the enthusiasm due to the increased, though

warranted, scrutiny. In order for cell-based therapy to have clinical applications, basic

criteria and standard must be established to determine if the phenomenon researchers are

characterizing is true HSC plasticity and cannot be attributed to artifact. As a result









several stringent criteria have been outlined which must be fulfilled in order to

demonstrate true plasticity.

The criteria demonstrating HSC plasticity is three-fold. First, the cell must be

capable of self-renewing and homing to the bone marrow thereby reconstituting

hematopoiesis for the lifetime of the organism. This is necessary so that short term

progenitors are not used as therapy which may slowly die off as progenitors differentiate

and are not replaced. Long-term repopulating self-renewing cells must be transplanted so

that the therapy would not fail and the disease or pathologic condition reemerge.

Secondly, the bone marrow contains a myriad of cell types ranging from those along any

point of hematopoietic development to the supporting cells of the stroma. During a bone

marrow transplant, a number of these cells could be transplanted with the bolus

containing the enriched HSC no matter stringent the purification parameters. These

"contaminating" cells could contribute to the tissue type where the donor-derived tagged

cells are found confounding results. In order to conclusively demonstrate the plasticity of

the HSC, clonal studies must be done. Through clonal transplants, a single cell must be

shown to be able to produce the blood along with the non-hematopoietic tissue. These

experiments exclude the possibility of several different cells accomplishing different

roles, and tissue which arises from the donor must necessarily be from the single cell.

Finally, for these cell based therapies to be practical it must be demonstrated that the

plasticity measured is robust and functional transdifferentiation into the non-

hematopoietic tissue. Many cells, especially those of the immune system, are capable of

assuming the general morphology or even surface marker expression of cells they are

nearby either due to stimulation or macrophage engulfment. It must be demonstrated that









the cells are physiologically performing the role of the tissue they are replacing, i.e. cells

that are residing in the pancreas having the morphology and characteristics of beta cells

must actually produce insulin to be therapeutic. In addition, a few isolated cells capable

of producing insulin will not rescue a person from diabetes, therefore the

transdifferentiation or plasticity must be robust producing a physiologically relevant

amount of tissue. Only when these three stringent criteria have been met can the cell be

classified as plastic. To date there has been relatively few examples fulfilling all three,

although those that have present some exciting potential.

One of the initial studies have shown that after long term stable hematopoietic

reconstitution by a single bone marrow HSC, donor-derived cells could be found in

multiple tissues including the brain, skeletal and cardiac muscle, liver, and endothelial

cells.82 This elegant work used a homing assay to isolate HSCs which presented stem

cell specific surface markers and then were able to successfully home to the bone marrow

niche. These homed cells were then isolated and single cells were transplanted into

lethally irradiated recipients. While this work was of note, there was as significantly low

level of contribution to the various tissues and there was no functional assay of the donor-

derived cells. It does, however, suggest the exciting possibility of regeneration of various

damaged tissues by HSC-derived progenitors. Two notable studies also demonstrated the

plasticity of the HSC in liver to replace hepatocytes injured chemically.83' 84 Excitingly,

these cells were able to restore liver function, however, clonal assays were not done in

these transplant studies. In addition, Orlic et al. demonstrated the functional recovery of

cardiac muscle through HSC transplantation.50 After these initial pioneering papers a

flood of work was embarked upon, however since then the tide was stemmed due to the









difficulty of meeting all three criteria." Grant et al. has developed a model mimicking

diabetic retinopathy, and using this model we have been able to expand the understanding

of HSC while fulfilling the three plasticity criteria.30

Diabetic Retinopathy

Diabetic retinopathy is the leading source of legal blindness among working-age

Americans. It is caused by damage to the small blood vessels in the retina as a result of

diabetes mellitus. It is estimated that over fourteen million people in the United States

have diabetes with approximately half of these individuals not yet diagnosed and unaware

of the condition. Ninety percent of patients with diabetes have noninsulin-dependent

diabetes mellitus (NIDDM) and control their blood sugar with oral medications or diet

alone. The other ten percent have insulin-dependent diabetes mellitus (IDDM), and must

use insulin injections daily to regulate their blood sugar levels. Although diabetic

retinopathy is frequently seen in both types of diabetes, patients with IDDM are at greater

risk for Diabetic Retinopathy complications. The risk increases over time for all patients

with diabetes. After five years, approximately one-quarter of patients with IDDM have

retinopathy and by fifteen years, nearly everyone with IDDM experiences retinal damage.

Diabetics as a group have twenty-five times the usual risk of blindness.

The entire vasculature of a diabetic individual experiences the pathologic changes

including plaque formation and swelling of the endothelial cells. These vessels have a

diminished capacity to carry blood, and consequently all downstream tissue becomes

ischemic. This ischemia causes changes in existing vasculature by stimulating

compensatory growth. This pathologic growth is unstable and the vessels are fragile. As

a result their rupture can cause leakage of blood into the vitreous and consequently vision

loss.









Once pathologic retinopathy has developed, laser photocoagulation is currently the

mainstay of treatment. Laser surgery has been used in the treatment of diabetic

retinopathy for more than twenty years and its benefit has been clearly established. The

abnormal neovascular vessels of proliferative diabetic retinopathy are treated with

panretinal laser photocoagulation (PRP). This type of laser involves treatment to the

peripheral retina which is not receiving adequate blood flow due to the vessel pathology.

By photocoagulating the ischemic regions the stimulus that drives the neovascular

process may be halted. This type of laser treatment is frequently successful in stopping

the growth of the abnormal vessels, but in some cases they may regress. It is not without

side effects as some loss of peripheral and color vision is normal following this type of

treatment. Ironically it is the existing PRP laser treatment in humans from which we

developed our mouse neovascularization model described in chapter 2 and used

throughout this body of work.

Angiogenesis vs. Neovascularization

Our diabetic model is an example of neovascularization. During

neovascularization, de novo blood vessels are formed which are not derived from

preexisting vasculature. The cells which contribute to neovascularization are derived

from a distant source, namely the HSC residing in the bone marrow. Contrastingly,

angiogenesis is the process of endothelial cell sprouting from pre-existing vasculature.15

Local endothelial cells, even with their diminished capacity to divide, are able to produce

enough daughter cells to supply blood vessel lining, i.e. normal endothelial cells turnover

is replaced by neighboring cells. Under conditions of severe injury or in some pathologic

condition such as diabetic retinopathy, these vessels are derived from the EPC. In vitro

studies have shown that EPC are capable of producing tube-like structures under culture









conditions and can be derived from bone marrow cells.18' 86, 87 Pro-angiogenic factors

such as VEGF and GM-CSF increase the number of circulating EPC in the adult and

have been shown to promote blood vessel growth.88'89 In addition,

hydroxymethlyglutaryl-CoA reductase inhibitors are efficient stimulators of EPC

transdifferentiation and formation of endothelial cells involving the Akt protein kinase

pathway.90 In vivo, several groups have shown that EPC contribute to blood vessels in

adult organisms to relieve cardiac ischemia, however these models used short-term

progenitor cells in an acute injury model.29'91 92 While clearly the EPC can functionally

provide therapy for ischemic injury, these studies did not demonstrate whether these EPC

were derived from the HSC or from some other cell such as the mesenchymal stem cell.

During development, the pluripotent progenitors which contribute to the formation

of both blood and blood vessels are the hemangioblasts.93-96 The hemangioblast

phenotype can also be derived in vitro from embryonic stem cells when cultured with

VEGF.93 The presence of an adult hemangioblast in vivo and the role bone marrow

derived cells play in neovascularization, however, is incomplete. The work described

here will elucidate the role HSC derived cells have in promoting or contributing to

neovascularization and describe the plastic nature of these cells in ischemic tissue.

Results

The methods used to obtain the following results are described in detain in chapter

two. Any alterations or additions of the model described will be noted.

The C57BL6.GFP Chimera

As described above, there are three stringent criteria for the demonstration of HSC

plasticity. Briefly, the criteria are 1) the cell must be self renewing and able to provide

all of the blood and blood products for the entire life or the organism, 2) the cell must be









able to do so clonally, and 3) the cell must product functional non-hematopoietic tissue in

a robust manner. The C57BL6.GFP chimera studies will directly address these three

criteria. To address question one, HSC were isolated from a donor GFP animal as

described. Figure 3-1 is an example of the enriched HSC. The row of panels was

obtained from a whole bone marrow preparation purified with a FICOLL gradient. A

vast majority of cells are lineage positive (>80%) and Sca-1 negative (>93%) indicating

that the bulk of the cellular mass in the marrow is progenitor cells. Once the cells have

been enriched for HSC with MACS and FACS, a high proportion of cells have the

expected surface marker phenotype of the HSC (>98% Sca-1 positive and >99% lineage

negative).

o10 4 o1 1







Gip Gfp Gfp

Figure 3-1. Reanalysis of HSC post-enrichment used for transplantation. HSC were
% % ,. -.2 326.
:: C ""









flushed from the bone marrow, enriched by MACS, stained for the SKL
surface markers, and enriched by FACS. Panel 1: Sca-1 expression of
enriched HSC achieving 98% purity. Panel 2: Cells expressing any of the
lineage markers were depleted to a 99% purity. Panel 3: 99% of the
enriched cells express the pan-hematopoietic marker CD45.

These cells were then transplanted into a lethally irradiated recipient and allowed to

long term engraft for three months. Once long term multilineage engraftment was

demonstrated in the peripheral blood of the primary recipient, the animal was sacrificed

and the GFP HSC isolated from the marrow. These cells were once again trlasplanted

into secondary lethally irradiated recipients and allowed to engraft for four months. This









combined total represents much longer than any short-term progenitor would be able to

provide hematopoiesis. Figure 3-2 depicts a representative FACS analysis of the

peripheral blood of a serially transplanted mouse with donor GFP HSC. Significant

proportions of the T-cell (CD4), B-cell (B220) and mylomonocytic (CD1 Ib) lineages are

donor derived (see methods chapter for description of GFP standardization). This

contribution could only be from a long term repopulating, and thus self-renewing HSC.



9 14 12 41



010 10 10 1(
10 101 102 103 4 10 1 10 103 10 10 101 10 10 104
Gfp Gfp Gfp

Figure 3-2. HSC can engraft multiple lineages long-term and self-renew. Enriched
HSC were transplanted into a primary recipient and hematopoietic
reconstitution was demonstrated long-term. HSC were then isolated from
the primary recipients and transplanted into lethally irradiated secondary
recipients. Peripheral blood was isolated from secondary recipients and
stained for various hematopoietic lineages. Panel 1: CD4 (T-cell) lineages
were donor-derived. Panel 2: B220 (B-cell) lineages were donor-derived.
Panel 3: CDllb (Mylomonocytic) lineages were donor-derived.

The second criteria addresses the clonality of the HSC in its ability to produce all

the blood lineages from once single cell. These experiments will also be crucial to

demonstrate the ability of the HSC to produce an alternative non-hematopoietic tissue

type. In these experiments, HSC were purified as above, except that during the final

transplanting into the lethally irradiated recipients, one single cell was isolated and

transplanted along with non-GFP rescue progenitor cells. Figure 3-3 is the peripheral

blood mononuclear cells stained with the same lineage markers, T-cell (CD4), B-cell

(B220) and mylomonocyte (CD1 lb). This figure demonstrates the clonal ability of the









HSC in hematopoiesis, or the capability of a single cell to provide all of the blood

lineages. Each of these cohorts was then placed into the neovascularization model.


7 9 9. 272




10 101 3 10 10 102 103 104 100 101 102 103 104
Gfp Gfp Gfp

Figure 3-3. HSC can produce all hematopoietic lineages clonally. Single enriched
HSC were transplanted into lethally irradiated recipients. Peripheral blood
was isolated and stained for various hematopoietic lineages. Panel 1: CD4
(T-cell) lineages were donor-derived. Panel 2: B220 (B-cell) lineages
were donor-derived. Panel 3: CD1 lb (Mylomonocytic) lineages were
donor-derived.

Assessment of GFP Retinal Blood Vessel Endothelial Cells

Once long term multilineage engraftment has been demonstrated in these animals,

exogenous growth factor (VEGF) was administered to prime the system for blood vessel

growth. As noted, VEGF is a potent stimulator of endothelial recruitment and blood

vessel formation. The VEGF is packaged into AAV which infects the cells of the retina

and causes overexpression and accumulation of the protein. Indeed, the vitreous of the

eye is almost completely lacking proteases, so there is ample signal for the endothelial

cell formation of blood vessels. After one month to allow for peak VEGF expression, the

major blood vessels of the eye are photocoagulated with a laser. This ischemic injury,

combined with the VEGF, elicits a dramatic neovascular response in the retina. One

month after photocoagulation the animals were sacrificed to measure the amount of HSC

contribution to the new vasculature. The mice were perfused with Hoechst stain to mark

the nuclei of cells and delineate vessel lumens. Eyes were removed for sectioning and

immunohistochemical analysis of the donor cells for both blood and endothelial cell









surface phenotypes. This was done to determine whether the cells were truly

transdifferentiated into endothelial cells, or if they were invading leukocytes or

macrophages. Eyes were sectioned along both sides of the optic nerve, and more than 30

sections were obtained from each eye. The sections were stained with hematoxylin,

Factor VIII, platelet endothelial cell adhesion molecule, or mouse endothelial cell

antigen-32.

Figure 3-4 shows the GFP cells which surround the lumen of the newly

formed vessels. These same sections when counterstained with the endothelial specific

markers demonstrate that the cells lining the lumen of the vessels are endothelial in

nature. Each row is of a different capillary tuft, and each is stained with a different

endothelial marker. The top row is stained with Factor VIII conjugated to PE. Panel C

shows that the vessel lumen is endothelial, as expected, and the GFP cells seen in panel B

colocalized with Factor VIII which merge yellow in D. Panel F shows another vessel

with donor derived cells (GFP in Panel F) which costain with PECAM in panel G.

Another vessel has the same donor derived endothelial phenotype expressing MECA-32

(Panel J and K). Finally, in these vessels when stained with CD45, a hematopoietic

specific marker, the GFP cells did not express CD45 and had entirely adopted the

endothelial phenotype.

This result was also readily observed on a whole mounted retina. Figure 3-5

shows an entire retina from an animal perfused with the red fluorescent dye as described

in the methods chapter. Under low power (Panel A), areas of donor derived GFP cells

are visible contributing to the vasculature in the treated eyes. The contralateral, untreated

eye has no such endothelial contribution, although areas where the blood was not









removed by the perfusion can be seen containing the GFP hematopoietic cells (Panel B).

Under higher power magnification, GFP cells can be seen wrapping around the vessels

containing the red dye in various stages of blood vessel formation (Panels C-F). Of note,

this HSC contribution to endothelial cells did not occur where no ischemic injury was

induced.


I f


Figure 3-4.


Donor-derived HSC contribute to endothelial cells of blood vessels in the
eye. Neovascularization was induced in HSC engrafted animals. Retinas
were sectioned and stained with endothelial specific markers. Panel A; A
treated animal was perfused with a buffer containing Hoescst dye to
delineate vessel lumen and a treated control retina was cross-sectioned.
Panel B: The same cross-section had GFP donor-derived cells lining the
blood vessel lumen. Panel C: The same cross-section was stained with an
antibody to Factor VIII conjugated to PE to stain endothelial cells. Panel
D: Merged images of B and C demonstrating endothelial cells which were
donor-derived. Panels E-H: Another cross-section was stained with
Platelet Endothelial Cell Adhesion Molecule-1 and illustrated in the same
manner as A-D. Panels I-L: Another cross-section was stained with
Mouse Endothelial Cell Adhesion-32 and illustrated in the same manner as
A-D. Magnification is x60.







































Donor-derived HSC produce functional endothelial cells surrounding
blood vessel lumens. Mice were long-term hematopoietic engrafted with
GFP HSC and placed into the neovascularization model. The animals
were perfused with TRITC labeled dextran, sacrificed, and retinas were
imaged by confocal microscopy. Panel A: A whole mounted retina is
demonstrated under low magnification (x4). The red fluorescence fills the
perfused, functional blood vessels. Small capillary tufts of donor-derived
GFP cells, which are magnified in C, D, E, & F, can be observed around
areas of photocoagulation. Panel B: From a contralateral, untreated eye,
circulating donor-derived GFP hematopoietic cells are present in the
lumen of a blood vessel. Magnification x40 Panel C: Donor derived-GFP
cells are associating with a perfused blood vessel (large arrowhead).
Other donor-derived cells have either not directly associated with vessel
lumens or have extravasated and not formed endothelial tubes.
Magnification x40. Panels D-F: High magnification images show GFP
cells surrounding vessel lumens (D&E) and forming early
neovascularization (F). Magnification x60.


Figure 3-5.









The HSC has Hemangioblast Function

The recruitment of HSC derived cells to regions of injury agrees with studies done

which have found limited contribution to non injured tissues." In these experiments, the

donor derived GFP cells were able to contribute to both the blood products and

endothelial cells of the vasculature in the same mouse, however the exact cell which

could accomplish these feats cannot be established by these experiments.

The classic definition of a HSC is a cell capable of long term hematopoietic

reconstitution after irradiation, or self-renewing. We have fulfilled this definition

through the series of transplantation studies described above, however the ability of a

single HSC to do so clonality, and thus ruling out any contribution by other

"contaminating" cells, was necessary to prove HSC plasticity. As described in the

methods chapter, a single HSC was enriched and isolated though micromanipulation.

The HSC was transplanted along with Sca-1 negative non-GFP bone marrow cells (short

term progenitors) and transplanted into a lethally irradiated recipient. Of the 80 mice

transplanted, peripheral blood long term multilineage engraftment was demonstrated in 3

animals which were then subjected to the ischemic neovascularization model. Each of

the three animals exhibited the capillary tuft growths seen in the previous experiments

that were entirely donor derived as demonstrated by GFP expression. In addition these

vessels were functional in their ability to hold the red fluorescent dye perfused into the

vasculature. Since these animals had both blood and blood vessels which were derived

from a single transplanted HSC there can be no contribution from another source and any

GFP cells must necessarily be derived from the HSC. As shown in figure 3-6, the HSC

demonstrated hemangioblast activity in their ability to produce both blood and blood

vessels in a clonal manner. Panels A-C are from a serially transplanted mouse. The red









perfused blood vessel (Panel A) colocalizes with the GFP donor-derived endothelial cells

(Panel B) to show donor derived neovascularization (Panel C). These vessels were

derived from a self-renewing HSC. Panels D-F are from a single cell transplanted

animal. The red perfused blood vessel (Panel D) colocalizes with the GFP donor-derived

endothelial cells (Panel E) to show donor derived neovascularization (Panel F). These

vessels arose from the HSC in a clonal manner, therefore the HSC can give rise to both

blood and blood vessels and function as a hemangioblast.

Discussion

The previous work demonstrates the true plasticity of the HSC and fulfills the three

criteria established to prove this capacity. The self-renewing capability was

demonstrated through serial transplantations and long term hematopoietic reconstitution.

The ability of the HSC to provide hematopoiesis along with non hematopoietic tissue in a

clonal manner was shown through single cell transplants. Both experiments

demonstrated the ability of the HSC to produce functional vessels in a robust manner.

Taken together, these experiments outline an alternative developmental fate of the HSC,

namely the EPC, and describe how this outcome can be induced through growth factor

administration and ischemic injury. The EPC was shown to be derived from the HSC,

and not the MSC as previously posited.97 This understanding is especially valuable in

current treatments where the EPC has been shown to have the ability to contribute to

therapeutic neovascularization in several studies of ischemic injury, some in human

clinical trials.51' 88 This work demonstrates that vessel growth is not only carried out by

local or circulating endothelial cell angiogenesis, but under conditions of injury the HSC

can provide neovascularization. New blood vessels formed were largely derived from the

recruitment of undifferentiated precursors cells from the bone marrow.


































The HSC is self-renewing and can clonally form endothelial cells. Both
serially transplanted long-term engrafted and single cell transplanted
animals were placed in the neovascularization model. Animals were
perfused with the TRITC-labeled dextran and retinas were imaged by
confocal microscopy. Panel A-C: A long-term engrafted serially
transplanted mouse retina was imaged. Panel A shows the red channel
only indicating perfused, and therefore functional blood vessels. Panel B
is the donor GFP HSC contribution to the neovascularization. A and B
were merged in C and yellow areas are donor derived cells colocalizing
with the perfused vessel. The HSC is self-renewing and can produce all
blood lineages and endothelial cells of the vasculature. Panel D-F: A
single-cell transplanted mouse retina was imaged. Panel D shows the red
channel only indicating perfused, and therefore functional blood vessels.
Panel E is the donor GFP HSC contribution to the neovascularization. D
and E were merged in F and yellow areas are donor derived cells
colocalizing with the perfused vessel. The HSC can clonally produce all
hematopoietic lineages and endothelial cells lining blood vessel walls.
Magnification x60.


The next series of experiments will ascertain the potential to modulate

hemangioblast function. Understanding the growth factors and biological conditions

during ischemia and how they regulate contribution to neovascularization by the HSC


Figure 3-6.






41


and HSC progenitors may provide methods to manipulate blood vessel formation.

Modulation of the HSC/EPC pathway may allow for tailoring of therapies to increase

neovascularization in ischemic conditions such as stroke, wound healing, or cardiac

muscle damage. Conversely, the ability to decrease pathologic or undesirable

neovascularization as seen in tumor neovascularization or diabetic retinopathy could stem

from a greater understanding of the HSC to EPC developmental fate.














CHAPTER 4
MODULATORS OF HSC/HEMANGIOBLAST ACTIVITY

Few topics have stirred more recent debate than the promise of hematopoietic stem

cells (HSC) exhibiting functional plasticity. Indeed, the candidacy of HSC for

therapeutic treatment of disease is contingent upon demonstrating their ability to fulfill

stringent plasticity criteria. Initial papers described HSC transdifferentiation into a

variety of non-hematopoietic tissues in various organs such as the liver, brain, cardiac

muscle, blood vasculature, intestine and pancreas.30 50 82-84,98 Using various tissue

specific markers and phenotypic characteristics these authors have described tissues to

which HSC are able to contribute demonstrating plasticity of the HSC in their

experimental settings. However, attempts to recapitulate these studies have found

limited HSC plasticity.85, 99 A possible rationale for these dichotic accounts is that these

studies employed differing methods to isolate HSC and examine the target organ of

potential transdifferentiation. Specifically, a variety of HSC isolation and purification

schema have been employed including: elutriation, or separation based on relative

density, serial transplantation where only those cells with the capability to home and long

term repopulate the bone marrow niche rescue a mouse, and cells isolated through

fluorescence activated cell sorting broken down into "side-population" studies of dye

exclusion, and single KTLS (c-kit+, Thy-11o, lin-, Sca-1+) cell transplantations.30' 82, 100, 101

Each method isolates functional HSC as defined by long-term multilineage hematopoietic

reconstitution in vivo. However, each technique may isolate "functional" HSC at

different developmental stages with respect to plasticity. It may be improper to compare









HSC isolated by physical means, i.e. elutriation and side-population, with those isolated

by binding of antibodies to cell surface receptors. The differing populations isolated and

the manipulations which the cells undergo may impact their behavior in the experimental

settings. These methods must be reconciled before a definitive answer can be reached.

Sharing a mesodermal kinship to the HSC are the endothelial cells (EC) of the

vasculature. During embryogenesis hematopoietic and endothelial precursors develop in

both spatial and temporal immediacy. In the adult, EC circulate in the peripheral blood

which are phenotypically similarity to mature EC.16 These cells have the ability to

contribute to new vessel formation either in place of or in addition to resident EC

proliferation. In addition, it was shown that these circulating cells contained a population

which were derived from the bone marrow called endothelial progenitor cells (EPC).17 It

is now accepted that these bone marrow EPC exist and contribute significantly to adult

blood vessel formation, and that these EPC are HSC derived.30

Several experimental systems which damage blood vessels have been able to

induce robust HSC transdifferentiation.30, 102 The preceding chapters described how adult

HSC exhibit hemangioblast function by producing both blood and blood vessels in a

novel model of retinal neovascularization. The model uses long-term bone marrow

chimeric mice that have been stably reconstituted with hematopoietic stem cells from

GFP donor mice (fulfilling the first plasticity requirement). These cells are positive for

the surface markers Sca-1 and c-kit and demonstrate robust GFP expression in blood

products after a four month period. This time is sufficient to eliminate any contaminating

progenitor cell which would have since died off and created a deficiency leukemia in

mice engrafted with these short-term progenitor cells. The long-term engrafted chimeras









then receive a combination of growth factor administration and laser induced ischemic

injury to promote new blood vessel formation in adult murine retinas. Briefly, Adeno

Associated Virus (AAV) expressing Vascular Endothelial Growth factor (VEGF) is

administered intravitreally and allowed one month to reach peak expression. The retina

is then photocoagulated and new vessels attempt to grow into the ischemic region.

Since HSC have the ability to long-term repopulate hematopoiesis, lethally

irradiated mice were then transplanted with a single HSC. These animals exhibited

significant GFP in peripheral blood and bone marrow all of which was derived from the

single transplanted HSC proving clonality in transplanted HSC hematopoiesis. Chimeras

derived from both serially transplanted and single GFP+ HSC produced whole GFP

vascular beds after acute injury and VEGF induction.30 The vessels produced were not

only robust, but were functional as determined by perfusion after cardiac administration

of a fluorescent dye.

The previous chapter has illustrated a new developmental outcome of the HSC: the

production of EPC in response to vascular injury. The work demonstrated that both

blood and blood vessels can be clonally derived from adult HSC via a combination of

growth factor administration and ischemic injury. Thus, adult HSC meet the definition of

a plastic stem cell in that they have the ability to act as a hemangioblast in vivo. Whether

the HSC participates in everyday maintenance vasculogenesis or partakes only in

response to chronic vessel injury remained to be determined and was one of the focuses

of this work. In addition, it was determined that some form of significant injury is

needed for induction of the HSC to EPC pathway, presumably since resident EC are

inadequate to seed and proliferate the damaged areas. While the HSC is now known to









produce EPC under injury conditions, the potential role of physiologic mediators which

impact vasculogenesis in relation to hemangioblast HSC activity merit examination.

Since their first description in 1989, the nitric oxide synthases (NOS) have been

shown to play a role in a myriad of biological functions. The free radical NO, produced

from the conversion of L-arginine to citrulline in the presence of oxygen, has been shown

to function in distinct processes such as inflammation, host defense, neurotransmission,

and smooth muscle contractility. Three distinct isoforms of the enzyme have been

characterized including nNOS which is expressed in neuronal tissues, iNOS, expressed in

a wide variety of tissues, and eNOS which is predominately expressed in the endothelial

cells of the vasculature. The nNOS and eNOS isoforms are constitutively active in their

expressing tissues, which the iNOS isoform is induced in response to proinflammatory

cytokines or endotoxins from foreign bacteria. This induction of iNOS produces a 100-

fold increase in NO as part of an immune response, and NO production is much higher

than is seen compared to the basal levels of the constitutively active isoforms.103 NO

produced by iNOS acts as an antimicrobial and antiviral agent by decreasing DNA

replication.

Nitric Oxide (NO) also mediates endothelial cell function and hence blood vessel

formation. It has been shown to influence neovascularization in several models of

angiogenesis. 104-106 The role of NO in promoting angiogenesis has been controversial in

part because of the complex regulation of NO generation and inactivation. In addition to

vasodilatation, increased local concentrations of NO stimulate proliferation and migration

of endothelial cells, both of which are essential for angiogenesis. 107-109 The NO produced

by the three separate isoforms are activated under distinct activities and have unique









regulatory controls.109 Since iNOS is activated under certain pathological conditions,

such our injury model, and eNOS is constitutively activated in endothelial tissues, these

isoforms may influence the process of neovascularization. The altered amount of NO due

to lack of these enzymes in the cell will affect hemangioblast recruitment and formation

of blood vessels.

Angiogenesis is initiated by vasodilation in order to open up vessels facilitating

introduction of cells in circulation to the site of blood vessel growth. NO is known to

have several angiogeneic affects, including increasing matrix metalloprotease expression

along with tyrosine phosphorylation of proteins in cells populating the sprouting capillary

region.74 Interestingly, in various neovascularization models NO has been shown to be

both proangiogenic and antiangiogenic.74' 104 The theory is that the two isoforms are

activated under differing circumstances and hence are thought to affect blood vessel

formation differently. Indeed, in vivo this is the case. Blood vessel formation due to

HSC contribution under conditions of ischemic injury are influenced by NO as produced

by the iNOS and particularly the eNOS isoforms.110-112

The endothelial NOS (eNOS) isoform is constitutively expressed at basal levels by

endothelial cells and is thought to promote branching, organization, and maturation or

endothelial cells during vessel development. eNOS deficient (eNOS-/-) animals exhibit

fetal growth restrictions, reduced survival, and an increased rate of limb abnormalities.113

They also demonstrate marked vascular pathology such as increased cardiomyocyte

apoptosis, congenital septal defects, postnatal heart failure, decreased capillary density

and vascular permeability.114 Endothelial cells from eNOS-/- animals demonstrate

decreased rates of angiogenesis with reduced branching in vitro.115 These animals also









exhibit an impairment of postnatal angiogenesis in response to growth factors and

ischemia.116 Correspondingly, eNOS has been shown to mediate the mitogenic effect of

VEGF on cultured microvascular endothelium.106 These findings led to the in vivo work

demonstrating that NO production is essential for angiogenesis in hindlimb ischemia, for

wound healing, and coronary collateral growth after myocardial ischemia.115' 117

VEGF has been shown to be a potent vascular permeability factor and plays a

leading role in angiogenesis, and our model takes advantage of this ability to promote

blood vessel synthesis.73 The angiogenic effect of VEGF under both pathological and

physiological conditions has been revealed to be predominantly mediated by eNOS.118

VEGF promotes NO production from eNOS in EC cells, and inhibition of eNOS by

pharmacological agents in vivo have decreased angiogenesis and vascular permeability

induced by VEGF.105 This demonstrates that eNOS is both a downstream mediator of

VEGF induced angiogenesis and an upstream promoter of VEGF expression. This

results in putative positive feedback loop between NO and VEGF which promotes

angiogenesis.119

The inducible NOS (iNOS) isoform is expressed by endothelial cells in response to

external stimuli such as VEGF, proinflammatory cytokines or lipopolysaccharide. iNOS

activation results in a 1000-fold greater generation of NO then eNOS activity alone.120

Its induction is thought to promote tube elongation during vessel development, although

evidence supports that it may have a contrasting anti-angiogenic effect.79 iNOS deficient

animals (iNOS-/-) are relatively healthy but do have a slight decrease in NO production

and vascular permeability during angiogenesis in collagen gels placed in a mouse cranial

window.106 During normal blood vessel formation the interplay between eNOS and









iNOS activity has been postulated to dictate vessel size and degree of branching. In this

chapter I will describe experiments where wild-type GFP+ HSC are transplanted into

eNOS-/- and iNOS-/- recipients to assess the effect of NOS dysfunction in tissue on

hemangioblast activity.

Results

iNOS and eNOS GFP chimeras demonstrated robust HSC engraftment.

To directly assess the role of NOS activity in the promotion of HSC trans-

differentiation into blood vessels, cohorts of wild-type (WT) C57BL6, iNOS-/-, and

eNOS-/- animals were generated. Animals were transplanted with 2,500 highly enriched

GFP+ HSC. It was necessary to use a highly enriched HSC population rather then a

single HSC due to the poor survival of eNOS-/- animals during transplant and the

difficulty in producing single cell transplanted animals in general. The enriched HSC

populations used were isolated using the same protocol previously employed for single

cell transplants in WT animals.30 Briefly, whole bone marrow was obtained from the

bone marrow of GFP animals. Cells were plated on tissue culture treated plates for 2

hours during which time the adherent cell population, which contains progenitor cells

such as the mesenchymal stem cell, stick to the plate. Non-adherent cells are collected

and stained with Sca-1, c-kit, and the lineage markers. Cells which were sorted by FACS

for the stem cell markers of Sca-1 and c-kit and were lineage negative were injected

intravenously through the retro orbital sinus. Long-term multilineage hematopoietic

engraftment was confirmed >3 months post transplant by flow cytometry analysis of

peripheral blood and is shown in figure 4-1. The first column in each cohort represented

is peripheral blood stained for B-cells expressing B220, with the second column stained

for macrophages expressing CD1 lb, and the third column T-cells expressing CD4. The









top row (C57BL/6) and the second row (GFP donor strain) are supplied for reference

controls to facilitate comparison between recipient and donor background fluorescence.

The third row is a typical C57BL/6.GFP chimeric mouse demonstrating robust

hematopoietic engraftment. The forth row (iNOS.GFP), and fifth row (eNOS.GFP) are

representative of engraftment levels of transplanted knockout animal's blood lineage

profiles. The bottom row is peripheral blood stained for VEGFR2 demonstrating that

GFP EPC are in the circulation of C57BL/6.GFP, iNOS.GFP, and eNOS.GFP animals.

Engrafted recipients were subsequently termed C57BL6.GFP, iNOS.GFP, or eNOS.GFP

chimeras. Recipients that were robustly reconstituted by donor HSC (>75% donor

derived myeloid cells) then underwent our model of ischemic injury to induce adult

retinal neovascularization (n > 10 for all cohorts). By waiting >3 months post transplant

before inducing retinal ischemia, it is assured that the ability of HSC exclusively to

regenerate blood vessels is being assessed. No other cell that can be directly isolated

from the marrow has been shown to be capable of long-term reconstitution in a transplant

setting. Any contaminating precursor cells would not have had the ability to repopulate

the bone marrow for this extended period of time and would have long since disappeared

from the circulation. Further proof of the plastic ability of the HSC is demonstrated in

previous work where we illustrate how a single adult HSC is capable of making both

blood and blood vessels in a transplant recipient eliminating the possibility of any other

contaminating cell. Also, this activity is serially transplantable producing functional

vessels in a robust manner.30







50



C57 =L6 U, i



OFF GFP GFP
8In
0" -. ......-'.




P FP GFP.
rL t I "
UFP QFP UFP





PFP GFP GFP


iPOS.glp


iC l

GFP


GFP


eNOS.gfcH I =-[ I s.'I







'L I L .. I -
GFP-- F G-FP
l I( it- l I 134 T 1 1 11' P Il4
3FP GFP GFP


NOS knockout animals exhibit long-term, multi-lineage, donor GFP
peripheral blood engraftment. Peripheral blood mononuclear cells were
analyzed by flow cytometry 3 months post-transplant. The first column is
B-cells expressing B220, the second column is macrophages expressing
CD1 Ib, and the third column is T-cells expressing CD4. The top row
(C57BL/6) and the second row (GFP donor strain) reference controls
show recipient and donor fluorescence. The third row is a representative
C57BL/6.GFP chimeric mouse demonstrating robust hematopoietic
engraftment. The forth row (iNOS.GFP), and fifth row (eNOS.GFP) are
representative of engraftment levels of transplanted knockout animals.
The bottom row is peripheral blood stained for VEGFR2. Circulating
VEGFR2 positive cells are in C57BL/6.GFP, iNOS.GFP, and eNOS.GFP
animals. Numbers in the top right corner are percentages of doubly
lineage stained and GFP positive cells. iNOS= Inducible Nitric Oxide
Synthase, eNOS= Endothelial Nitric Oxide Synthase.


Figure 4-1.









The NO pathway affects blood vessel formation

After induction of retinal ischemia by laser ablation injury, C57BL6.GFP chimeras

produced a variety of GFP+ blood vessels at the sites of injury ranging from small

capillaries to larger vessels. Size was most likely dictated by the degree of the laser injury

as seen in the original hemangioblast characterization (Fig. 3-5 C and Fig. 3-6 C & F).

Strikingly, the NOS.GFP chimeras produced a marker phenotype change from the wild-

type mice indicating a role for the NOS pathway in hemangioblast function. iNOS.GFP

chimeras produced primarily small, highly branched blood vessels that perfused readily

(Fig 4-2 E and G) when injured. These vessels were largely donor-derived as shown in

the red-green merged images demonstrating the colocalization of the perfused fluorescent

dye and the GFP cells. The contralateral eyes had little to no donor contribution as is

seen in Figure 4-2 D and F. This indicated that the eNOS isoform, which is still present,

is sufficient for maintenance of vascular health, and that iNOS plays a role in blood

vessel branching.

In contrast, eNOS-/- mice retinas exhibited a marked phenotype when compared to both

control and iNOS -/- animals done in parallel. Strikingly, eNOS.GFP chimeras only

produced relatively large and unbranched vessels of donor origins regardless of ischemic

insult (Fig. 4-3 D-F). These vessels tended to perfuse poorly in spite of their large size.

Of note, this phenotype was not due to the inability to visualize the red dye in large

vessels due to the fact that the fluorescent perfusant can be easily visualized in B6 control

vessels of similar size. In addition, a few small vessels were readily perfused and the

animal demonstrated the gross muscle contraction and liver color change indicative of

sufficient perfusing. This is consistent with the known vascular defects of eNOS--









animals. Whether this lack of vessel functionality is due to some vascular blockage of

some alternative defect is not known.


The iNOS pathway modulates hemangioblast neovascularization.
iNOS.GFP chimeric mice underwent the retinal ischemia model followed
by perfusion with TRITC-labeled dextran before eye enucleation and
confocal imaging of the retinas. All panels are red and green merged
confocal images. Panels D and F are retinas from control, untreated eyes.
There is little GFP contribution observed (yellow). Panels E and G are
from treated eyes where robust GFP contribution can be seen to
vasculature. Magnification is 60X and size bar is -10tM.


Figure 4-2.









Figure 4-3 demonstrates the large and unbranching characteristics of the donor-

derived vessels. These pictures are red-green merged confocal images and the lack of red

perfuasnt indicates how poorly these vessels function. Panels E and G are from treated

eyes where robust GFP contribution can be seen to vasculature forming large,

unbranched vessels that do not contain the TRITC-dextran. Panels D and F are retinas

from control, untreated eyes. There is significant GFP contribution observed with or

without ischemic treatment indicating that the eNOS pathway plays a significant role in

endothelial cell maintenance.

The profound contribution of HSC derived GFP+ cells to the untreated retinas of

eNOS-/- recipients strongly suggested that deletion of this gene induces chronic vascular

injury. While injury was known to be necessary for HSC hemangioblast activity, this

work demonstrates that a chronic lack of eNOS can also induce neovascularization. If

this postulate is true the transplanted GFP+ HSC should contribute to vascular

regeneration throughout the eNOS-/- recipient. The evaluation of neovascularization in

contralateral eyes demonstrates and agrees with current studies that determined some

type of injury is required for functional plasticity of HSC. In typical physiologic

conditions little or no HSC contribution to "normal" tissue occurs, but when acute

(ischemic injury) or chronic (eNOS knockout) pathologic conditions arise HSC readily

contribute to vascular tissue.

These experiments formally demonstrate that iNOS activity at the site of vascular

injury dictates the size and branch characteristics of new vessels formed in adult animals.

Furthermore, the new vessels are formed in all, or large part, from circulating endothelial

progenitors of HSC origin.












































The eNOS pathway modulates hemangioblast neovascularization.
eNOS.GFP chimeric mice underwent the retinal ischemia model followed
by perfusion with TRITC-labeled dextran before eye enucleation and
confocal imaging of the retinas. All panels are red and green merged
confocal images. Panels D and F are retinas from control, untreated eyes.
There is significant GFP contribution observed, however the vessels
formed do not contain the TRITC-dextran, therefore are poorly functional.
Panels E and G are from treated eyes where robust GFP contribution can
be seen to vasculature forming large, unbranched vessels which do not
contain the TRITC-dextran, therefore are poorly functional. Panel D is
60X. Panel E is 4X magnification. Panel F is 10X magnification. Panel
G is a composite of 60X images. Size bar is -10M unless noted
-100tM.


Figure 4-3.









The NOS pathway affects blood vessel branching characteristics.

To further examine the role of NOS in neovascularization, retinas from the non-

treated contralateral eyes were compared to the injured retinas of WT, iNOS-/-, and

eNOS-/- recipients. This was done in order to elucidate whether NOS could drive HSC

formation of vasculature without ischemic injury and growth factor administration.

iNOS-/- animals responded in a similar fashion to WT animals with production of GFP+

HSC derived vessels in the injured retina (Fig. 4-2 E & G), but little or no contribution

could be found in the retinas from the contralateral untreated eye (Fig. 4-2 D & F).

Unexpectedly, retinas from eNOS-/- recipients, which as described in other studies have

systemic vascular dysfunction, demonstrated robust GFP+ HSC derived contribution to

the preexisting vascular endothelium of both test (Fig. 4-3 E & G) and control eyes (Fig.

4-3 D & F).

After induction of retinal ischemia by laser ablation injury, C57BL6.GFP chimeras

produced a variety of GFP blood vessels at the sites of injury ranging from small

capillaries to larger vessels. In C57BL6.GFP chimeras, size was most likely dictated by

the degree of the laser injury (Fig. 3-5 D-F) and no GFP+ contribution to vasculature was

observed in control eyes (Fig. 3-5 B). iNOS.GFP chimeras produced primarily small,

highly branched blood vessels that perfused readily in treated eyes (Fig. 4-2 D & F).

These animals had limited donor EPC contribution in contralateral untreated eyes (Fig. 4-

2 E & G). Strikingly, eNOS.GFP chimeras only produced relatively large and

unbranched vessels regardless of ischemic insult (Fig. 4-3 D through G). The branching

characteristics of the three strains were markedly different suggesting that the NO

pathway functions in vessel organization. Total branch points of GFP vessels per 60X

field of view were counted for each genotype (Figure 4-4). C57BL6 model control









cohorts averaged about 18 branch points per visual field. iNOS-- recipients had nearly 3-

fold more branch points per field, while eNOS-- recipients averaged 61 times less.

60
48
S50

c 40

S10 30
0 17.8
T
20
E
c 10
0.29
0
WT iNOS eNOS
Strain

Figure 4-4. The nitric oxide pathway alters hemangioblast blood vessel formed
branching characteristics. Confocal Z-series images were compressed and
counted "blindly" for number of vessel branch points per image.
C57BL/6.GFP retinas averaged 17.8 branches per image (n=5).
iNOS.GFP retinas averaged 48 branch points per image (n=4).
eNOS.GFP retinas averaged 0.29 branch points per image (n=38). The
blood vessels of iNOS-- retinas were 2.7 times more branched than WT
animals (p< 0.0001) while eNOS-- were 61.5 times less branched than WT
(p< 0.0002).

These experiments formally demonstrate that NOS activity at the site of vascular

injury dictates the size and branch characteristics of new vessels formed in adult animals.

Furthermore, the new vessels are formed in all, or large part, from circulating endothelial

progenitors of HSC origin. In addition, a chronic lack of eNOS activity over time,

combined with our ischemic injury model, results in a proliferative retinopathy into the

preretinal space, the hallmark of proliferative retinopathy developed in diabetic patients.









NO production affect on vasculature in non-ocular tissue

The finding that HSC have the ability to contribute to vascular tissue in non-treated

eyes during a disease state lends to the examination of tissues far removed and unrelated

to the eye. To determine the extent of donor GFP+ HSC contribution to the overall

vascular system multiple tissues (spleen, thymus, brain, kidney, liver, muscle, skin, and

gut) from the C57BL6.GFP, iNOS.GFP and eNOS.GFP chimeras (n=10 per cohort) were

harvested. Each of these animals had demonstrated long-term, multilineage

hematopoietic engraftment and had undergone the retinal ischemia model. At one month

after the induction of retinal ischemia the animals were euthanized and perfused with

tetramethyl rhodamine isothiocyanate-conjugated dextran (TRITC, a red fluorescent dye)

through the left ventricle. Tissues were harvested and immediately placed in optimum

cutting temperature medium and frozen to preserve GFP. 10 micron thick sections were

then cut and mounted with DAPI to stain the nuclei. Sections were examined by

fluorescent microscopy for GFP+ contributions to the vasculature. Results for the spleen,

thymus and brain are shown (Fig. 4-5). In all cases the C57BL6.GFP and iNOS.GFP

yielded similar results: limited evidence for GFP+ cells being incorporated into blood

vessels in any tissue outside of the treated retina (Fig. 4-5 A-F, and data not shown).

This indicates that whole body irradiation alone is not sufficient for induction of HSC

contribution to vasculature in tissues which are not treated further. In contrast,

eNOS.GFP chimeras exhibited robust GFP+ contributions to the vasculature (as

determined by co-localization with the perfused red fluorescent dye) in all tissues

examined (Fig. 4-5 G-L, and data not shown). Lack of eNOS creates a pathologic

vascular condition where HSC are induced to contribute to vascular repair throughout an

organism.

















































Chronic vascular injury in eNOS.GFP chimeras induces widespread
hemangioblast activity from adult HSC. NOS knockout animals which
underwent the neovascularization model, and spleen (A, G, B &H),
thymus (C, I, D & J), and brain (E, K, F & L) were harvested from TRITC
perfused animals. 10M cryosections were prepared and mounted with
Vectashield plus DAPI. iNOS.GFP (A-F) and eNOS.GFP (G-L)
chimeras were examined by fluorescence microscopy. The donor GFP
HSC derived cells are green, and the TRITC-labeled dextran perfusant is
red. Panels A & G are magnification X40. All remaining panels are all
magnification X64.


Figure 4-5.









To ascertain the endothelial cell nature of the GFP+ cells surrounding the vessel lumens

tissue sections were stained for the pan endothelial cell marker MECA-32. Ten micron

frozen sections were stained with a primary antibody to MECA-32, followed by a Texas

Red conjugated secondary antibody and DAPI. Endothelial cells were then scored for the

presence of both MECA-32+ and GFP+ cells via fluorescent microscopy. Splenic

sections demonstrate the characteristic results observed in all tissues studied (Fig. 4-6).

Donor derived hematopoietic cells in the spleen serve as internal negative staining

controls for MECA-32 in each section. WT animals showed occasional GFP+, MECA-

32+ endothelial cells in the brain (closest organ to the site of VEGF administration) with

the majority of tissues such as the spleen (Fig. 4-6 A-D), kidney, liver, and muscle

showing no donor derived endothelial cells. iNOS-/- animals, which exhibit minor

systemic vascular defects, had occasional GFP+, MECA-32+ endothelial cells in the

spleen (Fig. 4-6 E-H) and other tissues.

Overall GFP+ HSC derived contribution to the vasculature of iNOS-/- animals,

outside the area of retinal ischemia, was at most 1% in >150 sections examined for

MECA-32+ vessels. Robust GFP+ donor derived endothelial cell production was

observed in eNOS-/- recipients which have been demonstrated to have chronic and severe

vascular pathology. Most vessels were quite large, and most showed extensive GFP+

HSC derived MECA-32+ endothelial cell contributions in the spleen (Fig. 4-6 I-P) and

other tissues examined. The HSC contribution to vasculature detected in untreated tissue

was analogous to that observed in the treated retinas demonstrating that chronic vascular

injury appears to be sufficient to induce the hemangioblast activity of adult HSC.






































Figure 4-6.


Donor-derived cells lining vascular lumens in eNOS.GFP animals are
MECA-32 positive. Splenic cryosections were prepared from
C57BL/6.GFP (A-D), iNOS.GFP (E-H), and eNOS.GFP (I-P) chimeras.
Sections were stained with anti-MECA-32 antibody and a Texas Red
conjugated secondary antibody to delineate vascular endothelium.
Sections were mounted with DAPI (A,E,I,M) to delineate nuclei with blue
fluorescence, examined for GFP expression (B,F,J,N) via green
fluorescence, or MECA-32 staining (C,G,K,O) via red fluorescence.
Merged images of the DAPI, GFP, and MECA-32 Texas Red stains are
shown in D,H,L and P. (A-L) Magnification X64. (M-P) Magnification
X32.


Quantitation and location of NOS produced in knockout animals.

To ascertain the influence and determine the expression of NOS in retinas lacking

one specific NOS isoform, retinas were dissected and stained with isoform specific

antibodies. iNOS-/- (Fig. 4-7 A-C top) and eNOS-/- (Fig. 4-7 A-C bottom) animals were


II IB rCI II ,









quantitated for NOS expression in parallel. Animals were sacrificed and the eyes

enucleated as described. The dissected retinas were then imaged through confocal

microscopy. Figure 4-7 (A top and bottom) demonstrates that in each knockout strain the

isoform which is deleted is not expressed in vivo at detectable levels. The iNOS -/- has

relative amounts of NOS expressed (B and C top), while the eNOS -/- retinas

demonstrate an increase in iNOS expression as seen throughout the large, and particularly

the smaller vessels (B and C bottom). This confirms that there is an upregulation of

iNOS expression in eNOS knockout retinas indicating a dysregulation in amount of NO

produced resulting in the pathologic blood vessel formation observed in these animals.

Discussion

Through growth factor administration and ischemic injury to the retina, HSC can

be induced to transdifferentiate into vascular endothelium. Furthermore,

transdifferentiation can also be observed during a pathologic disease state of chronic

vascular injury. The substantial role NO plays in vascular tone, and the presence of a

NOS isoform specifically found in endothelial cells hinted at a role of NO in blood vessel

formation and remodeling. NOS activity can also dictate the general size and branch

characteristics of new blood vessels formed in response to ischemic injury and growth

factor administration. Using the neovascular model of inducing HSC hemangioblast

activity to promote blood vessel formation in the adult retina, donor WT HSC

transplanted into iNOS-/- recipients produced highly branched vessels that are generally

smaller in size. These HSC are functioning in an environment where local NO

production is similar to what is seen in wild type, non-infection conditions due to the

eNOS isoform which is constitutively active in endothelial cells. A wide variety of

vessel sizes are formed which are functional as measured by perfusion of marker dye.





































Figure 4-7. Nitric oxide production is dysregulated in eNOS knockout animals.
iNOS-- and eNOS-- retinas were stained with NOS isoform specific
antibodies. Vessels were illuminated by agglutin staining (red) and
regions which were positive for the NOS antibody are green. In the top
row, panel A depicts an iNOS-- retina stained with iNOS specific
antibody. Panels B and C are iNOS-'- stained with eNOS isoform specific
antibody. In the bottom row, Panel A depicts an eNOS-'- retinas stained
with eNOS isoform specific antibody. Panel B and C are eNOS-'- stained
with iNOS isoform specific antibody.

The branch patterning is similar to normal mouse vasculature, although slightly

increased. Contrastingly, eNOS-/- recipients produced primarily unbranched vessels of

large size. This indicates that the local NO production due to the eNOS isoform activity

is necessary for normal hemangioblast derived blood vessel formation. Modification of

NO production via the eNOS isoform, which can now be specifically targeted with

pharmacological agents, could provide a means to influence neovascularization and









angiogenesis in pathologic diseases such as Diabetic Retinopathy, and Retinopathy of

Prematurity. This altered HSC response was in addition to the widespread vascular

remodeling by donor HSC seen throughout the eNOS-/- recipients even in non-injured

organs and tissues. The vessels of eNOS-/- recipients were difficult to perfuse indicating

their general vascular dysfunction. This further emphasizes the crucial nature of the

eNOS isoform's NO production to ensure proper vessel formation and functionality

foreshadowing studies occurring outside the realm of the eye on which our model

focuses.

This chapter reiterates work demonstrating that an injury state, whether it be acute

as seen in the photocoagulation of blood vessels, or chronic as seen in the vascular

pathology observed in the eNOS knockout animals, is required for HSC plasticity.85

Furthermore, local mediators, including VEGF and NO, can greatly influence not only

the size and amount of new blood vessels formed but also their functionality. The

regulation of NOS activity as a means to influence the remodeling of vascular beds may

provide specific treatment regimes. It may prove beneficial to selectively inhibit a

particular NOS isoform to correct an imbalance thus altering the development of new

blood vessels. Whether a deregulation of NOS or NO activity is a causal effect in human

proliferative retinopathy remains to be determined. If this is the case, pharmaceuticals

that affect these activities, some already in use for non-related disease treatment, may

provide an effective therapy or preventative for human diseases in relation to proliferative

or pathological blood vessel formation due to hemangioblast activity.














CHAPTER 5
LIMITATIONS OF STEM CELL RESEARCH AND ETHICAL CONSIDERATIONS

While the promise of stem cells as therapy is considerable, there are limitations to

their usage including their biological activity and ethical implications. The biological

limitations of stem cell plasticity are related to their ability of to transdifferentiate and

their potential to form tissues. The ethical implications are many, and I will address

some of them in this chapter.

Biological limitations

In the current research environment, there is currently heated controversy over the

reported plasticity of stem cells, particularly the HSC in relation to cardiac muscle, liver,

and the nervous system.121 The limitations of adult stem cells include :their diminished

capacity to proliferate when compared to embryonic stem cells. Autologous

transplantation of cells back into a patient will still retain genetic abnormalities.

Correspondingly, ex vivo expanded or genetically modified cells used for therapy may

produce unforeseen consequences. Some tissues arise through a complex developmental

fate, such as the pancreas which is derived from the infolding of several tissue layers

which will not be easily mimicked in vitro. The resident stem cells within a tissue type

are extremely rare. This paucity of cells makes them extremely difficult to identify,

isolate, and purify. In addition, it had proven difficult to culture adult stem cells

compared to embryonic ste cells in vitro limiting their use as potential therapy. Finally

fusion It is believed that cell fusion may be a potential method for introducing new

genetic material to correct mutated or malfunctioning genes that cause disease. Cell









fusion occurs when two or more cells combine to form one cell which then contains more

genetic material than normal. Fusion has been shown to occur in embryonic along with

adult stem cells.122, 123 In adult mice, fused liver cells may contain 80 chromosomes,

double the amount found in a normal mouse liver cells. Other cell types, including

megakaryocytes and muscle cells function with an increased ploidy as well. In most cell

types, however, aneuploidy would be detrimental often inducing apoptosis or cell

transformation. The resulting imbalance in gene dosage could lead to nonfunctional

tissue or cancer. It is not clearly understood, nor has it been definitively proven whether

fusion is a pathway for stem cell plasticity with research indicating that fusion can result

in, but is not necessary for plasticity.124-126 If the former is the case, then investigation

into the effect of producing cells with an increased ploidy at any time must be done. A

fusion event during contribution to the regenerated tissue could preclude stem cell based

therapy.

The first work characterizing fusion done by Terada et al. found that fusion events

in vitro were extremely rare.122 Consequently, the robust amount of donor derived

contribution is unlikely to arise from such rare events. Recent work from our lab

demonstrated that the cells derived are diploid, and any unresolved fusion of the HSC

would have resulted in an increase in ploidy shown in figure 5-1.127 The circulating

endothelial precursor cells derived from the donor marrow exhibit normal 2N ploidy

when stained with the DNA dye propidium iodide. This does not rule out the possibility

of a fusion event which was resolved resulting in normal ploidy, however, and special

specific experiments much be done to ascertain if this fusion resolution is occurring.

























S 150 200
Channels


25I I 50 I
250 0 50


150 200 250
Channels


Figure 5-1.


Propidium iodide staining of circulating EPC does not indicate abnormal
ploidy. Long-term GFP engrafted C57BL/6 recipients underwent the
neovascularization model. Animals were bled, and FICOLL enriched
peripheral blood was stained and FACS enriched for VEGFR2 expression.
Cells were stained with propidium iodide and analyzed by FACS. Left
panel depicts EPC from a nontransplanted C57BL/6 animal bled in
parallel. Right depicts a test animal. Both exhibit classical diploid
staining profiles.


Ethics

Any comprehensive analysis of the stem cell field would be remiss to not include

the ethical implications for their use in therapy. The field has proven to be a polarizing

issue which influences many religious and political referenda born simultaneously with

Dolly, the first cloned mammal. The debate arises from the use of embryonic stem cells

which at this time can only be isolated from an embryo resulting in the embryo's

destruction. These cells are attractive, however, because of their ability to produce all of

the cell types in an adult animal or provide an environment in which DNA can be

transferred in nuclear transfer. To date no single adult cell has been shown to have the

pluripotency of embryonic cells along with providing the intracellular environmental cues

necessary to reprogram transferred DNA. Adult stem cells do not have the same capacity


50 1


I L .- _1 1-RI-I-IIIIII&P M 0


~"""""'









to produce any tissue or cell types. This inherent extraordinary therapeutic potential

resulting from the destruction of an embryo leads to opposition based on the idea of when

life begins.

In the United States, many Fundamentalist Christian groups are strongly opposed to

embryonic stem (ES) cell research as the destruction of the embryo is considered

abortion, or murder. They believe that any and all research using human stem cells is

morally unacceptable. Other religions, however, are supportive of embryonic research.

Many Jewish groups of differing denominations do not view an early stage embryo as a

human being, therefore usage of embryonic tissue is not destruction of a human. Many

Humanists, Unitarian Univeraslists, and Muslim clerics have also come out in favor of

stem cell research. In addition, proponents point out that stem cell research uses

discarded embryos from in vitro fertilization, and that fertility clinics routinely destroy

thousands of embryos. These unused embryos would normally be discarded or kept

frozen indefinitely if not used in research. There is no general consensus among religious

groups which gives rise to many concerns over the use of ES cells.

Concerns Over Stem Cell Use

Certainly, stem cells are not the first human discovery to revolutionize scientific

knowledge and create waves of ethical debate. Since ancient times society has

admonished man for approaching these boundaries as exemplified in the Greek myth of

Icarus who did not heed his father's command; he reveled in the "unnatural" sensation of

flight and then plummeted to his death after the sun melted his wings. This Greek myth

embodies our apprehensions about interfering with nature. Galileo Galilei expanded the

frontiers of astronomy and posited that the Earth rotates on its axis and revolves around

the Sun. This led to his eventually condemnation for heresy. In Victorian times, society









grappled with the balance between the knowledge gained from performing autopsies for

crucial understanding of human anatomy versus the desecration of those who were dead.

Even recently, complete consent to produce recombinant DNA for lifesaving medications

such as insulin has been granted but only after vehement protestations over genetic

engineering. There are shared concerns among all instances of testing medical

boundaries, and the concerns of stem cell technology include issues of safety, efficacy,

and resource allocation. For decades, patients have undergone adult HSC transplantation

in the treatment of immune deficiencies and cancer. Although graft-versus-host disease

and posttransplantation infections are major risks of allogeneic bone marrow transplants,

investigators have worked to minimize these consequences and many patients accept

these risks in the hope of the lifesaving benefit of disease eradication. However, the field

of stem cell therapy is still in its infancy and researchers are incrementally improving

safety, efficacy, and applicability to a wider spectrum of disease. In all instances of

expanding the horizons of our knowledge a societal consciousness was at play, often

times encumbering progress and questioning techniques of intervention.

Stem cell therapy differs from previous technologies in how these potential sources

of regenerating tissue are tapped. Adult stem cells are typically acquired by harvesting

adult tissues. Patients give informed consent and usually undergo little risk at donation.

In contrast, human embryonic stem cells (hES) are obtained by culturing cells from the

inner cell mass of a blastocyst. This blastocyst is usually acquired from an unused human

embryo produced by in vitro fertilization or from an aborted fetus. The harvesting

process requires dissolving the blastocyst bringing into question the moral and legal

status of the human embryo.









Many religious perspectives consider the human fetus an individual human entity.

However, there is substantial debate regarding at which specific stage dignity is conferred

in development ranging from conception, to primitive streak development, implantation,

or birth. Taking into account the many perspectives on the moral status of the human

embryo weighed against the scientific promises of a healthier tomorrow through stem cell

technology, our society has attempted to define the legal status of the human embryo. In

the United States, the first mandate was outlined in 1973 when the US Supreme Court

ruled that a fetus is not a person in terms of constitutional protection (Roe v Wade, 410

US 113 [1973]). For a better examination of the decision's effect on research, the

National Institutes of Health (NIH) imposed a moratorium on fetal research, and

Congress founded the National Commission charging it to put together policy and

guidelines on fetal research. The commission published a report encouraging fetal

research due to its potential, provided that the research risks to the fetus were minimal

and were only those that would be accepted for a term fetus. Thus, despite Roe v Wade,

the commission extended protection to a fetus equal to adult patients in research. This

included fetuses planned for elective abortion.

The NIH moratorium was lifted in 1975, however during President Ronald W.

Reagan's second term Congress enacted legislation that further protected the fetus by

ending federal support of fetal research involving any level of risk. In 1996, Congress

extended this restriction by banning federal funding for "the creation of a human embryo

or embryos for research purposes." This led the NIH to distinguish between deriving and

using existing human embryos to support embryonic stem cell research. Under these

guidelines researchers using already established hES cell lines derived from private









sector support can receive public sector monies provided that the fertilized embryos

would otherwise have been discarded after IVF or were from already aborted fetuses,

donors are aware of the research use, and no payment was made to the donors.

President William J. Clinton created The National Bioethics Advisory Commission

(NBAC) to thoroughly review moral and legal issues of stem cell re search. This

commission largely framed its moral position based on a utilitarianism argument-the

good of many outweigh the status of one. In addition, it drew on medicine's aims to heal

and prevent disease urging consideration of a long-term benefit-to-harm balance. The

NBAC conclusion recommended allowing federal funding for hES research on excess

IVF embryos. Reasons supporting this position include the potential of ES cells in

regenerative and reproductive medicine and the need for federal support to avoid private

sector conflicts of interest which sometimes invokes secrecy, limiting the spread of

knowledge, and places shareholders interests ahead of public good. Taking all these

guidelines and perspectives into account, President George W. Bush made an executive

order on August 9, 2001 to limit federal funding of hES research to cell lines already

derived from 64 embryos.

The cost to society of foregoing use of this technology, either by failure to correct

genetic abnormalities or by improving the success of lifesaving organ transplantations,

may be equal to or greater than the perceived costs to the dignity of life due to destruction

of a human embryo. There must be a balance between the perceived costs to the dignity

of life held by those with the most sacrosanct concept of the moral status of embryos and

those who would directly benefit from stem cell based therapy. The climate in which

stem cells are explored can be nurturing or profoundly limiting. As medical scientists,









we must not make judgments or ethical decisions on our own; rather, we must ensure full

informed consent of the population as a whole. This approach may limit quick progress

and may disqualify avenues of research and therapy, but as responsible researchers we

must use the resources of society in a worthy manner to explore fully the tremendous

potential of embryonic stem cells.

The use of adult stem cells eliminates any ethical concerns as the cells used for

therapy can be obtained with only slight discomfort, and potentially from the individual

patient themselves. This is attractive due to the fact that there would be no human

leukocyte antigen mismatching, and therefore no need for immune suppressing drugs or

the fear of tissue rejection and graft versus host disease. If adult stem cells prove to have

the same potentiality as ES cells, either individually or as a collection, their use would

end the need for embryonic tissue and their subsequent destruction eliminating any

ethical concerns over the ends justifying the means.














CHAPTER 6
GENERAL CONCLUSIONS

The goal of this work was to characterize the hematopoietic stem cell's plastic

ability and describe biological pathways which can modulate this ability. In a historic

context, this work spans several stages of the he stem cell field as it matured from the

beginning flurry of activity into its current stage of careful evaluation. The pioneering

work demonstrated the exciting potential of the field as an alternative and powerful tool

for use as a cell-based therapy for many diseases ranging from cancer to diabetes, heart

attack, and Parkinson's disease. Those founders demonstrated that these cells,

specifically the hematopoietic stem cell, was capable of producing many different tissue

types. It was found that the HSC is capable of producing not only all of the blood

lineages, but also muscle, pancreatic cells, heart, intestinal epithelium, brain, and blood

vessel endothelium. These exciting results sparked a revolution in the scientific field

with new findings constantly being published in peer reviewed scientific journals along

with the front pages of popular newspapers and magazines. This initial impetus soon

played out, however, as the heralded plasticity of stem cells came under thorough and

critical scrutiny, and justifiably so. It became apparent that the cell types produced were

not necessarily functional, nor could there be certainty of the source of the donor cells-

several different cell types within the transplant could be individually contributing to the

observed tissue, therefore no one cell would be capable of forming several tissue types.

This critique was addressed in several studies which began the second stage of stem cell

research. Krause et al. did a single cell transplant homing assay to prove several tissue









types could arise from one cell.82 Grant et al. also did single cell transplants and

demonstrated the functional ability of endothelial cells which clonally arose from the

HSC to carry red fluorescent dye perfused into the circulator system.30 In addition,

Lagasse et al. rescued liver function in a metabolic liver disease with HSC.84 Clearly,

stem cells, particularly the HSC, are capable of providing functional rescue of disease

conditions in a clonal manner. With this understanding, we now embark on the third

stage of stem cell plasticity research-defining the genes and biological pathways

involved in regulating or controlling stem cell activity.

Many genes have been shown to maintain the "stemness" of stem cells by

controlling their self-renewing and proliferating capacity. In addition, we are starting to

understand factors, such as the nitric oxide pathway, which play a role in stem cell

homing and functional behavior.128 This work, along with other exciting work done in

our lab on chemokines such as stromal-derived factor-i's involvement in homing,

highlights the therapeutic benefit of not only stem cell research, but also fortifies the

foundation for cell-based therapy.129 This third age of directed stem cell repair of

damaged or non functional tissue has the potential for direct translation into disease

therapy along with opening exciting new avenues of original research. This body of

work chronicles the relatively new field of stem cell plasticity research from the initial

characterizing of HSC plasticity up to describing biological pathways which can

orchestrate HSC activity. The founding work and initial application to HSC plasticity

described here can lead to many novel stem-cell therapy strategies for debilitating

conditions and diseases. Additional effort will focus on direct translation of this

knowledge into human disease therapy.













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BIOGRAPHICAL SKETCH

Steve Guthrie was born and raised in Lancaster, Pennsylvania. He attended

Albright College in Reading, Pennsylvania where he graduated in 1998 with two majors

(biology and philosophy) receiving the Gary Kennis Philosophy Award and Ernest J.

Pastorello Biology Prize. He then moved to Gainesville, Florida, where he worked as a

lab technician for Dr. Alfred Lewin, and then as a biological scientist for Dr. Edward

Scott for 2 years. He joined the Interdisciplinary Program in Biomedical Sciences at the

University of Florida College of Medicine in 2000 where he began his doctoral study

under the guidance of Dr. Edward Scott in the Department of Molecular Cell Biology.

He was awarded a Grinter Fellowship, and received first place in his department, and

fifth place overall at the 2003 Medical Guild Research Day sponsored by College of

Medicine. Steve will be doing post-doctoral research at the University of Alabama at

Birmingham beginning Summer of 2005.