Precursor cells of oligodendrocytes in rat primary cultures

DEVELOPMENTAL

BIOLOGY

108,474-480

(1985)

Precursor Cells of Oligodendrocytes in Rat Primary Cultures A. ESPINOSA

DE LOS MONTEROS,

G. ROUSSEL,

Centre de Neurochimie

du

C. GENSBURGER,

J. L. NUSSBAUM,

CNRS, 5, rue Blaise Pascal 67084 Strasbourg

Received May 2, 1984 accepted in revised

fomn

November

AND G. LABOURDETTE

Cedex, France 16, 196.b

In monolayer primary cultures of brain from newborn rats, which contain astrocytes and oligodendrocytes, a new morphological cell type (flat black cells) was observed. Microphotographs of different areas of the monolayer, taken every 30 min, showed that these flat black cells can divide and that they undergo morphological transformation in vitro. They give rise to oligodendrocytes which were identified by their characteristic morphology but also by their content

of Wl

in culture.

Wolfgram

0 1985 Academic

protein.

These

findings

suggest

that

INTRODUCTION

Monolayer cultures from brain of newborn rats enriched in oligodendrocytes have been recently obtained using different experimental approaches (Labourdette et al, 1980; Meier et aL, 1982; McCarthy and De Vellis, 1980). Previous investigations have shown that the number of oligodendrocytes in culture largely depends on the composition of the culture medium (Labourdette et aL, 19’79; Raff et al, 1983) and on the initial plating density and that their proliferation can be stimulated by addition of brain extract (Roussel et al., 1983). The fundamental knowledge brought about by these cultures is that oligodendrocytes grown in the absence of neurons, and hence which do not myelinate axons, are able to synthesize myelin components. Presence of myelin-specific proteins was visualized using polyclonal antibodies (Roussel et uL, 1981); myelin-related enzyme activities were determined (Labourdette et al., 1979), and synthesis of specific glycolipids was demonstrated by the incorporation of precursors (Bhat et uL, 1981). The other important finding was that differentiated oligodendrocytes which synthesize myelin components like Wl Wolfgram protein (Roussel et ah, 1983), galactocerebrosides, and even myelin basic proteins (Bologa et uL, 1983) are still able to proliferate in vitro. Thus such cultures allow studies on the maturation and the proliferation of oligodendrocytes and on the relationship between these two essential phenomenons. In this paper we bring further evidence that a differentiation process is also occurring in primary culture. METHODS

Cultures were prepared from brain of newborn rats as described previously (Labourdette et uL, 1979). The 001%X06/85

$3.00

Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

the flat black

cells are precursors

for oligodendrocytes,

Press, Inc.

474

cuiture medium is composed of Waymouth MD 705/l supplemented with pyruvic acid (110 mg/liter), penicillin (50 u/ml), streptomycin (50 pg/ml), and 10% heat-inactivated calf serum. The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% COz, and the medium was changed every fifth day when they were maintained over a long period. For the photographic study, the flasks were taken out from the incubator at Day 9 and set under a phasecontrast microscope, located in a 37°C room. The cultures were first examined in order to select several areas containing a high proportion of putative precursor cells. The choosen areas were marked with a diamond mounted on the objective and numbered. Photographs were taken every 30 min for 29 hr. Oligodendrocytes were then identified as described previously with antiWl protein serum (Roussel and Nussbaum, 1981). For that purpose the cells were dried in a cold air stream, and fixed with ethanol/PBS (phosphate-buffered saline) and acetone at -70°C. They were then rinsed with PBS and additional photographs were taken to compare the same areas before and after fixation, since we observed that during the fixation process some cells, particularly oligodendroglial cells, are lost. Cells were incubated with a loo-fold dilution of the antiserum in PBS containing 5% normal sheep serum for 30 min. After exhaustive washing of the excess of anti-WI protein serum, the corresponding antigens were visualized by the use of a loo-fold dilution of FITC-labeled sheep (anti-rabbit IgG) antibodies (IPP). The flask was then washed several times with PBS and mounted in buffered glycerol. Microscopical examination was done with a Leitz Orthoplan microscope equipped with phasecontrast and fluorescence optics. After observation, the same areas were incubated successively with a 500-fold dilution in PBS of a mouse monoclonal GFAP antibody (Amersham) and a 50-fold

ESPINOSA

DE LOS MONTEROS

dilution in PBS of an anti-mouse y-globulin preparation coupled with Red Texas (Amersham). areas were viewed as described above.

ET AL.

sheep These

RESULTS

In previous investigations on primary cell cultures from newborn rat brain grown in Waymouth medium supplemented with 10% calf serum, we observed the appearance of Wolfgram Wl protein positive cells around the eighth day after plating (Roussel et aZ., 1981). The specificity of this antiserum supports their oligodendroglial nature. These cells in their majority are small, round, refringent, and they lay above the astrocyte layer. Some of these cells sometimes appear darker, but still are oligodendrocytes on the basis of their positive immunological reaction to Wl. In the course of these studies, two observations were done. First on Day 7 or 8 after seeding the proportion of refringent cells is very low in the culture but on Day 10 or 11 it increases notably (Fig. 1). A very high proliferation rate seemed unable to explain such an increase of the number of refringent cells. The second observation was that in areas where the oligodendrocytes were numerous, flat cells looking black in phase contrast were always present. In order to get more

Oligodendroq&e

Precursors

in

Culture

475

relevant information about these phenomenons, we decided to take photographs of the same areas at regular intervals of time. In preliminary experiments we found that to follow accurately the behavior of one cell the largest delay between two photographs had to be around 30 min. For the experiment reported six areas were selected. Figure 2 shows a montage of photographs of an area from a g-day-old culture taken over a 29-hr period. Five cells which were characterized as oligodendrocytes by their positive reaction to anti-W1 serum at the end of the experiment were not refringent at the beginning of the experiment. It was found that they arose from the morphological transformation of flat black cells. Careful examination permits to note the slow and continuous evolution of the flat black cells toward a more round-shaped cell state, becoming less dark after some hours. Thereafter, the cells become more and more lucent with a halo around them until they are refringent. Each cell evolves independently; this is clearly shown in Fig. 2 where the five cells do not become refringent at the same time and show a more or less strong immunofluorescence after immunoreaction with the anti-W1 serum. Moreover apparent transient reversion can occur in rare cases; i.e., the cell

FIG. 1. Primary cultures from brain of newborn rats grown in Waymouth medium supplemented with 10% calf serum. (a) At 7 DIV the astrocytic layer is still incomplete. Note the presence of numerous polygonal flat black cells above the astrocytes, most of them bearing expansions. A few round refringent cells are visible. (b) At 10 DIV, the astrocytic layer is complete, some black cells are still present; there are much more small round refringent oligodendroglial cells. Note the presence of a few dark oligodendrocytes (arrows). Phase contrast x300.

FIG. 2. Sequential pictures showing the transformtion of the flat black cells into typical round cells positive to Wl Wolfgram protein (oligodendrocytes). Pictures were taken from 9 DIV every 30 min. Delays shown are: (a) time 0; (b) 2 hr; (c) 5 hr; (d) 6 hr; (e) 8 hr; (f) 11 hr; (g) 13 hr; (h) 14 hr; (i) 15.5 hr; (j) 17.5 hr; (k) 20 hr; (1) 21.5 hr; (m) 23.5 hr; (n) 27.5 hr; (0) 28.5 hr; (p) 29 hr; (q) was taken after fixation, and (r) after immunoreaction with anti-W1 serum. X210. 476

ESPINOSA

DE LOS MONTEROS

ET AL.

Oligodendrocyte

FIG. 3. Double immunolabeling with a rabbit anti-W1 serum and a monoclonal fixation (b); staining with anti-W1 serum (c) and with monoclonal GFAP antibody most black cells are Wl- and GFAP-. Note one Wl-, GFAP+ black cell (large arrow) arrow). X400.

Precursors

in Culture

477

GFAP antibody. Phase contrast before (a) and after (d). Most round refringent cells are Wl+ and GFAP-, and one Wl+ (slightly labeled), GFAP+ dark cell (thin

478

DEVELOPMENTAL BIOLOGY

present in the square appears bright on Fig. 2k, but looks dark in Fig. 2p 2.5 hr later; at this time this cell has a small cell body and shows multiple long branched processes typical for oligodendrocytes. Finally, after immunoreaction, this cell is unambiguously positively labeled by the anti-W1 antiserum. Confusion with the original precursor flat black cell like those present in great number in Fig. la is not possible. The culture was also treated with a monoclonal GFAP antibody: most astroblasts were labeled as previously described (Roussel et al, 1981). On the area shown, one cell looking dark in phase contrast (Fig. 3) was positive and its prolongations were very fluorescent. Its morphology, with numerous and long processes contrasts with that of the other black cells precursors of oligodendrocytes. This cell could be a fibrous astrocyte. Another GFAP-positive dark cell was faintly labeled with anti-W1 serum. Several round refringent cells were positive to anti-W1 serum but did not contain GFAP. About 300 flat black cells were counted in the six areas at the beginning of the experiment (Table 1); from these 141 could be followed up to the end. The others could not be followed accurately for various reasons, i.e., cells moving out of the field, dead cells occulting the cells under investigation. From the 141 cells, ‘76 became refringent and were characterized as oligodendrocytes. The other 65 flat black cells remained black; from these, 55 were seen dividing once, 3 divided twice, and only 10 cells looked unchanged. To divide the cells first round up and then they give rise to two daughter cells which rapidly become black as their parent cell (Fig. 4). TABLE 1 BEHAVIOROFTHE FLATBLACK CELLS DURING THE 29-HR EXPERIMENT(DIV 9 ~010) Flat black cells Followed up to the end

Becoming round and refringent

Cell number

Percentage

Total number

141

100

Total number

76

54

Wl positive (oligodendrocytes)

45

32

31

22

Total number

65

46

Dividing once Dividing twice i Not dividing

52 3 10

37 2 7

Features

i Wl negative

Remaining flat and black

Note. In the six areas of the culture which were photographed, 304 black cells were present at the beginning of the experiment but only 141 could be followed up to the end.

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For the flat black cells which turned into round refringent cells, an average of 5-10 hr was required to achieve this morphological transformation. Concerning the time necessary for a newly appeared refringent cell to express the Wl protein at a detectable level, two observations were done; 3 out of 45 cells mentioned above which appeared 2 hr before the end of the experiment were already immunoreactive, but some round refringent cells present at the beginning of the experiment were still nonimmunofluorescent at the end of the experiment. Thus the delay for a round refringent cell to express Wl protein can be short but is highly variable from one cell to another. DISCUSSION

In a previous paper, we have described conditions to obtain primary cultures from brains of newborn rats which contain a high proportion of oligodendroglial cells (Labourdette et a& 1979). Wl-positive oligodendrocytes are found around the eighth day after plating; their maturation continues during the following days and can be immunologically demonstrated by their immunoreactivity toward anti-myelin basic proteins and anti-myelin proteolipids sera (unpublished data). The number of oligodendrocytes increases over a 3week period and their proliferation rate seems largely influenced by the culture conditions and by addition of brain extract (Roussel et ah, 1983). A time-dependent biochemical transformation has also been noted in embryonic mouse brain cell culture by others, although the delays were different (Bologa et aa, 1983). Oligodendrocytes are easily identified by their typical morphology. During a previous study flat dark cells were observed among the oligodendrocytes (Roussel and Nussbaum, 1982). They did not react with anti-W1 protein serum and only some of them were GFAP positive. We observed that these flat black cells were already numerous in 6- to 7-day-old cultures, when oligodendrocytes were still very low in number. In the present study we demonstrate that flat dark cells are immediate precursors of oligodendrocytes by the direct visualization of their morphological transformation into typical oligodendrocytes. Some flat black cells seem to evolve more or less rapidly but all cells evolve on the same schedule. The cells remaining flat and black proliferate actively; during the 29 hr of the experiment most of them divided at least once. However the number of these cells did not vary much since around half of them became oligodendrocytes. Therefore this resulted in an increase of the total number of oligodendrocytes.

ESPINOSA DE LOS MONTEROS ET AL.

Oligodendrocyte

Precursors

FIG. 4. Sequential pictures showing the division of a flat black cell (large arrow), arrows); (a) time 0; (b) 1 hr; (c) 2 hr; (d) 2.5 hr. Phase contrast X420.

An important question is to know if the black cells are glioblasts which are still able to differentiate into either oligodendroblasts or astroblasts, or if they are already committed to become oligodendrocytes. In this experiment we did not see a clear evidence of a black cell giving an astroblast although some cells looking dark in phase contrast contained GFAP and resembled fibrous astrocytes. Transient presence of GFAP in oligodendrocytes in vivo has been demonstrated (Choi and Kim, 1984); the same phenomenon could also take place in culture and cannot therefore be used as an indication of the undifferentiated glioblast nature of these cells.

in Culture

giving rise to two daughter

479

flat black cells (small

Indirect evidence was brought by Raff et al. (1983) that actual glioblasts would be present in cultures from optic nerve. In a very recent paper (Meller and Waelsch, 1984) black cells precursors of oligodendrocytes were described. However, these cells look different from ours and it was not demonstrated that these cells give rise to characteristic oligodendrocytes synthesizing specific markers. In vivo, in the newborn animal there are nervous precursor cells (De Vitry et aL, 1980) and in particular glioblasts which will give rise to astroblasts and oligodendroblasts (Skoff et al., 1976). In a culture from such newborn animals usually mainly astroblasts sur-

480

DEVELOPMENTAL BIOLOGY

vive and grow in vitro. However, in our culture conditions oligodendroblasts or possibly glioblasts could survive and proliferate. This occurrence would provide a good system to study in details the different steps and the parameters of gliogenesis from the glioblasts up to the differentiated oligodendrocytes and astrocytes. REFERENCES BHAT, S., BARBARESE, E., and PFEIFFER, S. E. (1981). Requirement for nonoligodendrocyte cell signals for enhanced myelinogenic gene expression in long-term cultures of purified rat oligodendrocytes. Proc. Nat1 Acad Sci USA 78, X83-1287. BOLOGA, L., BISCONTE, J. C., JOUBERT, R., MARGULES, S., and HERSCHKOWITZ, N. (1983). Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures. Ezp. Brain Res. 50, 84-90. CHOI, B. H., and KIM, R. C. (1984). Expression of glial fibrillary acidic protein in immature oligodendroglia. Science (Washington, D. C.) 223, 407-409. DE VITRY, F., PICART, R., JACQUE, C., LEGAULT, L., DUPOUEY, P., and TIXIER-VIDAL, A. (1980). Presumptive common precursor for neuronal and glial cell lineages in mouse hypothalamus. Proc Natl. Acud Sci. USA 77,4165-4169. LABOURDETTE, G., ROUSSEL, G., GHANDOUR, M. S., and NUSSBAUM, J. L. (1979). Cultures from rat brain hemispheres enriched in oligodendrocyte-like cells. Brain Res. 179,199-203. LABOURDETTE, G., ROUSSEL, G., and NUSSBAUM, J. L. (1980). Oligodendroglia content of glial cell primary cultures from newborn rat

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brain hemispheres depends on the initial plating density. Neurosd Leti 18.203-209. MCCARTHY, K. D., and DE VELLIS, J. (1980). Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. CeU Bid 85.890-902. MEIER, D. H., LAGENA~R, C., and SCHACHNER, M. (1982). Immunoselection of oligodendrocytes by magnetic beads. I. Determination of antibody coupling parameters and cell binding conditions. J. Neurosci+ Res. 7.119-134. MELLER, K., and WAELSCH, M. (1984). Cyclic morphological changes of glial cells in long-term cultures of rat brain. J. Neurocytol. 13, 29-47. RAFF, M. C., MILLER, R. H., and NOBLE, M. (1983). A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature (Lmdm) 303,390-396. ROUSSEL, G., LABOURDETPE, G., and NUSSBAUM, J. L. (1981). Characterization of oligodendrocytes in primary cultures from brain hemispheres of newborn rats. Den Biol 81,372-378. ROUSSEL, G., and NUSSBAUM, J. L. (1981). Comparative localization of Wolfgram Wl and myelin basic proteins in the rat brain during ontogenesis. Histochem. J. 13,1029-1047. ROUSSEL, G., and NUSSBAUM, J. L. (1982). Surface labelling of oligodendrocytes with anti-myelin serum in cell cultures from the rat brain. Light- and electron-microscopic immunocytochemical studies. Cell Tissue Res. 225,581-594. ROUSSEL, G., SENSENBRENNER,M., LABOURDE‘ITE, G., WITTENDORPRECHENYANN, E., PETTMANN, B., and NUSSBAUM, J. L. (1983). An immunohistochemical study of two myelin-specific proteins in enriched oligodendroglial cell cultures combined with an autoradiographic investigation using 3H thymidine. Dev. Brain Res. 8, 193-204. SKOFF, R. P., PRICE, D. L., and STOCKS,A. (1976). Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation. J. Comp. NeuroL 169,291-312.