In vitro development of dissociated and immunomagnetically- purified embryonic chick optic tectum cells

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Title:
In vitro development of dissociated and immunomagnetically- purified embryonic chick optic tectum cells
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Galileo, Deni Scott, 1961-
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Chick Embryo -- cytology   ( mesh )
Superior Colliculus -- cytology   ( mesh )
Anatomical Sciences thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 118-123.
Statement of Responsibility:
by Deni Scott Galileo.
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Typescript.
General Note:
Vita.

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Full Text











IN VITRO DEVELOPMENT OF DISSOCIATED
AND IMMUNOMAGNETICALLY-PURIFIED
EMBRYONIC CHICK OPTIC TECTUM CELLS











By


DENI SCOTT GALILEO


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


1988

















































Copyright 1988

by

Deni Scott Galileo










ACKNOWLEDGEMENTS

I would like to thank Dr. Paul Linser for both

direction and yet ample latitude to explore and satisfy my

own intellectual curiosities. I would also like to thank my

committee comprised of Drs. Francis Davis, Carl Feldherr,

and Chris West for guidance and constructive criticisms

throughout my dissertation research. A special thanks goes

to the University of Florida Division of Pediatric

Hematology and Oncology and especially Dr. Adrian Gee for

making this research possible through his commitment of

continuous gifts of microspheres, antibodies, and

encouragement to me. Also, I thank Dr. John Ugelstad of the

University of Trondheim, Norway, for a generous gift of

microspheres.

I would also like to thank Drs. Gudrun Bennett and

Gerry Shaw of the University of Florida as well as Dr.

Steve Pfeiffer of the University of Connecticut for gifts

of antibodies without which most of this work could not

have been done. Thanks are also in order to Drs. Paul

Begovac and Steve Dworetzsky for many worthwhile

discussions and sharing the common bond of being a usually

helpless guinea pig in the experiment of education that

runs continuously and often without controls in the big

orange and blue apparatus. I whole-heartedly thank anyone

that I have left out that has made my time in graduate

school either intellectually rewarding, pleasurable, or

iii










both. Alternatively, I wish to castigate those that have

made the learning processes of myself and others at the

University of Florida needlessly entangled in red tape,

rules, regulations, grades, prejudices, position, rigidity,

and everything else that runs counter to true learning.

Finally, I wish to thank Dr. Arlene Stecenko for

friendship and encouragement for the past 3 years and for

the ability to see that my quandaries could often easily be

resolved.










TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............................. ........ iii

LIST OF TABLES......................................... vi

LIST OF FIGURES..................................... .. vii

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

CHAPTERS

I INTRODUCTION (A NATURAL HISTORY)............... 1

II SEPARATION OF DAY 7-8 CELLS.................... 4

Introduction.......... ................. ...... 4
Materials and Methods.......................... 7
Results........................................ 19
Discussion.................................... 48

III SEPARATION OF DAY 12-13 CELLS.................. 69

Introduction .................................. 69
Materials and Methods.......................... 72
Results........................................ 76
Discussion............... .................... 99

IV CONCLUSIONS... ....................... .. ....... 115

REFERENCES............................................. 118

BIOGRAPHICAL SKETCH .... .............................. 124










LIST OF TABLES


Table Page

2-1 Effects of substrata on monolayers.............. 39

2-2 3H-thymidine incorporation: Day 7-8 cells...... 49

3-1 3H-thymidine incorporation: Day 12-13 cells.... 98

3-2 3H-thymidine incorporation: Monolayers .........102










LIST OF FIGURES


Figure Page

2-1 Chamber used for immunomagnetic separations... 13

2-2 Cell isolation in methylcellulose............. 21

2-3 A2B5 antigen modulation in vitro.............. 25

2-4 In vitro development of A2B5(+) cells......... 27

2-5 A2B5 antigen modulation via methylcellulose... 30

2-6 Calculated incomplete separations.............. 33

2-7 Remixed separated cells....................... 35

2-8 Effects of substrata: glass vs polyornithine.. 38

2-9 Neurofilament expression in vitro............. 43

2-10 GS expression in vitro....................... 47

2-11 3H-thymidine incorporation into monolayers.... 51

2-12 Summary diagram of results..................... 53

3-1 Markers for dissociated cells (graph)......... 78

3-2 Markers for dissociated cells (photos)......... 80

3-3 A2B5 antigen modulation in vitro.............. 85

3-4 In vitro development of A2B5(+) cells......... 89

3-5 Filament expression in vitro ............... 93

3-6 Oligodendrocyte development in vitro ......... 97

3-7 3H-thymidine incorporation into monolayers....101

3-8 Summary diagram of results .................... 104


vii















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


IN VITRO DEVELOPMENT OF DISSOCIATED
AND IMMUNOMAGNETICALLY-PURIFIED
EMBRYONIC CHICK OPTIC TECTUM CELLS

By

Deni Scott Galileo

August 1988

Chairman: Paul J. Linser
Major Department: Anatomy and Cell Biology


Immunomagnetic cell separation techniques were used

to purify embryonic chick optic tectum cells from 2

different developmental ages for in vitro development

studies. This negative cell selection method was based on

reactivity of monoclonal antibody A2B5 with cell surfaces.

Purified A2B5(-) cells obtained initially were >99.99%

pure. Surprisingly, A2B5(+) cells rapidly appeared in the

purified surface A2B5(-) cells in direct response to the

immunomagnetic depletion. After 1 day in culture, levels of

A2B5(+) cells in purified cultures equalled unpurified

levels (=50%). Similarly, visual densities of A2B5(+)

neurons were equal in purified and unpurified long-term

monolayer cultures.


viii










Degeneration of purified cells on the neuron-

selective substratum polyornithine suggested that these

cells contained a paucity of neurons initially after

separation. Immunohistochemistry combined with 3H-thymidine

autoradiography of cells and monolayers demonstrated that

new DNA synthesis was required for neither the acquisition

of surface A2B5-antigen, nor for differentiation into

neurons. These results suggest that in early embryonic

vertebrate brain (days 7 and 8) cells are present which are

capable of replacing depleted neurons in vitro.

With day 12 and 13 cells, nearly all purified A2B5(-)

cells were identifiable as glia by reacting with antibodies

against either glutamine synthetase or galactocerebroside.

Most (=80%) of the purified A2B5(-) cells in culture for

one day became A2B5(+). No increase in the percentage of

A2B5(+) cells from 45% was observed in unpurified cultures.

Long-term monolayer cultures from purified cells contained

many A2B5(+) cells with a flattened glial or round

morphology. These A2B5(+) cells frequently reacted with

antibodies against glial fibrillary acidic protein and

another glial marker, 5All, which indicated a partial glial

character. Additionally, flattened glial-like cells were

found to contain elaborate networks of anti-neurofilament-M

reactive filaments. The above combinations of markers were

not found in unpurified monolayers and are believed to be a

result of the immunomagnetic removal of neurons. It is

ix










hypothesized that the abnormal phenotypes in purified cell

cultures from day 12 and 13 cells represent unsuccessful

responses of the glia to replenish depleted neurons most

likely due to restricted developmental potentials.















CHAPTER I
INTRODUCTION (A NATURAL HISTORY)

The work presented in this dissertation is work that

began as an attempt to study in depth the phenomenon of

glutamine synthetase (GS) production in glia as mediated by

neuronal contact. This enzyme is produced in embryonic

chick retina cultures in glia that are in close apposition

to neurons (Linser and Moscona, 1979). Subsequently, it was

found that this phenomenon also occurred in cultures of

embryonic chick optic tectum cells (Linser and Perkins,

1987a) and probably occurs in the vertebrate central

nervous system in general. To study this phenomenon

directly, I wished to obtain purified glia that were not

producing any GS to which I could add back neurons and

trigger the gene expression at will.

I chose to utilize cell purification methods that

employed antibodies as the means of discrimination between

neurons and glia (immunoselection). I became aware of the

immunomagnetic cell separation procedures that use small

paramagnetic microspheres coated with specific antibodies

to remove a "target" cell population from heterogenous

populations. Although this method has been used in

different forms in several different systems, it appears

that it has most seriously been utilized by clinicians to

1










2

remove neuroblastomas and leukemias from human bone marrow

with unparalled efficiency (Treleaven et al., 1984).

Fortunately, one of only two laboratories in the United

States that uses this method clinically is at the

University of Florida in the Department of Pediatrics.

Through the aid of a mutual friend (science is as human an

endeavor as is anything else) I enlisted the aid of Dr.

Adrian Gee, the scientist in charge of such bone marrow

"purging" at U.F. He and his entire laboratory remained

committed to me for supplying microspheres and antibody

with which to coat them from the conception to completion

of my research.

Since then much time was spent on developing a simple

and effective procedure and separation chamber that would

be suitable for the separation of embryonic brain cells. At

first, it seemed that my procedure was not accomplishing

separations since apparent target cells were always in what

was hoped to be purified populations of nontarget cells. It

then became quite clear that these target cells were

appearing from nontarget cells after the separations. This

in itself was a unique and unexpected finding. It was

subsequently found that when different age embryonic cells

were separated that the target cells which appeared in the

purified populations apparently developed different

phenotypes according to the age separated. Early embryonic

cells appeared to be able to compensate for the depletion










3

of target cells (neurons), whereas older embryonic cells

could not. Herein lies the natural division of my work

into the two following chapters according to the results

obtained: separation of day 7-8 cells (Chapter II) and

separation of day 12-13 cells (Chapter III). Although these

results precluded my ability to obtain purified populations

of immature glia as I originally had hoped for, many unique

and interesting experimental phenomena occurred in the

purified cultures. These have led to a better (or more

confused, depending upon the point of view) understanding

of the potentials and restrictions of embryonic cells when

their development is perturbed in a controlled fashion.















CHAPTER II
SEPARATION OF DAY 7-8 CELLS

Introduction

During development of the chick optic tectum the

various differentiated cell types emerge in a temporally

stepwise but overlapping manner. The neurons are generally

the first cell type to exit the mitotic cycle (LaVail and

Cowan, 1971b) and to express a differentiation product such

as neurofilaments (Bennett and DiLullo, 1985). The glia are

generally later in becoming post-mitotic and show overt

signs of differentiation (Linser and Perkins, 1987a)

practically coincident with the completion of neurogenesis

(LaVail and Cowan, 1971b; Fujita, 1964). This timing of

overt differentiation, however, does not necessarily

reflect the timing of when different cells are determined

to become one cell type or another. Additionally, this

general pattern does not necessarily imply when a cell is

restricted in its ability to become anything else if its

microenvironment were to change.

The mechanisms that govern cell determination and

differentiation during brain development seem multiple but

are poorly understood. Interactions between cells appear to

influence development at several levels from physical

positioning of neurons (Levitt and Rakic, 1980) to the

4










5

expression of specific glial gene products (Fisher, 1984;

Linser and Moscona, 1979). Glutamine synthetase (GS), for

example, is produced in glia in culture when the glia are

in contact with neurons (Linser and Moscona, 1983; Linser

and Perkins, 1987a; Wu et al., 1988). Obviously, to study a

phenomenon such as this it would be of great advantage to

be able to purify the immature glia so that neurons could

be added back to elicit GS production. Such an ability

could also in itself reveal other phenomena that involve

cell interactions.

A major obstacle to studying interactions that take

place during early development, such as those that lead to

GS production, is that they occur when only few if any

cells are identifiable by commonly recognized

differentiation markers. Also early in development, most

cells do not differ enough from each other physically to

make use of such cell purification techniques as buoyant

density centrifugation (Campbell et al., 1977; Sheffield et

al., 1980). Methods that do not need overt physical

differences for separation are those that utilize

monoclonal antibodies that discriminate between the

surfaces of different types of cells (immunoselection).

Complement-mediated cell lysis has been used successfully

with embryonic neural tissues (Politi and Adler, 1987;

Nagata et al., 1986), but this method does not allow

recovery of both cell types, and not all antibodies fix










6

complement. "Panning" (Wysocki and Sato, 1978) is an

immunoselection method that allows for recovery of both

cell types; however, the purity of cells obtained is

marginally acceptable (=95%) for most applications.

One negative separation method that utilizes

monoclonal antibody binding to a target cell population to

remove it from a mixed population is immunomagnetic

purging. This method was developed for removing

neuroblastoma cells from human bone marrow (Treleaven et

al., 1984) and operates by attaching the target cells to

paramagnetic polystyrene microspheres via antibody linkages

and removing them with a magnet. Potentially all types of

monoclonal antibodies against cell surface constituents can

be used, and routine separations result in depletions of

target cells by 4 to 5 orders of magnitude (Philip et al.,

1987).

I have modified this method to work well with

dissociated embryonic chick brain cells. In hopes of

purifying immature glia for in vitro reassociation studies,

I removed the majority of identifiable neurons by using

the monoclonal antibody A2B5 (anti-ganglioside GQa,;

Eisenbarth et al., 1979). It was found that even though the

initial purification of A2B5(-) cells was complete, by 24

hours in culture approximately 50% of these cells had

become A2B5(+). This modulation of cell surface A2B5

antigen was found to be in direct response to the










7

depletion of A2B5(+) cells. Similarly, visual densities of

A2B5(+) neurons and neurofilaments were equivalent in

purified and unpurified cultures. New DNA synthesis was not

required for either modulation of surface A2B5 antigen or

differentiation of cells into neurons.



Materials and Methods

Animals

White Leghorn chick embryos were used throughout this

study. Fertilized eggs were purchased from the Division of

Poultry Science, University of Florida and stored at 150C

until initiation of incubation at 37.50C in a standard

humidified egg incubator. Time in days of incubation was

used as the index of developmental age. For the present

study, 7 and 8 day embryonic optic tecta were dissected at

the tectal commissure and isolated free of non-neural

tissues aseptically in calcium-magnesium free Tyrode's

solution (CMF; Linser and Moscona, 1979).

Cell Culture

Dissociated cells were prepared by incubating tecta

for 30 min. in 0.4% trypsin (Nutritional Biochemicals,

Cleveland, OH) in CMF at 370C, followed by dissociation

with a Pasteur pipette in Medium 199 (Hank's salts,

Degenstein formula; KC Biological, Lenexa, KS) containing

0.3 mg/ml soybean trypsin-inhibitor (Sigma Chemical Co.,

St. Louis, MO) and 0.03 mg/ml DNase I (Sigma) (SBTI-DNase).










8

Rotation-mediated suspension cultures of

reaggregating cells were made by placing 2x107 cells in 3

ml of Medium 199 supplemented with 10% fetal bovine serum

(FBS; Gibco Laboratories, Grand Island, NY), 100U/ml

penicillin + 100 gg/ml streptomycin sulfate (Gibco), and 10

gg/ml gentamycin sulfate (M.A.Bioproducts, Walkersville,

MD). Cells were rotated in capped 25 ml Ehrlenmeyer flasks

at approximately 75 rpm (1 inch radius) at 370C in a rotary

incubator (New Brunswick Scientific, Edison, NJ) and fed

approximately every other day with the same medium. Two

days previous to assaying cultures for glutamine

synthetase (GS), cultures were fed with the above medium

supplemented with 0.33 gg/ml hydrocortisone (Sigma). GS

levels were assayed by the modified colorimetric method of

Kirk as previously described (Linser and Moscona, 1979).

Adherent monolayer cultures were prepared by

incubating 106 cells in 1 ml of the above medium in 24 well

(200 mm2) tissue culture plates (Corning Glass Works,

Coming, NY). Cells were plated on either 1) the plastic

itself, 2) inserts of 12 mm dia. round glass coverslips, 3)

coverslips coated with 0.1 mg/ml poly-L-ornithine HBr

(m.w.= 100,000; Sigma), 4) coverslips coated with

polyornithine and then 1 mg/ml rat tail collagen (Sigma),

or 5) inserts of 7.5 mil thick Aclar fluorohalocarbon film

(Allied Chemical Corp., Morristown, NJ). Cultures were kept

in a standard tissue culture incubator at 370C in a 5% CO2/










9
air atmosphere. Monolayer cultures were fed with fresh

medium approximately every other day.

For cell isolation experiments, freshly dissociated

cells were suspended in a semisolid medium of 1.4% methyl

cellulose (Sigma) dissolved in the above culture medium at

a density of 107 cells/ ml. Cultures were kept in loosely

capped sterile Ehrlenmeyer flasks in a standard tissue

culture incubator in a 5% CO2 / air atmosphere. After

different lengths of time cells were recovered from the

semisolid medium by diluting the medium with several times

the volume of Tyrode's solution and were collected by

centrifugation.

Immunomagnetic Separations

Polyclonal sheep anti-mouse antibodies (kindly

provided by Dr. Adrian P. Gee, Div. of Pediatric Hematology

and Oncology, Univ. of Florida) used to coat the

microspheres were prepared by hyperimmunization with

purified mouse IgG (Organon Teknika- Cappell, West Chester,

PA). Useful antiserum was obtained after 5-6 immunizations.

Anti-mouse antibodies were affinity-purified on a column

made from mouse IgG (Cappell) bound to Affi-Gel 10 (Bio-Rad

Laboratories, Richmond, CA) and then mixed with Sepharose

CL-4B (Pharmacia, Inc., Piscataway, NJ).

Cells used for immunomagnetic separation were rinsed

in Tyrode's solution, incubated in 1/25 dilution of A2B5-

conditioned hybridoma medium in Tyrode's with 10% heat










10
inactivated FBS (HI-FBS) for 30 min. at 4C. Cells were

then rinsed 2x in Tyrode's and resuspended in SBTI-DNase.

The A2B5 hybridoma cell line was obtained from the

American Type Culture Collection, Rockville, MD, through

Dr. Michael F. Marusich, Univ. of Oregon, Eugene, OR.

Cells were mixed for 30 min. at 4C in SBTI-DNAse + 10% HI-

FBS with paramagnetic polystyrene microspheres (4.5 mn;

Dynal, Inc., Great Neck, NY) which were previously coated

with sheep anti-mouse IgG prepared as above. A 15-fold

excess of the number of microspheres/ the number of A2B5(+)

target cells was used which corresponds to a 7.5-fold

excess of microspheres/ total cells, since approximately

50% of dissociated cells were A2B5(+). The microspheres

were ethanol sterilized then coated with 30-40 gg antibody/

mg microspheres in a concentration of at least 0.2 mg/ml

antibody overnight at 4C with rotation in a microfuge

tube.

The cell-microsphere mix was then poured into the top

of the separation chamber which was prefilled with

Tyrode's, and unbound cells were eluted with Tyrode's by

gravity at a rate of approximately 1-2 ml/ min. in a

sterile hood until no more cells appeared in the eluant. In

a typical experiment, approximately 3x107 cells were

separated in 30 min. The separation chamber was constructed

from a plastic funnel and tubing (1/4" dia.) held on a ring

stand (Fig.2-1). Two magnet arrays were held against the










11

side of the tubing, one made of ferrite magnets above one

made of samarium-cobalt magnets. A pinch clamp at the

bottom of the tubing was used to control the rate of flow

induced by gravity.

Bound microsphere fraction cells were collected by

first removing the magnet arrays from the side of the

tubing and then washing out the bound cells and

microspheres into a test tube. Cells were released from the

microspheres by trypsinization as above followed by

addition of SBTI-DNAse and vortexing. Freed microspheres

were drawn away from the cells by placing the samarium-

cobalt magnet array against the side of the tube and the

suspended cells were aspirated out of the tube with a

pipette.

Cells to be assessed for cell surface A2B5 following

separation (approximately 2 hrs. after plating) were fixed

in 1% formaldehyde (ACS grade, Fisher Scientific,

Pittsburgh, PA) in phosphate-buffered saline (PBS) for 30

min., rinsed 3x in PBS, then incubated for 30 min. in 1/50

dilution of fluorescein-goat anti-mouse IgM (FITC-GAM IgM;

Boehringer Mannheim Biochemicals, Indianapolis, IN) in PBS

with 5% normal goat serum. Cells assayed for cell surface

A2B5 after one day in culture were incubated live in 1/25

A2B5-conditioned hybridoma medium +10% HI-FBS on ice and

then processed as above. Plating efficiencies of cells were

determined retrospectively from Ektachrome slides by
























Fig.2-1. Separation chamber used for the immunomagnetic
separations and Nomarsky micrographs showing cells before
and after the purification. Construction was of plastic
tubing and funnel held on a ring stand. Two magnet arrays
were used to ensure collection of all of the paramagnetic
microspheres. A pinch clamp was used to control the rate of
flow through the chamber. After ethanol sterilization in a
sterile hood the cell-microsphere mix was poured into the
funnel and the purified cells were collected in centrifuge
tubes from the bottom. Micrograph (a) shows smaller
microspheres binding to one cell (arrowhead) but not to
another. Micrograph (b) shows cells that were removed from
the mixture due to their coating of microspheres
(arrowheads). Bottom micrograph (c) shows 6 purified cells
without attached microspheres. Bar, 50gm.



















Iron
Magnets


4.


Samarium-
Cobalt
Magnets
L&










14

counting the number of cells that adhered to the

polyornithine coated coverslips initially after the

experiment and after 1 day in culture. No attempt was made

to determine cell numbers or densities in longer-term

cultures.

Miniaggregate cultures were made of both unpurified

and purified A2B5(-) cells in 24 well tissue culture plates

on a substratum of 2% poly(2-hydroxyethyl methacrylate)

(poly(HEMA)) (Interferon Sciences, Inc., New Brunswick, NJ)

(Folkman and Moscona, 1978). This substratum prevented cell

attachment to the plastic so that maximum intercellular

contact and interaction could take place. Cultures were

plated and fed as were adherent monolayers above. Two days

before the GS assay was performed, cultures were fed with

medium containing hydrocortisone as were rotation-mediated

aggregate cultures.

Vital Dye Experiments

Experiments were performed which made use of the vital

fluorescent carbocyanine dye DiO (3,3'-dioctadecyloxa-

carbocyanine perchlorate; Molecular Probes, Eugene, OR)

for cell marking purposes. These experiments were to

determine if A2B5(+) cells would appear if less than all of

the A2B5(+) cells were removed (incomplete separations),

and also to determine if remixing the separated cells

suppressed recruitment of new A2B5(+) cells (remixed

separations). In both of these, cells that were to be










15

labelled were incubated in 200 gg/ml dye solution for 30

min. according to Honig and Hume (1986). DiO stained cells

were viewed on an epifluorescent microscope with

fluorescein optics and DiO fluorescence was not visible

with rhodamine optics. Labelling efficiency with DiO was

nearly 100%.

For calculated incomplete separations, part of the

dissociated cells to be separated were incubated in A2B5 as

in a normal separation followed by incubation in the dye

DiO. These cells were then mixed with dissociated cells

that were not incubated in either of these in some ratio

(Fig. 2-6). This mix was then mixed with microspheres and

separated in the magnetic column as above. Eluted unbound

A2B5(-) cells were plated on polyornithine coverslips as

described above. After one day in culture, these cultures

along with control unseparated cultures were immunostained

live with A2B5 as described above. A rhodamine-goat anti-

mouse IgM (Fisher) secondary antibody was used to

discriminate the DiO staining. The percent of the DiO(+)

cells that were also A2B5(+) were scored.

In experiments where separated cells were remixed, the

separation was carried out as normal (complete) and the

bead-bound cell fraction was recovered via trypsinization

of the beads (Fig.2-7). Eluted A2B5(-) cells were meanwhile

incubated in DiO, and then the two separated cell fractions

were remixed in even proportions. After one day in culture,










16

remixed cultures along with straight purified and control

unpurified cultures were immunostained live with A2B5 and a

rhodamine-goat anti-mouse secondary antibody as above.

Scored were the percent of the DiO(+) cells that were also

A2B5(+) in remixed cultures and percent A2B5(+) in purified

and unpurified cultures.

Immunohistochemistry

Immunostaining with monoclonal antibody A2B5 was

performed by incubating coverslips in 1/25 dilution of

hybridoma supernatant in Tyrode's + 10% HI-FBS on ice for

30 min. Coverslips were then rinsed with Tyrode's 3x, fixed

with 1% formaldehyde in phosphate-buffered saline (PBS) for

30 min., and rinsed 3x in PBS. Either fluorescein-goat

anti-mouse IgM or rhodamine-goat anti-mouse IgM diluted

1/50 + 5% NGS in PBS for 30 min. was used as the secondary

antibody. This procedure results in specific labelling of

neurons in monolayer cultures without labelling the

flattened glial cells.

Immunostaining of cells and monolayers with anti-

galactocerebroside (GC) was performed essentially the same

as with A2B5 above. A dilution of 1/50 of a purified

monoclonal antibody against galactocerebroside (Ranscht et

al., 1982; kindly provided by Dr. Steve Pfeiffer, Univ. of

Conn. Health Center) was used. FITC-GAM IgG + IgM

(Boehringer) was used as the secondary antibody.










17

For localization of GS in monolayer cultures,

coverslips were reacted with A2B5 and then fixed in Bouin's

fixative. Followed by rinsing 3x in PBS, cultures were then

incubated in polyclonal antisera specific for GS (Linser

and Moscona, 1979) at a dilution of 1/100 in PBS + 5% NGS

for 30 min. After rinsing 3x in PBS, coverslips were

incubated in a mixture of 1/50 FITC-GAM IgM and Texas Red

goat anti-rabbit IgG (Fisher) + 5% NGS for 30 min.

followed by rinsing 3x in PBS.

For localization of intermediate filaments, coverslip

cultures were first immunostained with A2B5 and

formaldehyde fixed as above. Cultures were then

permeablized with 95% ethanol at -200C for several minutes

followed by rinsing in PBS. For glial filaments, cultures

were incubated in a 1/50 dilution of polyclonal anti-glial

fibrillary acidic protein (Dakopatts, Denmark) +5% NGS for

30 min. followed by rinsing 3x in PBS and incubation in the

appropriate mixture of secondary antibodies. For

neurofilaments, permeablized cultures were incubated in

either a 1/250 dilution of polyclonal anti-neurofilament-M

antiserum (Bennett et al., 1984) or a 1/100 dilution of a

purified monoclonal antibody NF-1 (Shaw et al., 1986)

followed by the appropriate mixture of secondary

fluorescent antibodies.












3H-Thymidine Incorporation

Incorporation of 3H-thymidine was performed on both

cells and monolayer cultures made from both unpurified and

purified cells. Cells were plated on polyornithine coated 8

chamber Lab-Tek tissue culture chamber/slides (Miles

Scientific, Naperville, IL) in culture medium containing 1

gCi/ml [methyl-3H]-thymidine (6.7 Ci/mmol; NEN Research

Products, Boston, MA) for analysis after 24 hours. For

analysis of monolayer cultures, cells were plated on

chamber/slides in the above medium and fed with fresh

medium with label approximately every other day.

Cultures were rinsed in Tyrode's 3x, fixed in 1%

formaldehyde, and immunostained with A2B5 as were coverslip

cultures. Chamber wells and gaskets were removed and slides

were dipped in Kodak NTB2 nuclear track emulsion (Eastman

Kodak Co., Rochester, NY) diluted 1:2 with water. Slides

were developed in Dektol developer (1:1) according to the

manufacturer's directions after 4-5 days exposure at 4C.

Micrography

All cultures were viewed and photographed using a

Dialux 20 (Leitz, Switzerland) epifluorescent microscope

equipped for mutually exclusive visualization of

fluorescein and rhodamine fluorescence through a 40x

Neofluar (Zeiss, West Germany) phase contrast objective

which had a numerical aperture of 0.75.










19

Results

Cell Isolation in Methyl Cellulose

Using the tissue dissociation method above, cells were

obtained that were nearly round and free of cell surface

debris. Approximate yields were 2x107 cells/ embryo or 107

cells/ lobe using 7 day and slightly higher (2.5x107/

embryo) for 8 day embryos. This yield was high compared to

dissociations of tissue that is several days older (Chapter

III). When suspended in the methyl cellulose, cells were at

least several diameters apart from each other with no

apparent physical contact.

The results of the effects of cell isolation on the

production of GS after reaggregation are shown in Fig.2-2.

GS was produced by cells isolated for 1 day at levels

equaling those after immediate reaggregation. Thereafter,

levels of GS fell rapidly, however levels were still

measurable after 2 days of isolation. This demonstrated

that these cells could be manipulated for up to 2 days

before reaggregation was necessary if GS production was to

be examined in culture.

Immunomagnetic Separations

Unless otherwise specifically stated, cell reactivity

with A2B5 antibody refers only to cell surface binding and

not to possible intracellular reactivity. The

immunomagnetic cell separation procedure described herein

produced initially extremely pure populations of A2B5(-)
























+1


CQ

O 0
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0a) ..4 4 a) 4-4 A


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22

cells from dissociated tecta. Purified cells were routinely

>99.99% A2B5(-) as assayed by indirect immunofluorescence.

Also, the vast majority of cells recovered from the

microspheres were low level A2B5(+). Yields of A2B5(-)

cells have ranged from 100% of theoretical yield (50% of

total cells) to less than 50% of theoretical yield,

depending on the batch of microspheres used. M450.40

microspheres gave the highest purified cell yields, while

M450.51 and Dynabeads M450 gave significantly lower yields,

presumably due to increased nonspecific binding to or

trapping of A2B5(-) cells.

For separation of cells via A2B5, I have found it

necessary to use a polyclonal antibody that was

specifically produced for the purpose of microsphere

coating. Several commercial affinity-purified polyclonal

anti-mouse antibodies have been tested for effectiveness

with this monoclonal without satisfactory purifications.

Microspheres precoated with anti-mouse IgG (Dynal) have

also been tested with similar unsuccessful results. The

results presented here represent only experiments where the

purity of A2B5(-) cells exceeded 99%.

A2B5 Antigen Modulation

Although the separations resulted in purified A2B5(-)

cells, it was found that many A2B5(+) cells appeared after

in vitro culture for only several hours. After one day in

culture, approximately 50% of the initially purified cells










23

had become A2B5(+) (Fig.2-3). This is the same level of

A2B5(+) cells in unpurified cultures after one day in

vitro. Plating efficiencies of both unpurified and purified

cells after one day in culture were nearly identical and

ranged from approximately 85-100% as determined by

retrospective counts from slides. Unpurified monolayer

cultures (1-2 weeks in vitro) contained A2B5(+) cells that

exhibited only neuronal morphologies that rested atop a

layer of A2B5(-) flat cells either singly or in

multicellular aggregates (Fig.2-4). Similarly, A2B5(+)

cells with neuronal morphologies with a layer of A2B5(-)

flat cells beneath them were present in monolayers from

purified cells. The clusters of A2B5(+) neurons in both

types of cultures appeared to be the same visual density

under high or low magnification. No difference in the

amount of A2B5(+) neurons in either type of culture was

seen. The cultures made from microsphere cells, however,

always contained visually larger and/or more numerous

aggregates of A2B5(+) neurons than did the unpurified or

purified cultures (not shown).

To assess the requirement of serum (FBS) in purified

cultures where A2B5 antigen was modulated, purified cells

were plated on polyornithine coated coverslips as above.

After one day in culture, cells were immunostained live

with A2B5 as above. To determine whether or not the

modulated A2B5 antigen was trypsin-resistant as was the































Fig.2-3. Quantitation of A2B5(+) cells when unpurified and
purified at the time of separation and after one day in
culture. The unpurified cells show a small but significant
increase in the number of A2B5(+) cells after one day. The
purified cells are initially almost entirely A2B5(-), but
after one day contain the same number of A2B5(+) cells as
unpurified cultures. Shown are means s.e.m. for 4
individual experiments.














=I IDay 0

60 -- M+1 Day


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30 --
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Unpurified Purified





















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28

antigen in freshly dissociated cells, purified cells were

plated on polyornithine coverslips as above. After one day

in culture, coverslips were subjected to the same

trypsinization regime as was used to dissociate tissue.

Cells were then immunostained for surface A2B5 antigen as

above. In both instances, it was found that there was no

reduction in the percentage of A2B5(+) cells (=50%). The

experiments that entailed trypsinization, however, resulted

in a large (unquantitated) release of cells from the

coverslip due to the trypsin treatment.

Subsequent to the finding of the appearance of A2B5(+)

cells in immunomagnetically-purified cell cultures, I

predicted that an analogous increase in A2B5(+) cells may

have occurred when unseparated cells were suspended in

methyl cellulose for a day. It was realized that suspension

in methyl cellulose was in effect also a separation that

would not allow the A2B5(+) and (-) cells to interact. This

prediction was found to be true both qualitatively and

quantitatively (Fig.2-5). Over 50% of the cells that were

A2B5(-) when suspended in the methylcellulose converted to

A2B5(+) after one day. Thus by two different techniques the

ability of A2B5(-) cells to become A2B5(+) when isolated

has been demonstrated with similar quantitative results.

Vital Dye Experiments

I made use of the a vital fluorescent dye, DiO, to

label certain cell populations for either controlled



























a) d i E- S
4-) 'd E-
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(d *D 0 >o
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31

"incomplete" immunomagnetic separations or remixing

experiments as diagrammed in Figs. 2-6 and 2-7. This was

done to determine if new A2B5(+) cells would appear if only

some of the A2B5(+) cells were removed, and also to

determine if appearance of new A2B5(+) cells in purified

cultures could be suppressed by adding back the cells that

were removed. The results of the incomplete separations are

shown in Fig.2-6. The abscissa is the percent cells

incubated in A2B5 before separation which is in effect the

percent of A2B5(+) cells removed, since the remaining cells

had not been exposed to the monoclonal antibody. The

ordinate expresses the percent of the known A2B5(-)/DiO(+)

cells that became A2B5(+) after one day in culture. It can

be clearly seen that the result of removing increasing

numbers of A2B5(+) cells was increasing recruitment of

A2B5(-) cells to become A2B5(+). Thus the increase of

A2B5(+) cells in purified cultures was in direct response

to the depletion of A2B5(+) cells.

Fig.2-7 shows the lack of effect of mixing back those

cells that were removed with purified cells on the

recruitment of newly appeared A2B5(+) cells. Here, complete

separations were performed and the purified A2B5(-) cells

were incubated in dye before remixing with cells recovered

from the microspheres. It is clear that mixing back those

cells that were removed had no effect on decreasing the

amount of newly appeared A2B5(+) cells from those that were
























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36

A2B5(-). Furthermore, when cells were allowed to develop

into monolayers, A2B5(+) neurons that had retained the dye

were found (not shown). Internalization of dye from cell

surfaces as well as a high dye background from labelled

flat cells made it impossible, however, to quantitate the

percentage of A2B5(+) neurons that were dye labelled.

Nonetheless, recruitment of A2B5(+) cells did not seem to

be suppressable with this experimental regime.

Effects of Substrata

The effects of different substrata on the development

of monolayer cultures was investigated to determine whether

purified cells might develop differently than unpurified

cells. Adler et al. (1979) showed previously that different

substrata had profoundly different effects on the

development of day 7 optic tectum cells. Here, when cells

were plated on either plastic, glass, or collagen

multicellular aggregates formed rapidly (<1 day) and then

attached to the substratum after about a week in culture.

Flat cells then migrated out of the aggregates on the

substratum eventually forming a mature monolayer culture

with networks of A2B5(+) neurons and neuronal aggregates

growing on top of the layer of A2B5(-) flat cells (Fig. 2-

8). This was true for both unpurified or purified A2B5(-)

cells. These different substrata appeared to be totally

nonselective for the growth of neurons and glia in culture

(Table 2-1).
























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Table 2-1
Effects of substrata on monolayers-


Cells Substratum Growth


Unpurified Glass All cells
Plastic IF
Aclar I
Collagen I
Polyornithine Neurons only

Purified Glass All cells
Plastic I
Aclar
Collagen
Polyornithine No growth

Microsphere Glass All cells
Plastic
Aclar
Collagen
Polyornithine Neurons only

Mixed Glass All cells
Plastic '
Aclar '
Collagen '
Polyornithine Neurons only



a Effects of substrata on the growth of neurons and glia
in unpurified and purified monolayer cultures. The only
substratum out of those tested which was cell type
selective was polyornithine which was selective for
neurons. Practically all cells degenerated on
polyornithine in cultures made from purified A2B5(-)
cells.










40

The substratum polyornithine resulted in much

different development of embryonic tectum cells in vitro.

As observed by Adler et al. (1979), when dissociated day 7

or 8 tectum cells were plated on polyornithine only the

neurons developed and the glia degenerated over a period of

about a week. This resulted in aggregates of neurons with

interconnecting processes attached to the coverslips amidst

the nuclei and debris of dead glia (Fig.2-8). When purified

A2B5(-) cells were plated on polyornithine virtually all

cells degenerated. Conversely, when cells recovered from

the microspheres (A2B5(+)-enriched) were plated on this

substratum many neurons developed. In fact, this fraction

of cells resulted in the most visually dense networks of

neurons. This suggests that the purified A2B5(-) cells were

deficient in neurons and that the microsphere cells were

enriched in them.

Filament Expression In Vitro

The expression in vitro of two neural tissue specific

intermediate filaments was examined by immunohistochemistry

of monolayer cultures. Glial fibrillary acidic protein

(GFAP) positive filaments were found to be present in the

population of A2B5(-) flat glial cells underlying the

neurons (not shown). Immuno-reactivity was seen in most if

not all flat cells but not in cells with a neuronal

morphology. Unlike GFAP, there were cells present in

dissociated 7 or 8 day tectum that reacted with antibodies










41

specific for neurofilaments (see Chapter 3). Twelve percent

of unpurified cells showed immuno-reactivity with a

polyclonal antisera specific for the phosphorylated form of

the middle weight chicken neurofilament triplet protein

(NF-M; Bennett et al., 1984). The pattern of staining was

mostly filamentous surrounding the nucleus. This pattern is

somewhat surprising since neurofilament reactivity was

confined to cellular processes (axons) in tissue sections

from the same age tecta (data not shown). This may suggest

that the dissociation procedure caused a retraction of

processes by neurons. When cells were double labelled with

A2B5, the large majority of NF-M(+) cells were also

A2B5(+). Twelve percent of the total dissociated cells were

NF-M(+). Ten out of 12 of these cells were also A2B5(+), so

83% of the NF(+) cells were also A2B5(+). Alternatively, 2%

of the total cells were A2B5(-) and NF-M(+), leaving 4% NF-

M(+) cells in the purified A2B5(-) population.

Neurofilament expression was examined in monolayer

cultures with both the polyclonal antisera and a purified

monoclonal antibody (NF1) specific for the phosphorylated

form of the rat heavy (200kD) triplet protein (Shaw et al.,

1986). Many of the A2B5(+) cells with neuronal morphology

in both unpurified or purified cultures contained

neurofilaments as evidenced by both antibodies (Fig.2-9).

Likewise, greater than 90% of neurofilament containing

neurons were A2B5(+). Cell monolayers that were made from























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44

microsphere fraction cells had visibly more dense

neurofilaments and A2B5(+) neurons (not shown). In

monolayers, more neuronal processes were found that

contained filaments positive for the polyclonal antisera

than for NF1, and processes that contained filaments that

were NF1(+) were almost always NF-M(+) as well. Thus, the

patterns of neuronal and glial intermediate filaments

appeared identical in purified and unpurified cells when

grown on a substratum of glass.

The pattern of neurofilament immunostaining was very

different, however, in purified versus unpurified cultures

when grown on a substratum of polyornithine (Fig.2-9). As

stated above, only a network of neurons survived when

unpurified cells were cultured on polyornithine. As in

neurons cultured on glass, many of the neurons on

polyornithine contained neurofilaments. Purified cultures,

on the other hand, degenerated on polyornithine and

contained a paucity of neurofilaments. The few cells that

survived and produced neurofilaments (or had them at the

start) were presumably those A2B5(-)/NF-M(+) cells (see

above) that eluted in the purified population.

Galactocerebroside Expression In Vitro

A monoclonal antibody specific for the membrane

molecule galactocerebroside (GC) was used to study the

development of oligodenrocytes in cultures of purified and

unpurified tectum cells. No GC(+) cells were present in










45

dissociated 7 or 8 day tectum. Similarly, no GC(+)

oligodendrocytes appeared in cultures of either unpurified

or purified cells (not shown). GC(+) cells appear by day 12

of development (see Chapter 3).

GS Expression In Vitro

The expression of glutamine synthetase was examined in

both aggregate and monolayer cultures to determine whether

or not purified and unpurified cultures were similar in

this respect. A quantitative assay revealed that both

purified and unpurified cells produced the same levels of

GS when allowed to reaggregate on poly(HEMA) (Fig.2-10).

Similarly, immunostaining for GS revealed the same staining

patterns in both purified and unpurified monolayer

cultures: GS was produced in glia under or near aggregates

of neurons (Fig.2-10). Thus, the production of GS in glia

in purified cultures parallels that of unpurified cultures

both quantitatively and with respect to position to

neurons.

DNA Synthesis In Vitro

The synthesis of new DNA was examined by combining

A2B5 immunostaining with 3H-thymidine autoradiography of

both newly plated cells and of monolayer cultures. Newly

plated cells were subjected to continuous labelling with

3H-thymidine for 24 hours, followed by immunostaining with

A2B5 and autoradiography. With both purified and unpurified

cells only a small number of nuclei were labelled (<5%;
































Fig.2-10. GS expression in vitro. Graph shows results of GS
assay performed on miniaggregate cultures on poly(HEMA) in
culture for = 1 week. Unpurified and purified cultures
produced identical levels of GS. Micrographs show
immunohistochemistry of monolayers (9 days in vitro) for
GS. GS was produced only in glia underlying neuronal
aggregates in both (a) unpurified and (b) purified
cultures. Bar, 50pm.







47

GS ASSAY


20-


10+


n=53 I
Unpurified Purified










48

Table 2-2) and all of these cells in both cultures were

A2B5(-). See Fig. 2-11 for examples of labelled nuclei.

Similarly, long-term (1-2 weeks) monolayer cultures

were subjected to continuous labelling with 3H-thymidine

followed by A2B5 immunostaining and autoradiography. The

A2B5(+) neurons that developed in both purified and

unpurified cultures did not contain labelled nuclei (Fig.2-

11). Out of several hundred neurons examined where cell

bodies could be clearly seen not one contained a labelled

nucleus. The majority of A2B5(-) flat cells, however, had

densely labelled nuclei. These nuclei were rather large in

diameter, quite flat, and oval shaped.

Discussion

A diagrammatic summary of the results is presented in

Fig.2-12. I have developed a method for the purification of

dissociated embryonic brain cells based on the removal of a

target cell population by specific antibody linkages to

paramagnetic microspheres. This method was developed to

purify embryonic glia so that phenomena such as the

induction of GS by neurons could be studied in vitro in a

controlled fashion. The cell isolation experiments in

methylcellulose demonstrated that embryonic tectum cells

could be manipulated for a significant length of time (=2

days) before they lost competence for GS production when

reaggregated. Thus it seemed entirely possible to develop a

method of separation that would be useful within this time
















Table 2-2
3H-thymidine incorporation:
Day 7-8 cells-


Cells Score % Labelled Nuclei


Unpurified 21/519 4.0%

Purified 28/566 4.9%


m 3H-thymidine incorporation into cells during 24 hours in
culture. Both unpurified and purified cultures contained a
small percentage of cells with labelled nuclei. All of
these labelled cells were A2B5(-).





















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54

frame. This approach would not be possible if the tissue

were retina, however, since the cells lose their

competence for reaggregation and GS production in a matter

of hours (Linser, 1987 and unpublished).

Separation Technique

The immunomagnetic separation technique that was used

was ideally suited for my purpose. Populations of extremely

pure A2B5(-) cells were obtained in a relatively short time

period. The ability to separate the tectum cells based

solely on surface antibody binding seemed necessary since

most 7 or 8 day cells are not sufficiently differentiated

to make use of other purification strategies.

Immunomagnetic cell separation has been applied to neural

tissues previously to purify oligodendrocyte precursors

(Meier et al., 1982; Meier and Schachner, 1982) with

success. This was a positive selection of the target cells

using large polyacrylamide-coated beads, however, in

contrast to the negative selection employed here. They

reported an enrichment of oligodendrocytes from 1.5% to 91%

purity and an average yield of 19%. The method described

here resulted in much higher purity of cells as well as a

higher yield. Also, cells could be recovered from the

microspheres with a purity comparable to the

oligodendrocyte selection above with much better yield.

Several major modifications of the technique used to

purge bone marrow (Treleaven et al., 1984) had to be made










55

for optimum separations of dissociated embryonic brain

cells. It was necessary to mix cells and microspheres in a

medium containing DNase. This was presumably due to the

release of DNA due to cell lysis. Various types of

separation chambers have been used for clinical

applications (Gee et al., 1987; Treleaven et al., 1984).

The chamber that was used for separation was much simpler

than those used for bone marrow purging. This open system

proved to be entirely satisfactory and contamination due

to this never occurred. Lastly, satisfactory separations

were obtained with a much lower microsphere/target cell

ratio than is used for marrow purging. This is believed to

be due to increased collisions between target cells and

microspheres as a result of a higher percentage of target

cells (50%) than in infected bone marrow (=1%).

A2B5 Antigen and its Modulation

A2B5 (Eisenbarth et al., 1979) was chosen as the

target cell antibody for the immunomagnetic separations.

This was because of its high specificity for neurons in

long-term monolayer cultures of dissociated differentiated

tectum cells. Other factors that led to its choice were

that it has been reported to be neuron specific in humans

(Kim, 1985; Kim et al., 1986) and to bind to most or all

neurons in chick brain (Schnitzer and Schachner, 1982). The

antigen it recognizes is also a protease resistant

ganglioside (Eisenbarth et al., 1979; Kasai and Yu, 1983).










56

When compared with immunostaining for neurofilaments of

freshly dissociated cells it was found that a small

percentage of NF(+) cells were A2B5(-). The majority of the

NF(+) cells, however, were A2B5(+) (83%) and were removed.

Thus the specificity of A2B5 for neurons initially is not

complete and all-inclusive but encompasses the majority of

identifiable neurons. No other markers that react with

differentiated neurons are known that react with 7 or 8 day

cells. Neuron specific enolase in chick brain does not

appear until later in development and apparently is not

produced in brain cell cultures (Ledig et al., 1985). Aside

from markers, culturing cells on neuron-selective

polyornithine suggests that at least most of the A2B5(+)

cells were neurons because the microsphere fraction was

enriched for neurons and the purified A2B5(-) cultures did

not contain them.

The appearance of A2B5 antigen on the surfaces of the

purified A2B5(-) cells was curious. This phenomenon was

first observed when the purified cells were incubated in

A2B5 and then the fluorescent secondary antibody. This was

done for fear that some A2B5(+) cells may have quickly

cleared the monoclonal from their surfaces after the

separation. Subsequent experiments revealed that no

reduction in the number of A2B5(+) cells occurred after a

day in culture (data not shown). Therefore, the correct

assay to assess the degree of depletion of A2B5(+) cells









57
was to incubate the purified cells in only the secondary

antibody since rapid turnover of the antigen did not seem

to occur.

As was stated earlier, the antigen recognized by A2B5

is a ganglioside (Eisenbarth et al., 1979; Kasai and Yu,

1983). These sialoglycosphingolipids are believed to be

synthesized in an progressive fashion from individual

sugars transferred from nucleotide conjugates (Ledeen,

1985). This apparently takes place in the Golgi apparatus

and probably the smooth endoplasmic reticulum through

membrane bound multienzyme complexes specific for synthesis

of each ganglioside. This occurs only in the cell soma of

neurons in the chick visual system (Landa et al., 1979)

after which the gangliosides translocate to nerve endings

via fast axonal transport (Ledeen, 1985). In fractionated

cells gangliosides are found predominantly in the

synaptosomal and microsomal fractions (Hamberger and

Svennerholm, 1971). Gangliosides exhibit turnover and are

degraded primarily in lysosomes in an ordered fashion and

evidence suggests that postsynthetic processing of them

does not occur (on the cell surface) between synthesis and

degradation (Ledeen, 1985).

The mechanisms that control the synthesis and export

of gangliosides to the cell surface not at nerve endings

are not well understood. From what is known about

ganglioside biosynthesis (Ledeen, 1985) it is probable that










58

the appearance of A2B5 antigen on purified cell surfaces is

not due to the modification of existing gangliosides on the

surface or even intracellularly. This cannot be ruled out,

however, because increases in the complex gangliosides

(including Goac) during development of the chick optic

tectum are correlated with decreases in simpler precursors

(GaD) (Rosner, 1980). Other possibilities that may explain

the appearance of A2B5 antigen are new synthesis and export

to the surface, or export of an intracellular pool. Since

it is known that chick brain cells have intracellular pools

of A2B5 antigen (see below; Schnitzer and Schachner, 1982)

this possibility is quite real. Export to the surface

could be via exocytotic vesicles or via a ganglioside

transfer protein found in brain (Gammon and Ledeen, 1985).

These types of export mechanisms could account for the

appearance of A2B5 antigen on the surfaces of purified

cells within the several hours that it has been seen to

occur (see Results).

The number of "recruited" A2B5(+) cells was a function

of the number of original A2B5(+) cells that were removed

as demonstrated by the calculated incomplete separations.

The question remains as to whether this triggered

modulation of A2B5 antigen was a result of the removal of

neurons or of A2B5(+) cells. Some (20%) of the A2B5(+)

cells that were removed are known to be neurons because

they contained neurofilaments. The remaining 80% were of










59

unknown type. They may, however, have been neurons that

either did not contain neurofilaments because of lack of

synthesis in the tissue or because of severing of axons

during dissociations. The results of culturing cells

recovered from the microspheres on glass and polyornithine

suggest that these are neurons because cultures from these

A2B5(+)-enriched cells appeared to be neuron-enriched.

Later on, there was a tight correlation between a cell

being A2B5(+) and having a neuronal morphology in long-term

monolayer cultures. Therefore, modulation of A2B5 antigen

on purified A2B5(-) cell surfaces may be a response to

neuronal depletion as effected by the removal of A2B5(+)

cells. Consistent with this hypothesis, surface A2B5

antigen modulation has been shown to occur with mouse

cerebellar astrocytes in culture in response to complement-

mediated depletion of neurons using an independent neuronal

marker (Nagata et al., 1986).

The inability to prevent recruitment of new A2B5(+)

cells by mixing back the cells that were removed implies

that the recruitment was very rapid and irreversible. There

exists the possibility that this was due to damage of the

cells that were removed from the microspheres by the second

trypsinization. This seems unlikely, however, since the

cells were trypsinized before dissociation. These recovered

cells also grew well in culture. It appears that the

events that led to the expression of A2B5 antigen on the










60

recruited cells were irreversible within the time frame in

which they were remixed (an hour).

A main question that remains is the significance of

A2B5 antigen and its appearance on the surfaces of purified

cells. Gangliosides are major constituents of the

glycocalyx of neural cells (Ledeen, 1985). Their general

stability in the membrane makes them ideal candidates for

roles such as adhesion and recognition. Gangliosides are

known to be the neural cell surface receptors for tetanus

toxin (GDib and GTxb) and for cholera toxin (GM1) (Ledeen,

1985). Gangliosides are also thought to influence in some

way the formation of synapses (Grunwald et al., 1985) and

the process of myelination (Ledeen, 1985). In culture,

purified gangliosides have been shown to mediate adhesion

of embryonic chick retina cells (Blackburn et al., 1986)

and to alter the morphology and growth of astrocytes from

fetal rat brain (Hefti et al., 1985). It is possible that

the ganglioside recognized by A2B5 on cell surfaces

similarly may function as a recognition molecule. It is

unlikely that it serves as an adhesion molecule since cells

that had bound antibody on their surfaces did not appear

to have diminished ability to form either heterotypic or

homotypic contacts with other cells. It may even be

proposed that A2B5 antigen is a molecule that is involved

in the communication between neurons and other cells since

A2B5 is neuron-specific in monolayer cultures, here, and










61

since its modulated appearance on cell surfaces depends on

the removal of cells already expressing it at their

surface.

Development of Purified Cells

It is clear that A2B5(+) cells were recruited in

purified cultures. It is also clear that neurons appeared

in purified cultures when grown on a nonselective

substratum. Initially, the small proportion of identifiable

neurons present before separation were depleted by 83% as

evident by NF immunoreactivity. Degeneration of purified

cells on polyornithine also suggests that neurons were

depleted because no cells survived. Presumably, they would

have grown if they were present in the purified cells. This

degeneration occurred even though the cells had become

A2B5(+). Thus, the presence of A2B5 antigen on a cell

surface does not in itself correlate with survival on

polyornithine. Microsphere fraction cells (A2B5(+)-

enriched), on the other hand, resulted in the visually most

dense network of neurons, which suggests that the majority

of neurons were A2B5(+).

When grown on a nonselective substratum such as glass

for = 1-2 weeks there appeared to be no decrease in the

density of A2B5(+) neurons or of neurofilaments in purified

cultures as compared to unpurified monolayers. If the

purified A2B5(-) cells were initially devoid of the

majority of neurons as is suggested above, then, neurons










62

must have come from preexisting nonneuronal cells to

exhibit the same density as in unpurified cultures (see 3H-

thymidine discussion below). The simplest explanation for

equal densities of neurons is that A2B5 expression on the

surfaces of cells is irrelevant to the development of

neurons in long-term monolayer cultures. A2B5 antigen may

have been modulated up and down on cell surfaces in both

unpurified and purified cultures. Then, the A2B5(+) neurons

at the culture endpoints may not have developed from the

A2B5(+) cells seen initially or after a day in culture. The

phenomenon of recruitment of A2B5(+) cells in purified

cultures may be separate from the appearance of neurons in

these cultures. No experiments were performed that could

conclusively demonstrate which of the possibilities had

occurred.

On the other hand, neurons as defined by morphology

and/ or neurofilament content were almost always surface

A2B5(+) (>90%) in long-term monolayer cultures. Conversely,

cells that expressed A2B5 antigen on their surfaces always

had a neuronal morphology in monolayer cultures.

Additionally, the presence of cells that were surface

A2B5(+) always preceded the development of neurons in long-

term cultures. A2B5(+) cells were present at the outset in

unpurified cultures, and were present after one day in

purified cultures. These correlations raise the possibility

that A2B5(+) neurons in long-term cultures developed from










63

the A2B5(+) cells that were seen after one day in culture.

The A2B5(+) neurons that developed in unpurified cultures

may have originated from the A2B5(+) cells that were

present when the tissue was dissociated (which were

presumably were the same cells that were A2B5(+) after a

day in culture). The fact that either no change or a slight

increase in the percentage of A2B5(+) cells occurred in

unpurified cells after one day in culture is consistent

with A2B5 antigen not being modulated in unpurified

cultures. Similarly, the A2B5(+) neurons that developed in

purified cultures may have originated from the recruited

A2B5(+) cells seen after a day in culture. If this

hypothesis were true, then, this would imply the existence

of a previously unidentified intermediate cell type in the

optic tectum neuronal lineage. This cell would have the

characteristics of being A2B5(+)/ NF-M(-) and would be

susceptible to degeneration on polyornithine.

The analysis of GS in culture revealed another manner

in which purified cultures were identical to unpurified

cultures. GS was produced in purified monolayer cultures in

an indistinguishable pattern from unpurified cultures.

Quantitatively, the expression of GS in the two types of

cultures was also the same. These results suggest several

possible explanations. One is that the purification did

not result in separation of neurons and glia. This has been

discussed above. Another is that a small number of neurons









64
may be able to induce production of GS in a certain number

of glia as well as a large number of neurons could. This

possibility is compounded by the fact that there are many

different types of neurons in the optic tectum (LaVail and

Cowan, 1971a) and it is not known which types are capable

of GS induction. Maybe the small percentage of A2B5(-)

neurons that were in the purified cells were the neurons

that induced GS. Another possible explanation is that

neurons were recruited in the neuron-depleted purified

cells in a rapid manner so that they could interact with

the glia to produce GS. This would have to have been within

about a day as was determined by the isolation of cells in

methyl cellulose. If the appearance of A2B5 antigen on cell

surfaces was an indication of commitment to becoming a

neuron as is suggested then the ability to induce GS may

also have occurred rapidly as did the expression of A2B5

antigen. However, this hypothetical change was not

sufficient to ensure survival of cells on polyornithine.

It is also worth mentioning that levels of GS produced

in aggregate cultures made from cells that were isolated in

methylcellulose were identical to levels produced by

immediately reaggregated dissociated cells. This is

surprising since it was shown that a much greater number

of A2B5(+) cells existed initially in cells from the

methylcellulose (=75%) than in immediately reaggregated

cells (=50%). So either the number of A2B5(+) cells has










65

nothing to do with the amount of GS produced or the proper

ratio of neurons to glia is somehow obtained. Although it

was shown that GS is produced in glia under clusters of

A2B5(+) neurons it could not have been determined whether

more GS activity was induced under large clusters than

under small clusters. The proper ratio of neurons to glia

could have been accomplished by division of the glia since

these cells have been shown to incorporate 3H-thymidine in

culture.

There appears to be an absolute correlation between

having cell surface A2B5 antigen and not synthesizing new

DNA in cultures of tectum cells. The lack of 3H-thymidine

labelled nuclei in neurons in cultures from unpurified day

7 or 8 cells was expected since other workers have found

that the majority of neurogenesis in the tissue has already

occurred by this time (LaVail and Cowan, 1971b; Puelles and

Bendala, 1978). The fact that no labelled neurons were

found suggests that all of the neurons that survived in

culture had completed their terminal S-phase by day 7.

Similarly, the lack of any labelled nuclei in recruited

A2B5(+) cells that appeared in purified cultures

demonstrated that new DNA synthesis was not required for

cell surface expression of A2B5 antigen. The antigen may

have been expressed via a mechanism as discussed in the

section above. The neurons that developed in these

cultures, likewise, did not require new DNA synthesis for










66
differentiation (i.e. the synthesis of neurofilaments).

This finding suggests that these neurons originated from

cells that were already born. This may have been from a

resting blast cell that was limited to the choices of

either differentiating into a neuron or dying.

Alternatively, these neurons may have originated from cells

that would have become glia had the need for more neurons

not occurred. This possibility would be exciting, since it

suggests that a change in phenotype or even cell type may

occur in post-mitotic cells if the neurons that developed

in purified cultures were recruited as is suggested.

Glial Development and Plasticity

Perhaps the most interesting question that the results

here pose is: From what population of cells were the

recruited neurons taken? Two theoretical possibilities

exist. The recruited cells either would have become

something else had there been no depletion, or they were

resting blast cells that normally would degenerate if not

needed. And since these cells were of neuroectodermal

origin, if they were not to become neurons then they were

to become glia. The results presented here do not support

one or the other possibility. Analysis of cell lineage in

the rat retina by using recombinant retroviral vectors has

shown that both neurons and glia are produced from common

progenitors throughout development (Turner and Cepko,

1987). However, results with purified cells from day 12 or









67

13 optic tectum suggest that the recruited cells in

cultures stem from the astrocyte lineage (Chapter 3). The

majority of the recruited A2B5(+) cells in these purified

cultures showed immunoreactivity for GS thus identifying

them as glia. There is also evidence in the chick

peripheral nervous system that certain glial precursors are

capable of being diverted to a neuronal lineage under

certain transplantation conditions (Le Lievre et al.,

1980).

The mechanism of recruitment is also unclear. One

possibility is that there exists either a negative feedback

system between A2B5(+) and A2B5(-) cells or a positive

feedback system between A2B5(-) cells only. With the

negative feedback system the A2B5(-) cells would have

recognized the loss of the A2B5(+) cells by some mechanism

and then reacted as a result of this. With the positive

feedback system the A2B5(-) cells would have sensed an

increase in the density of A2B5(-) cells in the purified

cultures and reacted to replenish A2B5(+) cells. The cell

isolation experiments, however, clearly rule out the latter

possibility since recruitment of A2B5(+) cells occurred as a

result of a loss of contact between all cells. What remains

in question then is whether the communication between

A2B5(+) and A2B5(-) cells is via cell contact or soluble

factors.









68
An interesting finding concerning glial development in

culture is the lack of appearance of oligodendrocytes in

culture. These results using an antibody for the marker

galactocerebroside (GC) essentially confirm the results of

Linser and Perkins (1987a) who failed to find cells

positive for the oligodendrocyte markers myelin basic

protein and S-100 (Linser, 1985). This is in contrast to

cultures made from day 12 or 13 tectum cells where GC(+)

cells are present and develop in vitro (Chapter III). Thus

it seems that either future cellular interactions were

disrupted that were required for oligodendrocytes to

develop, or that the culture conditions did not contain

some growth factors) that was required earlier in

development. It should be noted that these results are in

contrast to those with rat brain cells where

oligodendrocytes appear in cultures that do not contain

them initially (embryonic day 10) on time with those that

appear in vivo 13-14 days later (Abney, Bartlett, and Raff,

1981).














CHAPTER III
SEPARATION OF DAY 12-13 CELLS

Introduction

The previous chapter described an immunomagnetic

separation method to separate cells from early (day 7-8)

embryonic chick optic tectum. This method resulted in

extremely pure populations of cells that were negative for

the cell surface marker A2B5 (Eisenbarth et al., 1979). It

was found, however, that the A2B5 antigen was modulated on

the surfaces of about half of the purified cells in direct

response to the depletion of A2B5(+) cells. Neurons were

also apparently recruited in these cultures. Thus, day 7

and 8 optic tectum cells showed a remarkable ability to

maintain the correct number of A2B5(+) cells and neurons.

It was not clear, though, from what population of cells the

recruitment was occurring. This was largely due to the fact

that no glial differentiation markers which occur later in

development were present at days 7 and 8 to identify

definitive glia.

During development of the chick optic tectum, commonly

recognized glial differentiation markers do not appear

until relatively late in development (Linser and Perkins,

1987a). Glutamine synthetase (GS) is detectable by

immunohistochemistry in some astrocytes at day 9 and is

69









70

produced eventually in most if not all astrocytes. Glial

fibrillary acidic protein (GFAP) appears in a small

population of astrocytes beginning at day 16 of

development. The oligodendrocyte specific marker myelin

basic protein (MBP) appears on day 12 followed by S-100 on

day 16. This is in striking contrast to the neuronal

marker, neurofilaments, which appears beginning on day 3 of

development when neurons begin to exit the mitotic cycle

(Bennett and DiLullo, 1985). Unfortunately, however,

another widely used neuronal marker, neuron-specific

enolase, does not appear in chick brain until much later

(day 17) (Ledig et al., 1985).

The monoclonal antibody A2B5 which was used to

tentatively identify and remove neurons immunomagnetically

seemed at first to be a relatively stable marker with

freshly dissociated cells. It reacted with approximately

50% of dissociated cells from days 7-13. If it was assumed

that A2B5 was reacting with the same population of cells

throughout this range of ages, it would be interesting to

examine whether induced deficiencies of A2B5(+) cells and/

or neurons would be compensated for if day 12 or 13 A2B5(-)

cells were purified as they were with day 7 and 8 cells.

Since two known glial differentiation markers have appeared

in the tissue by this time (GS and GC), it might be

hypothesized that the capacity for neuronal "recruitment"

from other cells would be very limited. In several other










71

respects, as stated by LaVail and Cowan (1971a), age 12

tissue differs from earlier ages as follows: By this time

all mitosis in the neuroepithelium has ceased (Cowan et

al., 1968). The major 6 laminations of the tectum have been

arranged, and many cells have obtained their relative final

positions. Retinal axons by now have penetrated all parts

of the tectum, and by this age the superficial tectal

laminae are dependent upon retinal contact for survival

(Kelly and Cowan, 1972).

Therefore, I have immunomagnetically purified A2B5(-)

cells from day 12 and 13 cells for purposes of studying

their development in vitro as compared to unpurified cells.

A2B5 was compared to the differentiation markers present at

this time (NF and GS) as well as to the oligodendrocyte

marker galactocerebroside (GC) in freshly dissociated

cells. Monolayer cultures of purified and unpurified cells

were analyzed by immunohistochemistry for these markers and

for glial filaments (GFAP). 3H-thymidine autoradiography

was also performed on cells and monolayers to examine the

role of new DNA synthesis in recruitment and

differentiation. Reaggregation of cells to elicit GS

production as an indicator of neuronal-glial interaction

was not performed, since this age tissue is beyond the age

at which this can be done successfully (unpublished).

Similarly, the effects of polyornithine substrata on glial

degeneration (Adler et al., 1979) could not be utilized,










72
because glia can survive on this substratum by this age

(see Results).

I have found that glia which have been deprived of

neuronal contact can alter their immuno-phenotype

drastically. Day 12 and 13 tectum cells have a very limited

ability, if any, to replenish depleted neurons in vitro.

Instead, phenotypes were found in purified cultures that

were intermediate between, or showed characteristics of

both, neurons and glia. These were not found in unpurified

cultures and apparently represented reactions to depletion

of neurons. Taken with previous findings in Chapter II, the

inability to replenish neurons coincident with the

appearance of abnormal phenotypes suggest that the non-

neuronal cells in day 12 and 13 tissue gliaa) are

restricted in their potential to become neurons in vitro.

Materials and Methods

White Leghorn chick embryos were used throughout this

investigation. Fertile eggs were purchased from the

Division of Poultry Science, University of Florida, and

stored at 150C until initiation of incubation at 37.50C in

a humidified egg incubator. For this series of

investigations, 12 and 13 day embryonic optic tecta were

dissected as described in Chapter II.

To obtain sufficient quantities of dissociated cells

treatment of tissue with two different proteases was

required. Tissue was minced finely and then incubated in










73

5mg/ml neutral protease (Dispase; Boehringer Mannheim

Biochemicals, Indiannapolis, IN) for 1 hour with aggitation

followed by incubation in 0.4% Trypsin (Nutritional

Biochemicals, Cleveland, Ohio) for 30 min., both at 370C.

Dissociation of tissue into single cells was as described

in Chapter II.

Adherent monolayer cultures were prepared on glass or

polyornithine as described in the previous chapter.

Briefly, 106 cells/ well were plated on coverslips in 24

well tissue culture plates in Medium 199 supplemented with

10% fetal bovine serum. Monolayer cultures were kept in a

standard tissue culture incubator in a 5% C02/ air

atmosphere. Cultures were fed with fresh medium

approximately every other day.

Cell "isolation" in methylcellulose was accomplished

by suspending dissociated cells in a semisolid medium of

1.3% methylcellulose in Medium 199 according to the

previous chapter.

Immunomaanetic Separations

Immunomagnetic separations were carried out exactly as

described previously and so will not be described further

here. Cells assessed for cell surface A2B5

immunofluorescently following a separation were fixed with

1% formaldehyde in phosphate-buffered saline (PBS) for 30

min., rinsed, then incubated for 30 min. in a 1/50 dilution

of fluorescein-goat anti-mouse IgM (FITC-GAM; Boehringer










74

Mannheim) in PBS with 5% normal goat serum added. Cells

assayed after one day in culture were incubated live in

1/25 A2B5-conditioned hybridoma medium + 10% heat-

inactivated fetal bovine serum for 30 min. on ice and then

processed as above. Plating efficiencies of cells were

determined retrospectively from Ektachrome slides by

counting the number of cells that adhered to the

polyornithine coated coverslips initially after the

experiment and after 1 day in culture. No attempt was made

to determine cell numbers or densities in longer-term

cultures.

Immunoradiometric Assay for A2B5

An immunoradiometric assay (Hunter, 1978) was used to

quantitate cell surface binding of A2B5 both immediately

after the separation and after 1 day in culture. For this,

25"I-labelled antibodies were prepared by the lodo-Gen

method (Pierce Chemical Co., Rockford, IL) according to the

manufacturer's directions using polyclonal goat anti-mouse

IgG +IgM + IgA (Organon Teknika- Cappell, West Chester,

PA). Cells assayed for cell surface A2B5 were processed

exactly as those for the immunofluorescent assay, with the

iodinated secondary antibody in place of the fluorescent

secondary antibody. Processed coverslips were placed in

vials and bound radioactivity was measured with a Beckman

Gamma 7000 (Beckman Instruments, Norcross, GA). Specific

DPMs were obtained by subtracting mean nonspecific DPMs










75
from total mean DPMs of triplicate or quadruplicate

coverslips. Nonspecific background DPMs were obtained by

incubating unpurified cells in an irrelevant monoclonal

antibody specific for pipefish vitelline envelope (provided

by Dr. P.C.Begovac, Whitney Laboratory) for 30 min. on ice

and then processing them identically as those incubated in

A2B5.

Immunohistochemistry

All immunohistochemistry of cells and monolayer

cultures was performed as described in the previous chapter

except as noted below. 5A11 (Linser and Perkins, 1987b)

immunohistochemistry of monolayers was carried out

identically to that for A2B5. Immunostaining of cells for

glutamine synthetase was accomplished by permeablization

after formaldehyde fixing with 95% ethanol for 2 min. at

-200C, rinsing, and incubation in a 1/100 dilution of

polyclonal anti-GS (Linser and Moscona, 1979) for 30 min.

Other NF antibodies rasied against rat neurofilaments and a

monoclonal antibody specific for GFAP (DA3, NN18, anti-MSH,

A5; kindly provided by Dr. G. Shaw, Univ. of Florida) were

also used to immunostain monolayers.

3H-Thvmidine Incorporation

New DNA synthesis in cell cultures was investigated by

3H-thymidine autoradiography combined with immunohisto-

chemistry as described in the previous chapter.









76

Results

The dissociation protocol used for younger tissue

(Chapter II) did not produce satisfactory numbers of single

cells when using older tissue (day 12 and 13). Therefore,

the double protease treatment was adopted. This

still resulted in only approximately 5x106 cells/ embryo.

The double protease treatment was used only because a

greater number of viable cells could be obtained for

experiments and the results herein are not believed to

reflect an effect caused by the tandem proteases (see

Discussion).

Markers for Dissociated Cells

Unless otherwise specifically stated, the reference as

to whether a cell is A2B5(+) or (-) refers to cell surface

binding of this antibody only. With dissociated day 12 and

13 optic tectum cells, discrete populations were labelled

by antibodies against two recognized glial antigens and one

neuronal antigen, as well as with A2B5 (Figs.3-1,3-2).

Approximately 10% of dissociated cells were reactive with

the polyclonal antibody against neurofilaments. The

majority of these were A2B5(+). It was more difficult to

attempt to quantitate this because, unlike younger cells,

there was an absence of reactive filaments in the cell

cytoplasm around the nucleus. NF-M(+) (Bennett et al.,

1984) cells were less distinct and mostly showed

immunoreactivity in what was apparently membrane blebs of



























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82

retracted or severed axons on the surfaces of the cells.

Therefore, the most accurate count of NF-M(+) cells was

obtained with the day 7 and 8 cells described in the

previous chapter. Even this data may be an underestimate of

the number of NF-M(+) cells for the above reasons.

Polyclonal antibodies specific for the definitive

glial marker GS reacted with approximately 40% of

dissociated day 12 and 13 cells. These cells were A2B5(-)

and so the purified population of A2B5(-) cells (see

analysis of purification below) was approximately 80% GS(+)

(Figs.3-1,3-2). Thus, the astrocytes that were identifiable

as such by immunoreactivity with anti-GS were concentrated

in the purified population.

Dissociated cells at this time also were reactive with

a monoclonal antibody specific for the oligodendrocyte

marker galactocerebroside (GC; Ranscht et al., 1982).

Approximately 6% of dissociated cells were GC(+). Similar

to the analysis of GS, all of these cells were A2B5(-) and

so the purified A2B5(-) cells were 12% GC(+) (Figs.3-1,3-

2). Taken with the results for GS, >90% of the purified

cells could be identified as glia by these two

differentiation markers. A small population of the purified

cells (=4%) could be identified as neurons that contained

neurofilaments.











Effects of Substrata

As in the proceeding chapter, both unpurified and

purified A2B5(-) cells were cultured on substrata of either

glass or polyornithine. It was found that although there

were slight differences in the development of monolayers

with regards to the extent of cell aggregation before

spreading, polyornithine was not found to be selective for

neurons as it was with day 7 and 8 cells. No cell phenotype

that was observed to develop on glass was absent from

monolayers on polyornithine. Thus, the degeneration of

glial precursors on polyornithine was not manifested in

day 12 and 13 cells.

A2B5 Antigen Modulation

By the immunofluorescent assay described here and in

the preceedind chapter, the immunomagnetic separations

resulted in extremely purified populations of A2B5(-)

cells. Unpurified cells were approximately 45% A2B5(+)

(Fig.3-3). Purified cells were virtually free of A2B5(+)

cells, with a typical purity of >99.99% A2B5(-). After one

day in vitro, however, large numbers of A2B5(+) cells

(=80%) were present in the cultures of initially purified

cells (Fig.3-3). No increase in A2B5(+) cells was observed

in cultures made from unpurified cells. Thus, A2B5 antigen

is apparently modulated on the surfaces of the majority of

A2B5(-) cells in purified cultures, but not on the surfaces

of A2B5(-) cells in unpurified cultures. Plating

























Fig.3-3. Analysis of initial purification of A2B5(-) cells
and A2B5 antigen modulation on purified cell surfaces by
immunofluorescence (a) and immunoradiometric (b) assays.
The immunofluorescent assay (a) indicated that the purified
population of cells was initially devoid of A2B5(+) cells.
After 24 hours in culture, however, the purified population
was =80% A2B5(+). The unpurified population did not show
any increase in A2B5(+) cells. The immunoradiometric assays
(b) confirmed that the purified cells were devoid of cell
surface A2B5 antigen initially (left graph). After one day
in culture the purified cells expressed levels of A2B5
antigen approaching those of unpurified cells (right
graph). Shown in all graphs are means s.e.m. DPMs are
higher in the +1 day immunoradiometric assay due to use of
125I-labelled antibody with 3x the specific activity.














E Day 0
M+1 Day


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Unpurified Purified



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6000-
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3000-
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+1 DAY
IRA









86
efficiencies of unpurified and purified cells after a day

in culture were nearly identical and ranged from

approximately 60-90% as determined by retrospective counts

from slides.

To be certain that the observation of the modulation

of A2B5 antigen by immunofluorescence was accurate,

immunoradiometric assays were performed to quantitate cell

surface A2B5 antigen. Assays identical to the

immunofluorescent ones were performed using an iodinated

secondary antibody. The results of this assay are shown in

Fig.3-3 and confirm the immunofluorescent results of

depletion of A2B5(+) cells in purified populations. This

assay also established that the immunofluorescent assay was

a sensitive and satisfactory method for assessing the

presence or absence of A2B5(+) cells. Similarly, the

immunoradiometric assay confirmed the presence of near

control levels of A2B5 antigen on the surfaces of purified

cells after one day in vitro (Fig.3-3). There was no

quantitative increase in the levels of cell surface A2B5

antigen in unpurified cultures after one day.

Similar to the results with day 7 and 8 cells, an

increase in the number of A2B5(+) cells (=75%) was observed

after dissociated cells were kept suspended in isolalation

in a semisolid medium containing methylcellulose for 24

hours.









87
Development of A2B5(+) Cells In Vitro

Although up to this point the phenomenon of appearance

of A2B5(+) cells in purified cultures of day 12 and 13

cells appeared similar to recruitment in purified day 7 and

8 cells, the appearance of A2B5(+) cells in long-term

monolayers differed markedly. A2B5(+) cells that appeared

in purified cultures for the most part did not have a

neuronal morphology. They exhibited a flattened glial

morphology (Fig.3-4) after about a week in culture and

rested atop the A2B5(-) flat cells and were then round

after several more days or when the cultures had become

confluent. The appearance of these A2B5(+) cells with

nonneuronal morhpology in purified cultures was not

prevented by adding back the cells that were removed by the

microspheres (data not shown). A2B5(+) cells with

nonneuronal morphology also appeared in cultures made from

dissociated cells that were kept in methylcellulose for a

day (Fig.3-4). This was in contrast to the appearance of

A2B5(+) cells in unpurified monolayer cultures. These cells

exhibited a purely neuronal morphology in vitro and not the

nonneuronal morphologies observed in the purified cultures.

Nonneuronal A2B5(+) cells were also absent from cultures

made from cells recovered from the microspheres and this

fraction appeared to contain the largest and most numerous

aggregates of neurons (data not shown). Some A2B5(+) cells

with neuronal morphology were observed in purified























Fig.3-4. In vitro development of A2B5(+) cells in monolayer
cultures made from unpurified (a-c) and purified (d-f)
cells. Shown are phase/ fluorescent pairs with the
antibody staining shown on the right. A2B5(+) cells in
unpurified cultures appeared solely as neurons after about
a week (a) or 2 weeks (b) in vitro. In these cultures, the
antibody 5All reacted only with the flat A2B5(-) glia (c).
In purified cultures, the A2B5(+) cells appeared
predominantly with a flat glial morphology after about a
week in culture (d) and were rounded up after several more
days or when the monolayers approached confluency (e). Some
A2B5(+) neurons with processes also developed in these
cultures. The A2B5(+) nonneuronal cells reacted with 5All
(f, arrowheads) revealing a partial glial phenotype. The
micrograph pair in (g) shows several A2B5(+) nonneuronal
cells in a monolayer culture that was made from unpurified
cells that were suspended in methyl cellulose medium for 24
hours prior to plating. Thus these cells can be generated
in culture by 2 different isolation procedures. Bar, 50m.








89











a d6











CAW.R










90

cultures. These may have been the small number of

NF(+)/A2B5(-) neurons that were detected in the initially

purified cells (see above). The A2B5(+) cells in purified

(long-term) monolayer cultures with nonneuronal morphology

reacted with the monoclonal antibody 5A11. This antibody is

known to be specific for glia in retina (Linser and

Perkins, 1987b) and similarly reacted only with the

surfaces of A2B5(-) glia in the cultures of unpurified

optic tectum cells (Fig.3-4). Purified monolayer cultures

contained many 5All(+) round cells. These were presumably

the A2B5(+) round cells seen in duplicate cultures, but

double-label analysis could not be performed because both

A2B5 and 5All are an IgM. Thus the A2B5(+) cells in

purified cultures not only developed a nonneuronal

morphology in culture but expressed a surface antigen

(5All) that normally seems to be restricted to glia.

Intermediate Filament Expression In Vitro

Two types of intermediate filaments were investigated

in cultures of unpurified and purified optic tectum cells.

Glial filament expression was examined with both a

commercial polyclonal antibody against bovine glial

fibrillary acidic protein (GFAP) and monoclonal A5 (Debus

et al., 1983). A5 did not react with anything in our

cultures and so the following results are from using the

commercial polyclonal antibody. In unpurified monolayer

cultures GFAP reactivity was confined to the A2B5(-) flat