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University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 1 Hypoxia as a Priming Modality for Pre Transplant Neural Progenitor Cells Alex Tiemeier College of Public Health and Health Professions University of Florida Neural Progenitor Cells (NPC s ) are self sustaining, multipotent cells that differentiate into central nervous system (CNS) c ell types. NPC s from the adult subventricular zone (SVZ) are attractive candidates for cell replacement therapies. However, their utilization is limited due to reduced cell survival following transplant. In addition, the ability to drive neuronal cell phenotype would address the limitation of local i nterneuron replacement in these therapies. The goal of this research wa s to administer in vitro and/or in vivo hypoxia treatments to NPC s destined for transplant into an injured host to address the limitations of reduced cell survival and neuronal producti on. The hypothesis for this research wa s that the administration of hypoxic pre treatments to NPC s w ould increase their rate of expansion and neuronal production. The results suggest ed that in vitro hypoxia alone promoted a greater yield of neurospheres ( NS ) while in vivo hypoxia promoted increase s in NS yield, diameter and neuronal yield The results also suggested a synergistic effect for NS yield when in vitro and in vivo hypoxia treatments were combined. Neural progenitor cell (NPC) transplantation is an evolving experimental technology that shows promise with regard to restoration of function following nervous system insult ( Eftekharpour et al., 2008) NPC s harvested from the adult subventricular zone ( SVZ ) are of particula r interest for transplant cells as they possess the following qualities: 1) ample proliferative capacity, 2) neural multipotency, 3) the absence of ethical issues and 4) the absence of tumorigenicity upon transplant. Possessing ample pr oliferative capacity and neural multipotency indicates that these progenitor cells are capable of cell division, are self sustaining, and can exit the cell cycle to differentiate and mature into a variety of cell types (Reynolds & Weiss, 1992). A reduction in tumorigenicity refers to the fact that becaus e these cells are proliferative, but not uncontrollably so, their potential to form tumors post transplant is highly reduced. In fact, in animal models of adult NPC transplant, this potential has been confirmed (i.e. Karimi Abdolrezaee et al., 2006). The SVZ, the focus of this work, is a known niche for neurogenesis. The SVZ is an active region of the brain during embryonic development that produces neurons and glial cells (astrocytes, oligodendrocytes and mic roglia). After development the SVZ reduces in size but remains an active source of adult neurogenesis in the adult mammalian brain. The SVZ is comprised of four important cell types: ependymal cells, neuroblasts, astrocytes, and transitory amplifying prog enitor s (TAPs) (Doetsch et al., 1997). Astrocytes in the SVZ produce neural progenitors that transiently divide and mi grate toward the olfactory bulb where they become mature interneurons within several weeks (Alvarez Buylla & Lim, 2004; Lledo & Saghatelya n, 2005). These NPC s can be isolated and cultured in vitro as free floating colonies called neurospheres ( NS) (Reynolds & Weiss, 1992). NS can be passaged to asse ss their proliferative capacity and they can be plated and stained to assess their multipoten cy or the generation of different mature central nervous system, ( CNS ) cell types (Reynolds & Weiss, 1992; Louis et al., 2008). Oxygen is utilized and tightly monitored in over one hundred enzymatic reactions within mammalian tissues, including the CNS (Erecinska & Silver, 2001; Chen et al., 2007). It is widely known that fluctuations in oxygen concentration can cause changes among bod ily responses ; however little is known about how it affects the fate of progenitor cells with the CNS. Santilli et al. (2010) reported that hypoxic (low levels of oxygen) conditioning triggers a switch from mitochondrial respirat ion to anaerobic glycolysis, which greatly influences the growth and differentiation of neural stem cells ( NSCs ) Much of the existing literature reports on progenitor cells that are incubated in an environment with a partial pressure range for oxygen ( PO 2 ) environment because it is the standard or normal cell culture PO s (Zawada et al., 1998). However, the PO 2 within the brains of mos t mammalian species ranges from 0.55% (4.1 mmHg) in the midbrain to 8.0% (60 mmHg) in the pia (Chen et al., 2007 ; Studer et al., 1999). Therefore, more studies have begun to focus on the incubation of progenitor cells at lower partial pressures because the actual PO s s within the brain are significantly lower. This reduced level is the focus of hypoxic preconditioning (HP). Cell cultures are briefly exposed to a hypoxic environment that ranges from 1% to 5%, which as stated earlier falls into t he normal physiological range for the mammalian brain (Zawada et al., 1998). Oxygen levels that are near or within the physiological range improve
University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 2 ALEX TIEMEIER both proliferation and differentiation of progenitor cells, compared to levels outside the physiological rang e (Santilli et al., 2010 ; Studer et al. 1999 ; Chen et al., 2007). A small body of work has begun to determine the role of systemic hypoxia conditioning, or in vitro intermittent hypoxia (IH) on progenitor cells in the brain. This work has shown that a brief pulse of IH results in increased proliferation of progenitors found in the dentate gyrus of the hippocampus and in the SVZ (Zhu et al. 2005). These finding are of p articular interest to this work as sy stemic IH is proposed as a potential therapeutic intervention for conditioning pre transplant or post transplanted progenitor cells for CNS injury. Thus, the ability to conduct proof of principle experiments on the ability to alter the behavior of subseque ntly cultured NPCs could have a direct impact on CNS transplant strategies. Hypoxic preconditioning has been shown to induce the following effects: increased proliferation of progenitors, reduced apoptosis, elevated absolute number and proportion of dopami nergic neurons, and promotion of a higher tolerance of embryonic stem cells to cell death both in vitro or after transplantation into the ischemic rat (Zawada et al., 1998; Studer et al., 1999). Therefore, the purpose of using in vitro HP and of systemic IH is to enhance the proliferation, absolute number, survival and regenerative capability of NSCs before they are transplanted into an injured host (Theus et al ., 2008). CENTRAL HYPOTHESIS A ND AIM The c entral hypothesis of this work wa s that in vitro and/or in vivo hypoxic conditioning will generate a more robust transplant cell population. This hypothesis was tested by addressing the following aim: t o measure cell proliferation and neuronal differentiation following hypoxic conditioning on postnatal NPCS derived from the SVZ All culture and subsequent analyses were conducted as previously published (Ross et al., 2008). Cells were propagated as NS and total yield was recorded to assess the presence of neurospheres forming cells (NFC s ). In addition, the size (diameter) of NS was recorded to assess the proliferative capacity of individual NS. Finally, following plating and differentiation, immunocytochemistry was co nducted to evaluate the percentage yield of neurons in these cultures. MATERIALS AND METHOD S Neonatal C57BL/6 mice (P4) were housed at the Animal Care Services, and all procedures were in compliance with the regulations of the Institutional Animal Care and Use Committee. Systemic hypoxia (IH) protocols were conducted us ing a whole body plethysmograph The protocol consisted of 1 minute exposures alternating between 21% O 2 and 10% O 2 for 20 cycles (40 minute duration overall). In vitro hypoxia (r ange of 0.5 3.0%) was administered in a Billups Rothenberg chamber according Hypoxia was administered for either 24, 48 or 72 hours of maintained hypoxia, or a protocol that was intermittent (i.e. 24 hours of alternating hy poxia, then 24 hours normoxia for 72 hours total) To culture NS, SVZ tissue blocks were dissected, dissociated and plated overnight in growth media. Non adherent NFCs were incubated in trypsin and plated at clonal density (10,000 cells/c m 2 ). Resultant NS were passaged every 5 7 days and then assessed for frequency (total NS yield) and size (NS diameter) as a measure of NFC number and NS proliferative capacity, respectively. To assess multipotency, NS were transf erred to poly L ornithine coated coverslips and differentiated for 2 3 days without growth factors. For immunocytochemistry, cells were fixed for 30 minutes (4% paraformaldehyde) and blocked for 1 hour. Primary antibody (mouse anti III tubulin 1:1000) wa s applied overnight at 4 C. Coverslips were incubated with fluorescence conjugated secondary antibody (goat anti mouse 1:500). Slips were mounted in Vectashield containing DAPI counterstain. Fluorescence micrographs were obtained with a Leica DM5000B epifluorescence microscope (Spot CCD digital camera, Leica Application Suite Version 3.50 ) This is s ummarized in Figure 1.
H YPOXIA AS A P RIMING M ODALITY FOR P RE T RANSPLANT N EURAL P ROGENITOR C ELLS University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 3 Figure 1 Schematic representation of experimental methods. This flow chart presents a generalized view of the experimental procedures that were utilized. From left to right: in vivo hypoxia of an animal the isolation of NS, and in vitro hypoxia of the isolated NS. RESULTS In the first experiment, in vitro hypoxia was tested to determine if there was an effect on total NS yield and size. NFC s were plated at clonal density and then cultured in either 20% O 2 or hypoxia exposures. Total NS yield was greater following 48 or 72 hours of hypoxia exposure while no difference was seen after 24 hours or intermittent exposures (Figure 2A). Under these conditions, no significant changes in NS diameter were noted ( Figure 2B). Next, the same NS growth dynamics as well as neuronal differentiation were assessed in normoxic pups and in pups subjected to IH conditioning. Cells from both animal populations were subjected to 24, 48 72 or intermittent in vitro hypoxia. Thes e results revealed that, regardless of the in vitro culture conditions, cultures from pups preconditioned with IH yielded a greater number of NS. The greatest number of NS was obtained from animals that received both in vitro and in vivo intermittent hypox ia (Figure 3A). In this experiment, in vitro hypoxia alone again did not appear to have a significant effect on NS diameter ; however systemic IH appeared to result in greater diameter at 48 and 72 hours, with no real benefit seen with the additional in vitro intermittent protocol (Figure 3B). Finally, beta 3 tubulin expression was assessed in these cultures to determine any effect of individual or combined therapy on neuronal yield (representative image of a hypoxic NS, Figure 4A). Overall, in vitro hypoxia alone did not result in a significant trend of increased beta 3 tubulin expression (Figure 4B, blue series). However, in IH conditioned pups, neuronal yield was greater than in control pups in all treatment conditions except for the 48 hour condition, wh ich showed similar yield (Figure 4B, red series).
University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 4 ALEX TIEMEIER Figure 2 The effect of in vitro hypoxia exposure on NPC yield and diameter. Part A is a histogram that displays the total number of NS formed after they were administered their respected in vitro hypoxia treatment. Part B is a histogram that shows the diameter for NS formed from each in vitro hypoxia treatment. The treatments analyzed were 24 hrs, 48 hrs, 72 hrs, and intermittent in vitro hypoxia (IH).
H YPOXIA AS A P RIMING M ODALITY FOR P RE T RANSPLANT N EURAL P ROGENITOR C ELLS University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 5 Figure 3 The role of in vitro and/or in vivo hypoxia on NPC yield and diameter. Part A is a histogram that compares the total number of NS formed between normoxic and hypoxic animals. Normoxic animals only received in vitro conditioning while hypoxic animals received both in vivo and in vitro Part B is a histogram that compares the diameter for NS formed between normoxic and hypoxic animals.
University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 6 ALEX TIEMEIER Figure 4 The effect of in vivo and/or in vitro hypoxia on neuronal yield. Part A is a representative image of a hypoxic NS, depicting the presence of beta 3 tublin (red areas). Part B is a histogram that compares the percent of beta 3 tublin positivity between NS derived from normoxic and hypoxic animals. DISCUSSION Data collected from in vitro hypoxia administration suggest that in vitro hypoxia alone when given for 48 or 72 hours resulted in a greater yield of NS (Figure 1A). This finding indicates that 24 hours or the intermittent protocol in this experiment were inadequate to alter the NS behavior and that perhaps a longer intermittent protocol could affect these changes. Figure 1B reflects that, for the tested doses, in vitro hypoxia was unsuccessful in significantly affecting NS diameter. Although this conclusion seems to contradict previous scholarly findings, it is important to note that it was only one experiment. Further experiments are necessary to analyze the hypoxic doses utilized to find the ideal do se and time course of administration Data from in vivo hypoxia treatment appears to promote a positive effec t on NS yield, diameter, an d neuronal yield. This supports the idea that systemic hypoxia can significantly influence NPC biology within their endogenous CNS niche and provides momentum for further research into the ability of systemic hypoxia to act as a priming modality for transplant cells either pre harvest
H YPOXIA AS A P RIMING M ODALITY FOR P RE T RANSPLANT N EURAL P ROGENITOR C ELLS University of Florida | Journal of Undergraduate Research | Volume 13, Issue 3 | Summer 2012 7 or post transplant The most intriguing aspect of these results is the suggestion of a synergistic effect from combining in vitro and in vivo hypoxia treatments Looking at these figures one can see a synergistic effect for NS yield and diameter b ut only a trend toward synergism with neuronal yield. It is worth noting that a synergistic effect was seen for all doses for NS yield ( Figure 3A, red series). In addition, NS diameter appeared to be greater at 48 hr s 72 hr s, and with intermittent in vitro hypoxia (Figure 3B, red series). Finally, neuronal yield was greater from animals treated with hypoxia; however no significant trend in neuronal yield can be ide ntified. Although synergistic effects were not seen on every figure for every dose, these data suggest that combining both hypoxia treatments may significantly improve NS expansion and neuronal differentiation dynamics These preliminary results support further investigation of combined hypoxia treatments to prime cells destined for transplantation approaches REFERENCES Alvarez Buylla A, Lim D A. (2004). For the long run: m aintaining germinal niches in the adult b rain. Neuron 2004; 41 (5), 683 86. doi:10.1016/S0896 6273(04)00111 4. Chen H L Pistollato F, Hoeppner D J, Ni HT, McKay R D, Panchisio n D M. Oxygen tension regulates survival and f ate of m ouse central nervous system precursors at multiple l evels. Stem Cells 2008; 25 2291 2301. doi:10.1634/ stemcells.2006 0609. Doetsch F Garcia Verdugo JM, Alvarez Buylla A. Cellular composition and three dimensional organization of the subventricular germinal zone in the adult mammalian brain J Neurosci 1997 ; 17:4046 61. Eftekharpour E Karimi Abdolrezaee S, Fehlings M G. Current status of experimental cell replacement approaches to spinal cord injury. J Neurosur g. 2008 ; 24 (3 4), 1 13. doi:10.317 1/FOC/2008/24/3 4/E18 Erecinska M, Silver I A Tissue oxygen tension and brain sensitivity to hypoxia. Respir Physiol 2001; 128 (3), 263 76. doi:10.1016/S0034 5687(01)00306 1. Karimi Abdolrezaee S Eftekharpour E, Wang J, Morshead CM Fehlings MG Delayed transplantation of adult neural p r ecursor cells promotes remyelination and functional neurological rec overy after spinal cord injury J Neurosci 2006 ; 26(13):3377 89. Lledo PM, Saghatelyan A. Integrating new neurons into the adult olfactory bulb: Joining the network, life death decisions, and the effects of sensory experience. Trends Neurosci 2005; 28 (5), 248 254. doi:10.1016/j.tins.2005.03.005. Louis SA, Rietze RL, Deleyrolle L, Wagey RE, Thomas TE Eaves AC Reynolds B A Enumeration of neural stem and progenitor cells in the neural colony forming cell assay. Stem Cells 2008 ; 4 988 96. Retrieved from http://www.ncbi.nlm.nih.gov/sites/entrez Reynolds B A, Weiss S. Gener ation of neurons and astrocytes from isolated cells of the adult mam malian central nervous system. Science 1992 ; 255 ( 5052), 1707 10. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1553558. Ross HH Levkoff LH, Marshall GP, C aldeira M, St eindler DA, Reynolds BA, Laywell ED Bromodeoxyuridine induces senescence in neural stem and progenitor cells. Stem Cells 2008 ; 12: 3218 27. Santilli G, Lamorte G, Carlessi L, Ferrari D, Nodari L R Binda E, Delia D, Vescovi AL, De Filippis L Mild h ypoxia e nhances p roliferation and m ultipotency of h uman n eural s tem c ells. PLoS One 2010 ; 5 ( 1), e8575. doi:10.1371/journal.pone.0008575 Studer L Csete M, Lee S H, Kabbani N Walikonis J, Wold B, McKay R. Enhanced proliferation, survival, and dopaminergic differentiation of CN S pr ecursors in lowered oxygen J Neurosci 1999 ; 20(19):7377 83. Theus MH, Wei L, C ui L, F rancis K, Hu X, Keogh C, Yu SP In vitro hypoxic preconditioning of embryonic stem cells as a strategy of promoting cell survival and functional benefits after transplantation into the ischemic rat brain Exp Neurology 2008; 210:656 670. Zawada WM, Zastrow DJ, Clarkson ED, A dams FS, Bell KP, Freed CR Growth factors improve immediate survival of embryonic dopamine neurons after transplantation into rats. Brain Res 1998 ; 786: 96 103. Zhu LL Zhao T, Li HS, Z hao H, Wu LY, Ding AS, Fan WH Fan M. Neurogenesis in the adult rat brain after intermittent hypoxia. Brain Research 2005 ; 1055 (1 2), 1 6. doi:10.1016/j.brainres.2005.04.075
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