Growth regulator from spinal cord: produced in cultures of glial cells

Developmental Brain Research, 2 (1982) 397--409 Elsevier/North-Holland Biomedical Press

397

G R O W T H R E G U L A T O R FROM SPINAL CORD: PRODUCED IN CULTURES OF G L I A L CELLS

L. J. KAGEN, S. LOVECE MILLER and A. LABISSIERE The Hospital for Special Surgery affiliated with the New York Hospital and the Department of Medicine, Cornell University Medical College, New York, iV. Y. 10021 (U.S.A.)

(Accepted April 24th, 1981) Key words: growth inhibitor -- myogenesis-- glia -- astrocyte

SUMMARY Spinal cord cell cultures produced a regulatory factor which inhibited myogenesis. After serial passage (× 3) production of this factor continued as neuronal cells disappeared and large, pale polygonal cells rich in cytoplasmic microfilaments with morphology of astrocyte precursors became predominant. These glial cells responded to dibutyryl cyclic AMP by assuming a star-shaped appearance with multiple, radiating cytoplasmic processes. Cultures active in production of the growth regulator also produced nonneuronal-type enolase and glutamine synthetase. It is suggested that the growth regulator is produced by astrocytic glia in culture.

INTRODUCTION Cells derived from embryonic chick spinal cord, in tissue culture, produce a low molecular weight factor which inhibits myogenesis and interferes with proliferation of fibroblasts i0,ii. Because this activity was sensitive to the action of proteolytic enzymes, the regulatory factor was tentatively felt to be an oligopeptidelL Using choline acetyl transferase and catecholamine binding activities as markers, the neuronal content of cultures was found not to be related to the production of the low molecular weight growth regulator i3. In this report we wish to describe efforts to characterize the spinal cord cells in tissue culture which produce the regulator and to indicate their glial origin. In particular, cultures actively producing the factor were dominated by cells of apparent astrocytic lineage. 0165-3806/82/0000-0000/$02.50 © Elsevier/North-HollandBiomedicalPress

398 MATERIALS AND METHODS

Cell cuhures Cell cultures were prepared from carefully dissected spinal cord of I 1-day-old white Leghorn chick embryos as previously described 12. The tissue was treated for 5 min in 0.25 ~o trypsin in calcium- and magnesium-free Earle's balanced salt solution at 37 °C. After addition of chilled, complete medium, the resulting suspension was col lected by centrifugation, and inoculated into plastic tissue culture dishes (35 10 ram, Falcon Plastics, Oxnard, Calif.) at 1 ~ l06 cells/dish, containing Eagle's medium, 2 ml, supplemented with 10 ~,~fetal calf serum and 2 mM glutamine. Cultures of spinal cord cells for serial passage were grown to confluence, harvested by trypsinization, and reinoculated into fresh medium weekly.

Inhibition of cell growth and myogenesis This was assayed in target cultures obtained from thigh muscle of I 1-day-old chick embryos 12. Inhibition was assessed by qualitative microscopic observation of cell growth and myotube/myofiber formation, estimation of total protein synthesis, estimation of myoglobin synthesis, and estimation of protein content of cytosot fluids in target cultures exposed to preparations of the spinal cord inhibitor as describedV.~.

Glutamine synthetase Cell pellets harvested from tissue culture dishes were reconstituted in I ml Earle's balanced salt solution containing 1 ~ Triton X-100, homogenized over ice, and clarified by centrifugation 90,000 g, 30 min. The supernatant fluid samples, and dilutions prepared from them, were added (0.35 ml) to imidazole buffer 80 mM, pH 6.76 containing 240 mM glutamine, 40 mM KHzAsO4 and 0.8 mM ADP (0.5 ml) with 0.15 ml solution of hydroxylamine, 200 raM, with MnCle, 33.3 mM, and incubated at 37 °C for 20 min. The reaction was stopped by addition of 1.0 ml 2.3 ~/oFeCI3 with 5 '!'i, trichloroacetic acid in 0.033 N HC1. After centrifugation, enzyme activity was assayed from the resulting optical density at 500 nm. This method was modified from previous descriptions16,4z,4~.

Chromatographic separation of enolase activities This was based upon the method of Marangos et al. 22. Cell pellets from tissue culture, and other tissue samples, were homogenized over ice in Tris-phosphate (10 mM) buffer with 2 mM MgSO4, pH 7.4 and clarified by centrifugation (90,000 g, 30 min). The supernatant fluid, after dialysis against the Tris-MgSO4 starting buffer, was applied to a column of DEAE-cellulose. Stepwise elution was carried out with the starting buffer, followed by Tris-Mg buffer with 0.5 M NaCt. Recovered fractions were concentrated by lyophilization to 1 ml, and dialyzed against Tris-phosphate, MgSO4 starting buffer.

399

Enolase activity Enolase activity was assayed as described 40 using spectrophotometric detection of phosphoenol pyruvate formation at 240 nm. The assay mixture contained imidazole (50 mM), MgSO4 (1 mM), D(+)-2-phosphoglyceric acid (1 m M ) and KC1 (0.4 M). Scanning electron microscopy This was performed on cells cultured on glass cover slips fixed in sodium cacodylate buffered 2 . 5 ~ glutaraldehyde for 15 min at 37 °C followed by 45 min at r o o m temperature. After 3 rinses, they were treated with veronal-acetate buffered 1 osmium tetroxide and then dehydrated in a graded series o f ethanols followed by amylacetate solutions. After drying and gold coating they were examined in an Etec scanning electron microscope. Transmission electron microscopy Transmission electron microscopy was performed on trypsinized cell pellets treated as above, dehydrated in a graded series o f ethanol solutions and embedded in Epon. Thin sections, stained with uranyl acetate and lead citrate were examined in the Philips 201 electron microscope. RESULTS

Production o f myogenesis inhibitor by primary and serially passaged spinal cord cells Cultures o f chick embryonic spinal cord cells produced myogenesis inhibitor fac-

TABLE I

Susceptibility of target cultures of muscle cells to spinal cord inibitor produced by spinal cord cultures after serialpassage Target cultures. Inoculum 1 x l06 cells/dish, harvested for assay after 4 days in culture, 24 h after exposure to [14C]lysine(0.5/~Ci/ml). Cord inhibitor obtained from cultures inoculated with 1 x 106 cells/dish harvested after 7 days in culture. Numbers in parenthesis indicate protein content of producer spinal cord cells mcg per culture. Qualitative estimation of myotubes, by phase-contrast microscopy 3 + : abundant myotubes in every field; 1 + : rare myotube found after examination of many fields; 0: no myotubes av. for data -4- standard deviation shown.

Muscle cultures

Proteinsynthesis Inhibition (epm/eulture) (%)

Protein content (l~g/eulture)

Inhibition (%)

Myotube formation

Untreated 275,609 q- 7597 + cord inhibitor (80 ~ by vol.) Derived from primary culture (148) 55,125 ± 4438 80.0 Second passage (144) 62,279 -4- 2010 77.4 Third passage (104) 87,745 ± 14903 68.2

203.5 -k 0.0

-

3+

68.3 ± 6.1

66.5

0

72.9 ± 2.3 100.6 4- 15.1

64.2 50.6

0 1+

400

Fig. 1. Scanning electron microscopic view of primary culture of unseparated cells from embryonic spinal cord, demonstrating the presence of several cell lypcs and neurite network r Arro~s at neurites. N, neuronal cell body; G, glial cell. 1100.

tor in essentially equivalent amounts after primary culture or 2 serial passages (Table 1). Crude supernatant fluids harvested at 7 days from primary cultures, and from second and third passages inhibited both protein synthesis in target muscle cultures and cell growth, as indicated by protein content of target cultures. Myofiber formation was also completely inhibited as shown.

Morphology of spinal cord cells in primary and passaged cultures Primary cultures of spinal cord cells contained several recognizable cell types

401

Fig. 2. Scanning electron microscopic view of cell culture at third serial passage demonstrating presence of large fiat glial cells. Left panel: confluent carpet of 4 glial cells. Area of cell abutment indicated by arrows, x 1800. Right panel: surface view of extended glial cell with processes and surface microvilli, x 2200.

Fig. 3. Details of ultrastructure of glial cell by transmission electron microscopy illustrating masses of microfibrils (F) both in parallel and in loose, apparently random array. Mitochondrion (M). Left panel x 89,000; right panel x 82,000.

402

Fig. 4. Details of ultrastructure or' glial cell by transmission electron microscopy illustrating clect~~ lucid nucleus (N) and cytoplasm containing masses of fibrils (F). Left panel 17,300;right pa~lcl 54,700. Right panel demonstrates vacuoles, distended endoplasmic reticulum (ER) and surface mic~'¢,viHi. (Fig. 1). Neurons with networks o f long neurites were seen in every field. In additiol~, nonneuronal cells were also c o m m o n , particularly large, flat polygonal cells, with short surface microvilli and terminal filapodial processes, often exteading over the surface of neighboring cells. After serial passage, cultures contained a more uni['orl~ cell type. Neurons disappeared and the large, flat glial cells formed a confluent carpet (Fig. 2) with cell boundaries indistinctly seen at intimate areas of cell contact. The ultrastructure of the large glial cells is shown in sections by transmission electron microscopy (Figs. 3 and 4). The nucleus was generally electron-lucid. Microfilaments were a b u n d a n t in the cytoplasm in two forms. A r o u n d the nucleus and in other portions o f the cytoplasm, strands crossed over one another in loose, apparent random, arrays. Near processes, as well as in other parts of the cytoplasm, dense arrays of filaments were stacked in parallel bundles. In addition, distended rough endoplasmic reticulum was usually present. Vacuoles were found near the cell membrane. These filament-packed glial cells persisted, upon serial passage, to be the d o m i n a n t surviving cell in cultures which produced the growth regulator.

403 TABLE lI Reaction o f cell cultures to D B c A M P to form star-shaped cells with branching cell processes Cultures at times indicated were exposed to dibutyryl cyclic A M P 1 m M in culture medium in the absence of fetal calf serum. Observations made to 5 h. ÷ , indicates formation of star-shaped cell with many branching processes; such morphological conversion was observed in approximately 30~40% of cells; 0, no such reaction observed. Culture

Spinal cord, primary Spinal cord, third passage Skin Thigh muscle

Age (days in culture when treated)

Reaction to DBc.4MP

after change to serumfree medium only

5 5 5 2

÷ ÷ 0 0

0 0 0 0

Response to DBcAMP Cultures were exposed to dibutyryl cyclic adenosine monophosphate, 1 mM, at the times shown (Table II) in the absence of fetal calf serum. The large fiat polygonal glial cells reacted within hours to form many long, often branched processes, or cytoplasmic strands, extending from small dense cell bodies (Fig. 5). The cell bodies of these star-shaped cells appeared less adherent to the plastic dishes than were the original fiat cell types. The rapidity of the response, and its enhancement by serum deprivation leaves unsettled the issue of whether the DBcAMP treatment was toxic, or whether the response represented a physiological reaction. As shown in Table II, glial cells were particularly sensitive to this stimulus, and similar changes to star-shaped cells were not observed in other cell types derived from skin or thigh. Enzyme content of cultures of glial cells Enolase. Enolase was detected both in primary cultures of mixed spinal cord cells and after multiple passage where the large fiat glial cells predominated. After multiple passage, there was approximately twice as much enolase present per mg cell protein. In all cultures tested, enolase activity was eluted from DEAE-cellulose, ion-exchange columns at low ionic strength, as expected for the nonneuronal form of the enzyme (Table III). For comparison, results of chromatography and enzyme assay of homogenates of brain tissue are shown. In the adult, 43.4 ~ of the recovered enolase activity eluted in the position expected for the neuronal specific form, and 56.6 ~ in that for the nonneuronal type. Brain tissue of the embryo, however, possessed only 1.7 ~ of its enolase activity in the neuronal specific form, with the remainder being the nonneuronal type. Glutamine synthetase. Glutamine synthetase activity was detected in both primary and third passaged spinal cord cell cultures, with greater activity per mg cell protein present in the primary cultures (Table III).

404

Fig. 5. F o r m a t i o n o f small cell elements with long branching processes 4 h after exposure of large flat glial cells to D B c A M P , 1 rnM. Several large flat glia which have not u n d e r g o n e morphological change as yet are also seen. Both fields 1800.

405 TABLE III Enzyme content of cell cultures and brain tissue

Enolase: mg phosphoenolpyruvate formed at 6 min/mg cell protein. Non-neuronal: activity eluting with Tris-phosphate 10 mM with 2 mM MgSO4, pH 7.46. Neuronal-specific activity eluting with Tris-phosphate 10 mM with 2 mM MgSO4 and 0.5 M NaCI. Glutamine synthesase:/~M L-glutamic acid-~'-mono-hydroxamate formed per h/mg cell protein. Culture

Spinal cord Primary Third passage Brain tissue Adult Embryo (11 days of incubation)

Enolase

Glutamine synthetase

Non-neuronal type

Neuron-specific type

6.15 12.06

0 0

6.23

4.73

2.84

0.05

1.31 0.42

DISCUSSION Cell cultures derived from embryonic spinal cord continued to produce an inhibitor of myogenesis and fibroblast proliferation after serial passage, when they were depleted of neurons and contained large, fiat polygonal cells characterized by filopodial processes, filaments both in bundles and random arrangement, submembrane vacuoles and surface microvilli. These morphological characteristics were very similar to those seen in cultures of astroglial cells, and thought to be characteristic of astrocyte precursors or astroblasts18, 44. After addition of maturation factors or derivatives of cyclic adenosine Y,5'-monophosphate, astroglial precursors displayed alterations in shape to those more closely resembling mature astrocytes. The precursor cells, which have also been referred to as epithelial glioblasts, have been described as maximally stretched out, and smoothly blending with surrounding cells is, both properties evident in the ceils described here. Finger-like processes, or microvilli, extending above the cell surface, and a cytoplasm rich in filaments arrayed in bundles near processes, and without noticeable orientation elsewhere, have been described 3. In sparse cultures, these cells are spread out with a broad, continuous, fan-shaped ruffled edge. Macropinocytotic vacuoles are seen, as well as intercellular bridges4, 42. The morphological characteristics exhibited by the glial cells reported here, corresponded to those of the type C colonies of astrocyte precursor cells described by Federoff s. Their cytoplasmic characteristics were similar to those described and the nuclei also were electron-lucid. Morphological changes have been induced in astroblasts by dibutyryl cyclic AMP, monobutyryl cAMP, as well as by theophylline, and serum deprivation17,29, ag. DBcAMP-treated astroblasts also develop increases in glial fibrillary acidic protein and cytoplasmic filaments30, 3v. Similar changes have been observed in cultured cells

406 derived from glial tumors a,7,='l. It has not as yet been clearly agreed how closely these changes represent an in vitro counterpart of maturation since, to some degree, serum deprivation or biochemical toxicity might be responsible for cell retraction, and changes in adhesiveness to the culture substrate might produce some of these findings Nonetheless, the glial elements studied here responded as described for astrocyte precursors 9 whereas similar changes under these conditions were not observed in myogenic cells or in fibroblasts of muscle or skin cultures. The glial cells studied here possessed enolase of the nonneuronal type, and glutamine synthetase. Both these constituents have been used as biochemical markers of glia. Enolase (2-phospho-D-glycerate hydrolase) exists in mature brain tissue in isoenzymic forms. Neuron-specific enolase, NSE, is present in neurons as well as in certain cells of the amine precursor uptake and decarboxylation system ~a.24,aa. Nonneuronal enolase, NNE, is localized in glial cells within the nervous system a4 and absent from neurons and endothelial cells. In mammalian spinal cord the concentration of nonneuronal enolase is approximately 1.4-4 times as great as that of the neuronal form 2a. Neuron-specific enolase has been demonstrated in cultures of murine spinal cord cells aS. The isoenzymic forms of enolase are separable by ion exchange chromatographic procedures. With DEAE-cellulose, the nonneuronal form is of low affinity, whereas the neuron-specific form elutes with higher salt concentration z'-'. This observation forms the basis of the assay of enolase in the present studies. In adult brain tissue the ratio of the nonneuronal to neuron-specific enolase was 1.3. In the embryo, however, this ratio was 56.8, suggesting a deficiency of the neuronal form. Embryonic mammalian brain has been reported deficient in neuron-specific enolase, and it has been hypothesized that neurons switch from production of the NNE to the NSE form during differentiation and development 'z
407 T a k e n together, the evidence thus far indicates production o f the spinal cord growth regulatory substance by glial cells. The most likely cell type involved was the astrocyte precursor. The ability o f glia to affect the growth and metabolism o f other cell types has been described in several instances. Astroglia release factors that p r o m o t e the growth and survival of rat hippocampal neurons in vitro 1. Substances released f r o m glia in culture, stimulate neuronal development in experimental systems20,2s, as. The production of the enzyme ~,-glutamyl transpeptidase in endothelial cells of brain microvasculature was induced by cocultivation with neoplastic glial cellsL Glycopeptides, which inhibit protein synthesis, have been identified on the cell surface o f murine cerebrum 14. These inhibitors had considerably larger molecular size 15 than the spinal cord growth regulator studied here. Shimada 41 reported suppression o f myogenesis by spinal cord cells as well as by other heterotypic cells f r o m cartilage, retina, liver, lung and heart when cocultivated with muscle. This p h e n o m e n o n was prevented by interposition o f a Millipore filter between the myoblasts and the heterotypic cells.

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