Brain Research, 150 (1978) 225-238 © Elsevier/North-HollandBiomedicalPress

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Research Reports

INDUCED GLIAL DIFFERENTIATION OF FETAL RAT BRAIN CELLS IN CULTURE: AN ULTRASTRUCTURAL STUDY A. HAUGEN and O. D. LLERUM

The Gade Institute, Department of Pathology, University of Bergen, 5016 Bergen (Norway)

(Accepted November 10th, 1977)

SUMMARY Primary and secondary cultures of fetal rat brain ceils (FBC) from 18th day of gestation have been investigated by scanning and transmission electron microscopy. Primary cultures consisted of a monolayer of fiat, undifferentiated epithelioid cells, with some oligodendrocytes, astrocytes and immature neuronal cells. In secondary cultures, cells with glia morphology disappeared. Following addition of extracts from adult rat brains to secondary cultures, a dramatic change of the epithelioid cells took place. They detached from the plastic surface, extruded long cytoplasmic processes with numerous microvilli and cytoplasmic blebs as well as parallel arrays of microtubules and filaments. The differentiated cells resembled astrocytes, and characteristic glia filaments were also observed. An increase of ribosomes and rough endoplasmatic reticulum suggested enhancement of protein synthesis. At the same time S-100 protein and glial fibrillary acidic protein accumulated within the cells. The morphological changes were mostly reversible within 48 h of removal of the brain extract.

INTRODUCTION The development of the fetal rat brain in the last third of pregnancy is dominated by the outgrowth and differentiation ofglia cells from the subependymal layer1. Since direct in vivo studies of differentiation of the various types of brain cells in fetal life is technically difficult, many workers have attempted to get around this problem by use of cell cultures. By different procedures glia cells and neurons from fetal brains may be kept in tissue cultures for a limited period of time, although a large number of undifferentiated epithelioid cells may dominate the cultures in later passages4,S,30,al,38. There are, however, several ways to restart differentiation of the cells, e.g. as recently shown by Lim and Mitsunobu 16. They found in the adult brain a substance of high molecular weight, which was capable of inducing astrocyte morphology of fetal brain

226 cells in cultures within a short period of time. In this paper we have investigated u[tr:lstructural aspects of fetal brain cells in culture during the initial stages after induction ~t" morphological differentiation. This has been done as part of a larger study of chemicz,1 carcinogenesis in fetal brain cellsZ°,21. MATERIALAND METHODS

Cell cultures Brains dissected from 18th-day BD IX-fetuses6 were placed in a Petri dish containing medium (see below) and cleaned of any adhering tissue, cut into small pieces and dissociated with 0.25 ~o trypsin. The fetal brain cells (FBC) were cultured in tissue culture dishes (Costar, 205, Broadway, Cambridge, Mass.) in Dulbecco's modified Eagle medium (Flow, Glasgow, Scotland) supplemented with 10~ calf serum and penicillin (50 U/ml) and streptomycin (50 #g/ml). The cultures were kept at 37 °C in a humidified atmosphere of 5 ~ COz in air. The medium was changed twice a week, and subcultures were made with 0.25 ~ trypsin and 0.05 ~ EDTA. Second passage ceils were plated into culture dishes containing glass coverslips, and a monolayer of flat epithelioid cells was obtained for electron microscopic studies. Living and fixed cultures were observed and photographed through phase optics under a Leitz inverted microscope using Kodak panatomic-X film. Brain extract (BE) Whole brains from 6-8-month-old BD IX-rats were washed with Tyrode's solution, minced and homogenized at 1 °C with a Sorvall homogenizer. The homogenate was then spun at 100,000 x g for 1 h, and dialyzed 5 days in TyrodO 6. The supernatant fraction was divided into small portions, frozen and stored at --70 '~C. The brain extract (BE) was added to a final concentration of 25-30 % of medium. The control cultures received an equivalent volume of Tyrode's solution. Processing of cells for transmission (TEM) and scanning electron microscopy (SEM) TEM. Cultures were fixed during 1 h in 2 ~ glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 37 °C. They were postfixed for 30 rain in 1 °/oOsO4 in 0.1 Mcacodylate buffer (pH 7.4) and serially dehydrated in ethanol. In situ embedding in Epon 812 was performed by using graded additions of Epon-ethanol mixtures. The final polymerization was carried out at 40 °C. The Epon was then separated from the plastic dish by torsional bending of the dish. The cell monolayer remained in the Epon layer and could be viewed by placing the Epon sheet cell-side-up under the phase contrast microscope. Cells to be sectioned for electron microscopy were marked by scoring a circle around them. The scored area was cut out with a saw. This in situ embedding method allowed us to section the cells in controlled manner parallel or side (perpendicular) to the substratum. Sections were cut on a Reichert ultratome Om U3, and they were examined in a Philips EM 300, double-stained with uranyl acetate and lead citrate. SEM. The cultures were fixed in the same manner as for TEM. After dehydration through graded water-alcohol series to 100 ~ ethanol, the preparation was critical point dried with CO~, according to the technique described by Anderson ~. The cover-

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Fig. 1. Scanning electron micrograph of primary.FBC at:day 10 of culture showing oligodendrocyte" like cells on an underlayer of flat epithelioid ceils, x 1700.

Fig. 2. Electron micrograph of oligodendrocytes in primary culture, day 10. x 9800.

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Fig. 3. SEM picture of epithelioid cells in secondary FBC culture. No blebs and only occasional microvilli are observed. The margins of a cell are indicated with arrows. N, nucleolus. :, 2100. slips were m o u n t e d on stubs with silver-conducting p a i n t and coated in a v a c u u m evap o r a t o r with gold. T h e y were e x a m i n e d in a Philips S E M 500 microscope. M i c r o g r a p h s were o b t a i n e d o n K o d a k T r i - X - p a n p r o f e s s i o n a l film. T h e sequence o f changes in p r i m a r y a n d s e c o n d a r y cultures o f F B C , as well as after a d d i t i o n o f b r a i n extract, was studied in a total o f 10 separate experiments, each with litter o f 6-8 fetuses. A f t e r e x p l a n t a t i o n the cells were allowed to adhere to the plastic dishes a n d to grow out to form a m o n o l a y e r , which usually t o o k a b o u t one week. The first passage was p e r f o r m e d on the average at 14 days. E a c h e x p e r i m e n t c o m p r i s e d 6-10 parallel dishes. RESULTS

Primary cultures The m o n o l a y e r consisted o f flat, epithetioid cells. O n this layer a varying n u m b e r o f the cells with n u m e r o u s c y t o p l a s m i c processes adhered. The surface o f the cells was

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Fig. 4. Closely packed bundles of filaments in secondary epithelioid cellssectioned horizontally near the anchored part of the cell membrane, x 7200. remarkably free of microvilli and blebs (Fig. 1). By transmission electron microscopy many of them were dark, and resembled oligodendrocytes with large quantities of ribosomes or polyribosomes and extensive cytoplasmic microtubules (Fig. 2). Astrocy~e like cells containing fibrils and glycogen granula were also found. A few immature neuronal cells (neuroblasts) with neuritic processes containing parallel filaments and microtubules could also be identified. By prolonged culture in the same dish ( > 4 weeks), as well as by passaging the cultures, cells with glia and neuronal morphology disappeared.

Secondary cultures These consisted mostly of epithelioid cells as previously described (Fig. 3). By transmission electron microscopy these cells had a characteristic morphology with an abundance of closely packed filaments, with a diameter of approximately 7 nm (sheath bundles as seen in Fig. 4). They were especially numerous near the contact to the plastic, just beneath the cell membrane. Microtubules were scanty and formed no characteristic pattern. Besides round or oval nuclei, elongated mitochondria, collections of free ribosomes were observed, as well as some rough and smooth endoplasmatic reticulum. Tight junctions were present between neighbouring cells. Collagen fibres were absent.

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Fig. 5. SEM picture at early stage of morphological differentiation, i.e. 20 h after addition of BE. The cells contract and gradually form cytoplasmic processes. , 1700.

Effects of brain extract After addition of 25-30 % BE to the medium, the sheath bundles characteristic for secondary epithelioid cells disappeared within 2-3 days. At the same time the cell membrane detached itself from the plastic surface to form a contracted round perikaryon. Simultaneously many branched cytoplasmic processes were formed in all directions (Fig. 5). Most of the cells were transformed into cells which strongly resembled astrocytes (Fig. 6A and B). During the transformation, the surface of the cells became covered with stublike microvilli and blebs, and often the terminal parts ot the processes were covered with numerous blebs (Fig. 7). The area of intracellular contact was markedly decreased. Numerous filopodia sprang out from the cell margin (Fig. 6B). On the plastic surface from which the cell membrane had detached, small star-like residual bodies remained. By transmission electron microscopy a striking decrease in number of microfilaments occurred in perikarya, but in the cytoplasmic processes microtubules and numerous microfilaments were observed (Fig. 8A). In the longitudinally sectioned processes, tubules and filaments were organized in parallel directions. In the perikarya a characteristic change of the number and distribution of microtubules took place. In particular they were seen near the part of the cell membrane which was attached to the substratum (Fig. 8B). An increase of free ribosomes and rough

Fig. 6. A and B : end stage of morphological differentiation. The cells have astrocyte morphology with numerous microvilli on the surface. A x 1700, B x 900. In A, some non-differentiated epithelioid cells remain underneath.

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Fig. 7. Detail from cytoplasmic process with microvilli and blebs at the marginal point. The material underneath represents remnants of thin cytoplasm. 14,000. endoplasmatic reticulum was regularly seen, indicating accelerated protein synthesis (Fig. 9). In some cells numerous glia fibrils were observed having a diameter of approximately 10 nm (Fig. 10). Depending on a variable dose of BE, some of the number of epithelioid cells did not change morphology. In all cases, however, 70 °J,~of the cells were changed into gliallike morphology. When BE-exposed cells were changed to fresh medium without BE, their morphology more or less returned to the epithelioid pattern within 2 days. The transformation was thus dependent on a continuous presence of BE (Fig. I 1A and B). DISCUSSION The primary outgrowth of FBC from the 18th day of gestation resulted in the formation of undifferentiated, flat epithelioid cells as well as glia, mainly oligodendrocytes and some immature neurons, which later degenerated and disappeared. On addition of brain extracts, a drastic change of the epithelioid cells occurred, which resulted in numerous cells that by light as well as electron microscopy strongly resembled astrocytes (see refs. 5, 14 and 22). The whole cell was becoming rearranged with partial loss of attachment to the substratum, and contraction and extrusion of long cytoplasmic processes in all directions. The cells contained numerous microvilli and blebs as well

233

j~

~i~ ~ !~

~ ~ili~i~i~i ~i ~i~ii~i~i~i~......

Fig. 8. A: microtubules and microfilaments in cytoplasmic processes of BE-treated cells, x 30,000. perpendicular section of cell membrane showing arrays of microtubules lying near (i.e. under) the plasma membrane, x 50,000.

B:

as microfilaments, although characteristic glia filaments were seen in only a few cells (Fig. 10). The increase of free ribosomes and rough endoplasmatic reticulum indicated an active protein synthesis. In fact, as recently reported by Lim and Mitsunobu 17, we could also show that our cells at the same time accumulate the nervous system specific S-100 protein as well as the glial fibrillary acidic protein (GFA) (A. Haugen et al., in preparation). Thus the induction of differentiation at this developmental stage was not only a morphological event, but also within 2 days implied the formation of a protein which is not synthesized so early during fetal development 12. It is a well-known phenomenon that primary explantation of fetal brain tissue in cell culture results in the formation of a monolayer of flat epithelioid cells upon which a varying number of glia cells

234

Fig. 9. Electron micrograph from a horizontal section of a cell 48 h after addition of BE. Note the numerous blebs on the surface. 9000.

Fig. 10. Occurrence of glia fibrils in BE-treated cell with astrocyte morphology, x 17,000.

235

Fig. 11. Reversibility of BE action. A: FBC after two days o| exposure to BE. B: the same culture at two days after removal of BE. Phase contrast, x 350.

236 adhere 4,24,31,34,aa. If the brains are removed early during pregnancy, outgrowth of neurons may also be seen 4,a°. It has been postulated that the epitheIioid cells are primitive neuroectodermal cells 24,a4,aS. In fact, these cells may be induced to differentiate into glia morphology by various means. Thus dibutyryl-cAMP may do this within it few hours 15,31. Moreover the high molecular substance in BE described by Lira and Mitsunobu has the same effect. They concluded that this might be mediated by the adenylcyclase system 15,17. The direct formation of cells with electron microscopic characteristics of astrocytes strongly supports the conclusion that the epithelioid cells really are gila precursors. However, this does not exclude the possibility that they also may be precursors of neurons. Thus neuron formation from such fetal brain cells in culture has been induced by fetal brain extract 3° as well as by adding horse serum and fluorodeoxyuridine s. In addition Shapiro 31 could show that apart from inducing glia morphology, dibutyrylcAMP also increased the levels of the neuronal marker enzyme acetylcholinesterase by three times. Therefore, electron microscopical characteristics of glia cells, which we observed by induction of differentiation, does not exclude that flat epithelioid cells may also be capable of differentiation into neuronal elements. The lack of morphological differentiation, which fetal brain cells exhibited under the presently used culture conditions, in some respects resembles that of undifferentiated malignant gliomas. By use of various drugs, such as actinomycin-D, amethopterine, 5-bromodeoxyuridine, as well as dibutyryl-cAMP and sodium butyrate alone, undifferentiated glioma cells in culture have rapidly changed into astrocyte morphology TM aa. Also in this case the cytoplasmic processes contained numerous filaments and parallel microtubules aa. The physiological significance of such a dramatic formation of microvilli and blebs at the cell surface, and especially in the cytoplasmic processes, is at the present moment not understood ~. It has been suggested, however, that it is a way to store a surplus of plasma membrane and avoid reduction of the surface area of the cellL This might in turn allow an increased transport of nutrients u,aT. Such periodic zeiotic blebs have earlier been observed periodically in most cells in culture, depending on the cell cycle phases 2~. In addition blebs have been described in several kinds of cells during various experimental conditionsa, t3. They might also be due to a localized weakening of the cell membrane 28. In our investigation they contain ribosomes and sometimes endoplasmatic reticulum as well (Fig. 9). Concerning the internal organization of the epithelioid cells, the numerous stress fibres may be of importance for the cellular attachment (see ref. 36). The bundles of microfilaments have earlier been reported to contain actin 19 and thus may be contractile elements of the cells. The importance of the microtubules and microfilaments in defining cell morphology is well establishedg,lo,2s,2L This is also in accordance with our findings after addition of BE, where bundles of microfilaments accumulated in intimate association with the plasma membrane of the cytoplasmic processes, and microtubules accumulated in their centre. In any way it suggests an important role of the active molecule in BE in promoting organization of microfilaments and microtubules within the cell. In this respect the data are in agreement with the findings of Lira and Mitsunobu 16 and

237 show directly h o w the w h o l e cell is r a p i d l y r e o r g a n i z e d a n d undergoes s t r o n g m o r p h o l o g i cal alterations within a s h o r t p e r i o d o f time. Parallel to o u r article, L i m et a1.18 p u b l i s h e d a very t h o r o u g h study on electron m i c r o s c o p i c characteristics o f B E - e x p o s e d cells at a later stage o f m o r p h o l o g i c a l differentiation, i.e. at d a y 7. U n d e r their e x p e r i m e n t a l c o n d i t i o n s the increase o f glial filaments was m o r e p r o n o u n c e d , b u t c y t o p l a s m i c blebs were absent. I t is also r e m a r k a b l e t h a t the changes o f the cell shape are n o t irreversible, b u t r e t u r n t o w a r d s the original m o r p h o l o g i c a l p a t t e r n when the differentiation i n d u c t o r is no longer present. This opens the possibility o f m o n i t o r i n g , in cell culture, i m p o r t a n t events in b r a i n d e v e l o p m e n t . ACKNOWLEDGEMENTS This investigation was s u p p o r t e d by the N o r w e g i a n C a n c e r Society. W e t h a n k Mrs. Eva Bohlin a n d Miss G r o O l d e r 6 y for expert technical assistance.

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