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Astrocyte growth enhanced by culture supernatant of a cloned rat thymic myoid cell
304
Brain Re~earch, 34S ( i,4~5) 3()4--31i~, ~'Isc~icr
BRE 11163
Astrocyte Growth Enhanced by Culture Supernatant of a Cloned Rat Thymic Myoid Cell AIKO TADA-KIKUCHIl, ISAO KAMO 1, TSUNETOSHI ITOH2 and IKUYA NONAKA 1 lDivision of Ultrastructural Research, National Centerfor Nervous, Mental and Muscular Disorders, 4-1-10gawa-higashi, Kodaira, Tokyo 187, and 2Departrnent of Anatomy, Tohoku University School of Medicine, 2-1 Seiryo-cho, Sendai 980 (Japan)
(Accepted February 12th, 1985) Key words: astrocyte - - thymic myoid cell - - myelo-lymphoid cell - - cytokine - - proliferation - - differentiation
A conditioned medium (CM) from a cloned rat thymic myoid cell (IT-45R92) was used to investigate a possible relationship between the thymus and brain. CM stimulated the DNA synthesis of the cultured perinatal rat brain cells and spleen myelo-lymphoid cells but not that of the other types of cells including dorsal root ganglia, spinal cord, heart muscle, skeletal muscle, kidney, liver, skin, and thymic fibroblasts. CM appeared to stimulate glioblasts to differentiate into mature astrocytes that have a characteristic intracellular fibrous structure and a glial fibrillary acidic protein. These results indicated that thymic myoid cells release (a) 'cytokine(s)' for the astrocytes and myelo-lymphoid cells, similar to a 'lymphokine' (glia cell stimulating factor, GSF) produced by murine and human lymphocytes stimulated with mitogens. INTRODUCTION
immature glioblasts into astrocytes.
The thymus is the central organ for the completion of maturation of precursor T cells. Many different types of thymic non-lymphoid cells are considered to provide important signals for certain stages of T cell maturation. During identification of cell types responsible for thymic lymphoid cell proliferation, we found that rat thymic myoid cells had a significant activity for proliferation of myelo-lymphoid cells (manuscript in preparation). The thymus is also known to share several characteristics with the central nervous system (CNS), such as growth factors (interleukin 1 (IL 1) and colony stimulating factor (CSF)3, 4 and cell surface antigens (Thy 1, M R C OX2, etc) 1,5,8,14,17-19. Those close relations between the thymus and brain enable us to investigate whether the myoid cells have any effects on the growth of the brain cells, and if any, what types of cells are in response to such effects. We report here that CM of a cloned rat thymic myoid cell 1T-45R92 (ref. 7) contains a 'cytokine' which stimulates D N A synthesis of brain cells as well as lymphoid cells and promotes the differentiation of
MATERIALS AND METHODS A cloned myoid cell IT-45R92 was maintained routinely in an intensified R P M I 1640 ( G I B C O R P M I 1640 intensified with M E M essential and non-essential amino acids (1 ×), sodium pyruvate (1×) and vitamin ( I x ) ) + 10% fetal bovine serum (FBS, GIBCO). A conditioned medium (CM) was collected from the supernatant of single cell cultures of IT45R92 2 - 3 days after the culture reached confluency and stored at - 2 0 °C until use after removal of cell debris. The brain (cerebrum and cerebellum), posterior ganglia and various other non-neuronal tissues (spleen, heart muscle, skeletal muscle, kidney, liver, skin and thymic fibroblasts) of Wistar rat fetuses on 17th gestational day or 1-3-day-old newborns were dissected into 1 mm 3 pieces and were digested with 500 units of Dispase (Godoshusei) in M E M + 20% FBS for 30 min at 37 °C. Single cell suspensions were obtained after removal of Dispase by centrifugation and successive filtration through double gauze, 100 and 200 nylon mesh sieves. For morphological stud-
Correspondence: A. Tada-Kikuchi, Division of Ultrastructural Research, National Center for Nervous, Mental and Muscular Disorders, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187, Japan.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
305 ies, the brain cells ( 1 - 5 x 105) s u s p e n d e d in 0,1 ml of R P M I 1640 + 15% FBS were s e e d e d onto r o u n d glass coverslips of 10 m m in d i a m e t e r p l a c e d in Falcon plastic dishes. Within 24 h after seeding, the coverslips carrying confluent cells (referred to as the m o t h e r culture) were washed with a fresh m e d i u m ( R P M I 1640 + 15% FBS) and transferred to the center of 6 coverslips placed in a new plastic dish containing 4.5 ml of a 1:1 mixture of C M and the above intensified R P M I 1640 ( M C M ) . F o r control, the fresh m e d i u m was used in place of CM. H a l f volume of culture m e d i u m was changed every 3 days. Cells migrated from the m o t h e r culture onto the surrounding coverslips. T h e y were used for immunofluorescence staining and a u t o r a d i o g r a p h y studies. F o r examining the proliferating activities, the cells ( 1 - 1 0 x 104) were s u s p e n d e d in 2 ml of M C M or fresh medium and i m m e d i a t e l y seeded into 24 well plastic plates (Coaster). The cells were cultured for 72 h and a d d e d with 0.5 k~Ci/ml of tritiated thymidine ([3H]TdR, spec. act. 47 Ci/mmol) for a n o t h e r 24 h until cell harvest. The amounts of [3H]TdR i n c o r p o r a t e d into the cells were expressed as cpm/culture. All cells were cultured at 37 °C in a humidified a t m o s p h e r e of 5% CO 2 in air. F o r a u t o r a d i o g r a p h i c studies, coverslips carrying migrated cells from 5 - 2 1 day cultures were first treated for 24 h with 1 ~tCi/ml [3H]TdR in M C M . The cells were then fixed with acid alcohol at - 2 0 °C for 10 rain, incubated with 0.1 ml of rabbit anti G F A P serum (1:40, supplied by Dr. S.U. Kim) and 0.1 ml of r h o d a m i n e - c o n j u g a t e d I g G fraction of goat against rabbit l g G (1:40) each for 20 rain at r o o m t e m p e r a ture before dipping in Sakura N R - H 2 emulsion. RESULTS AND DISCUSSION The effects of C M on cultured brain cells from rat newborns ( 1 - 3 days old) were r e p e a t e d l y tested. W h e n 1 x 105 brain cells were cultured for 96 h in CM, those cells were greatly stimulated to incorporate [3H]TdR into the D N A (Fig. 1A). The increased D N A synthesis of the brain cells by C M was always o b s e r v e d after the lag phase of 24 h. W e then tested organ specificity of the growth stimulating activity of CM. The heart muscle, skeletal muscle, kidney, liver, thymic fibroblasts, skin, spinal cord, dorsal root ganglia, spleen and brain cells were p r e p a r e d from one litter of rat fetuses at 17th gestation day and cul-
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Fig. 1. Effects of CM on [3H]TdR incorporation into rat cells. A: new-born brain cells (1 x 105) cultured in 2 ml of either MCM or control medium. Values between MCM and the control in each experiment were statistically significant (P < 0.01). Each column indicates mean of 4 cultures (+ S.E.M,) Dotted column, control; open column. MCM. B: Cells from various organs of fetuses on gestation day 17 cultured with or without CM. Only brain and spleen cells showed high [3H]TdR incorporation by stimulation with CM (P < 0.01). For cultures of brain, spleen and liver cells; 1 × 105 cells/culture. For cultures of spinal cord, dorsal root ganglia, heart muscle, skeletal muscle, kidney, skin and thymic fibroblast cells; 1 × 104 cells/culture. Each column indicates means of 3 cultures. Dotted column, control: open column, MCM. tured with or without CM. The growth p r o m o t i n g activities of C M were specific for the brain and spleen cells with 3 times of proliferation but not for cells from the o t h e r organs (Fig. 1B). In many cases, cells from other organs except for l y m p h o i d and brain were adversely affected with CM. This may be due to
Fig. 2. Cellular morphology of newborn rat brain cells responding to CM. A: cells cultured in control medium for 5 days. B and (~: cells cultured in CM for 5 days. D - F : intracellular changes occurred in CM cultures of 7-21 days. Arrows in A and B indicate the. edgc of ~ round glass coverslip in a plastic dish of 60 mm in diameter. Ba: dendritic cells. Bb: cells with long processes. C: spindle type cells. D: flat cells in a cluster. E: astrocytes with a characteristic intracellular fibrous morphology (arrows), F: many astroeytes inc~rpor~ting [3H]TdR (heavy grains in the nuclei) were shown to be stained with anti G F A P antibody.
307 nutritional exhaustion of CM and/or inhibitors produced by myoid cells. Unresponsiveness of spinal cord and dorsal root ganglia cells seems to be caused by the maturity of those cells at the 17th gestation day, while the cerebral hemispheres and cerebellum are composed mainly of undifferentiated cells. CM also induced morphological changes in rat brain cells. The characteristic changes were as follows (Fig. 2); when the mother culture coverslips were transferred into the MCM, the first visible changes occurred as early as 8-16 h after transfer with appearance of round cells piling up on the mother culture. Those cells contained many large granules and resembled the immature macrophage-granulocyte lineage. Also, the cells started to migrate from the mother culture to the secondary coverslip. In controls, most of the cells remained on the mother culture (Fig. 2A). The cells first attaching to the secondary coverslips appeared to be dendritic (Fig. 2Ba) or spindle shaped cells (Fig. 2C, different field of 2B). Those adherent cells gradually transformed their morphology and another two cell types became prominent: cells connected with each other by long processes (Fig. 2Bb) and flat cells forming clusters (Fig. 2D). Those cells finally changed their intracellular morphology: they developed characteristic intracellular fibrous structures (Fig. 2E, arrows). We followed those morphological changes until day 21 after culture start. In order to investigate target cell specificity, cells migrated onto the secondary coverslip were treated with [3H]TdR and anti-GFAP antibodies. All anti-GFAP-positive cells incorporated [3H]TdR in the nuclei (shown as heavy grains) in response to CM (Fig. 2F, cells harvested on day 12). This tendency was also observed at two other different time points, cells harvested on day 5 and 21 (data not shown). These results suggested that target cells of CM were a glioblast-astrocyte lineage. However, mature astrocytes gradually ceased appreciable cell proliferation in MCM after morphological changes. Taken together, these results strongly indicate that CM stimulates exclusively the growth of glioblasts REFERENCES 1 Barclay, A.-N. and Ward, H.A., Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain recognized by monoclonal antibody MR( OX2. Eur. J. Biochem., 129 (1982) 447-458.
rather than that of mature astrocytes. Our present studies seemed to indicate another possible link between the thymus and CNS 16. However, the exact roles of thymic myoid cells in vivo remain unclear. A putative 'glial cell growth-promoting cytokine' produced by thymic myoid cells might be involved in generation of astrocytes in the CNS and etiology of some autoimmune disease 19. CNS also contains cells with myogenic potential. Generation of astrocytes and striated muscle cells in optic nerve cell cultures 2° may suggest a close relation between myoid cells and astrocytes in their development. In fact, our preliminary results indicated that cells with myoid potential from newborn rat brain cells stimulated the growth of glial cells. In this regard, various glial cell growth and/or maturation factors have been reported in cell extracts of brain 9-]2,~5, pituitary 2, submaxillary gland 13 and rat astrocytoma C6 m. Also, a platelet-derived growth factor (P-DGF)6 and an epidermal growth factor (EGF) 1] have been reported to affect the growth of cultured astrocytes. Besides those factors, a 'lymphokine' named GSF has been reported in culture supernatant of T and B lymphocytes stimulated with mitogens-~, 4. As for glial cell stimulating activity, CM has quite similar potency to lymphocyte-derived GSF. However, our preliminary results indicated that CM contains an IL 1, CSF and lectins beyond a glial cell growth-promoting activity. We are currently investigating whether an astrocyte differentiating activity in CM is carried by one of the above characterized molecular entities or other uncharacterized entities. ACKNOWLEDGEMENTS We thank Drs. Eiko Okada-Kobayasjai, Seung U. Kim and Kamesaburo Yoshino for helpful support. Also, we appreciate the excellent technical assistance of Ms. Aiko Sato. This work was supported by Grant no. 58570369 from the Ministry of Education of Japan.
2 Brockes, J.P., Lemke, G.E. and Balzer, D.R. Jr., Purification and preliminary characterization of a glial growth factor from the bovine pituitary, J. Biol. Chem., 255 (1980) 8374-8377. 3 Fontana, A., Dubs, R., Merchant, R., Balsiger, S. and Grob, P.J., Glia cell stimulating factor (GSF): a new lym-
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phokine. Part 1, Cellular sources and partial purification of routine GSF, role of cytoskeleton and protein synthesis in its production, J. Neuroimmunol., 2 (1980) 55-7t. Fontana, A., Otz, U., De Weck, A.L. and Grob. P,J., Gila cell stimulating factor (GSF): a new lymphokine. Part If. Cellular sources and partial purification of human GSF, J. Neuroimmunol., 2 (1980) 73-81. Garson, J.A., Beverley, P.C.L., Coakham, H.B. and Harper, E.I., Monoclonal antibodies against human T lymphocytes label Purkinje neurones of many species. Nature (London), 298 (1982) 375-377 Heldin, C.-H., Wasteson, A. and Westermark, B., Growth of normal human glial cells in a defined medium containing platelet-derived growth factor, Proc Natl. Acad. Sci. U.S.A., 77 (1980) 6611-6615~ Itoh, T., Establishment of a myoid cell clone from rat thymus, Cell Tiss. Res., 231 (1983) 39-47. Jones, B., Functional activities of antibodies against brainassociated T cell antigens If. Stimulation of T cell-induced B cell proliferation, Eur. J. lmmunol., 12 (1982) 30-37. Kato, T., Fukui, Y., Turriff, D.E., Nakagawa, S., Lim, R., Arnason, B.G.W. and Tanaka, R., Gila maturation factor in bovine brain: partial purification and physicochemical characterization, Brain Research, 212 ( 1981 ) 393-402. Kato, T., Yamakawa, Y., Sakazaki, Y., Ito, J., Kato, H., Tsunooka, H., Masaoka, A. and Tanaka, R., Glial cell growth-promoting factor in astrocytoma (C6) cell extracts, Dev. Brain Res., 2 (1982) 596-601. Leutz, A. and Schachner, M., Epidermal growth factor stimulates DNA-synthesis of astrocytes in primary cerebellar cultures. Cell Tiss. Res., 220 (1981) 393-404.
12 Lim~ R., Turriff, D.E., Troy, >.S., Moore. t~ ~,;~,.and Eng. L.F., Glia maturation factor: effect on chemicai differen!i:~tion of glioblasts in culture, Science, 195 (1977) 195- ]9~'~. 13 McClure, D.B., Ohasa, S. and Sato, (;.lt.~ ~:~tc.,'.~)rsh! !he rat submaxillary gland that stimulate growth ,.ff cuitmcd glioma cells: identification and partial charautc.~ization, J. Cell. Physiol., 107(1981) 195-2[17, 14 Oger, J., Szuchet, S., Antel. J. and Arnason. B.G.W.. ,\ monoclonal antibody against human ] suppressor lymphocytes binds specifically to the surface of cultured oligodendrocytes, Nature (London), 295 (1982)66--(4~; 15 Pettmann, B.. Sensenbrenner. M. and i,abaurdettc. G., [solation of a glial maturation factor from b e d brain. FEBS Lett., 118 (198(I) 195-199. 16 Pierpaoli, W. and Besedovsky, tt.O., Role ~f ,A~cthymus in programming of neuroendocrinc functions. ,,hn E.~p. hnmunol., 20 (1975) 323-338. 17 Pruss, R.M., Thy-I antigen on astrocytes in Icing-term cultures of rat central nervous system, Nature (London), 280 (1979) 688-690. 18 Shuller-Petrovic, S., Gebhart. W., Lassmann, H., Pumpold, H. and Kraft. D., A shared antigenic determinant between natural killer cells and nerve tissue, Nature ([.otldon), 306 (1983) 179-181. 19 Shimabukuro, K., Mizuno, Y.. Kurita, K.. lsuchiya, M. and Yoshimatsu, H., Thymus in Hashimoto's disease and focal chronic lymphoid thyroiditis, Keio J. M e d 24 (1975) 275-287. 20 Wier, M.L. and Lennon, V.A.. Differentiation ot skeletal muscle from dissociated optic nerve cells, lrmntmocytological observations, J. Neuroimmunol. , 1 ( 198 l ) 61 -68.
Brain Re~earch, 34S ( i,4~5) 3()4--31i~, ~'Isc~icr
BRE 11163
Astrocyte Growth Enhanced by Culture Supernatant of a Cloned Rat Thymic Myoid Cell AIKO TADA-KIKUCHIl, ISAO KAMO 1, TSUNETOSHI ITOH2 and IKUYA NONAKA 1 lDivision of Ultrastructural Research, National Centerfor Nervous, Mental and Muscular Disorders, 4-1-10gawa-higashi, Kodaira, Tokyo 187, and 2Departrnent of Anatomy, Tohoku University School of Medicine, 2-1 Seiryo-cho, Sendai 980 (Japan)
(Accepted February 12th, 1985) Key words: astrocyte - - thymic myoid cell - - myelo-lymphoid cell - - cytokine - - proliferation - - differentiation
A conditioned medium (CM) from a cloned rat thymic myoid cell (IT-45R92) was used to investigate a possible relationship between the thymus and brain. CM stimulated the DNA synthesis of the cultured perinatal rat brain cells and spleen myelo-lymphoid cells but not that of the other types of cells including dorsal root ganglia, spinal cord, heart muscle, skeletal muscle, kidney, liver, skin, and thymic fibroblasts. CM appeared to stimulate glioblasts to differentiate into mature astrocytes that have a characteristic intracellular fibrous structure and a glial fibrillary acidic protein. These results indicated that thymic myoid cells release (a) 'cytokine(s)' for the astrocytes and myelo-lymphoid cells, similar to a 'lymphokine' (glia cell stimulating factor, GSF) produced by murine and human lymphocytes stimulated with mitogens. INTRODUCTION
immature glioblasts into astrocytes.
The thymus is the central organ for the completion of maturation of precursor T cells. Many different types of thymic non-lymphoid cells are considered to provide important signals for certain stages of T cell maturation. During identification of cell types responsible for thymic lymphoid cell proliferation, we found that rat thymic myoid cells had a significant activity for proliferation of myelo-lymphoid cells (manuscript in preparation). The thymus is also known to share several characteristics with the central nervous system (CNS), such as growth factors (interleukin 1 (IL 1) and colony stimulating factor (CSF)3, 4 and cell surface antigens (Thy 1, M R C OX2, etc) 1,5,8,14,17-19. Those close relations between the thymus and brain enable us to investigate whether the myoid cells have any effects on the growth of the brain cells, and if any, what types of cells are in response to such effects. We report here that CM of a cloned rat thymic myoid cell 1T-45R92 (ref. 7) contains a 'cytokine' which stimulates D N A synthesis of brain cells as well as lymphoid cells and promotes the differentiation of
MATERIALS AND METHODS A cloned myoid cell IT-45R92 was maintained routinely in an intensified R P M I 1640 ( G I B C O R P M I 1640 intensified with M E M essential and non-essential amino acids (1 ×), sodium pyruvate (1×) and vitamin ( I x ) ) + 10% fetal bovine serum (FBS, GIBCO). A conditioned medium (CM) was collected from the supernatant of single cell cultures of IT45R92 2 - 3 days after the culture reached confluency and stored at - 2 0 °C until use after removal of cell debris. The brain (cerebrum and cerebellum), posterior ganglia and various other non-neuronal tissues (spleen, heart muscle, skeletal muscle, kidney, liver, skin and thymic fibroblasts) of Wistar rat fetuses on 17th gestational day or 1-3-day-old newborns were dissected into 1 mm 3 pieces and were digested with 500 units of Dispase (Godoshusei) in M E M + 20% FBS for 30 min at 37 °C. Single cell suspensions were obtained after removal of Dispase by centrifugation and successive filtration through double gauze, 100 and 200 nylon mesh sieves. For morphological stud-
Correspondence: A. Tada-Kikuchi, Division of Ultrastructural Research, National Center for Nervous, Mental and Muscular Disorders, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187, Japan.
0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
305 ies, the brain cells ( 1 - 5 x 105) s u s p e n d e d in 0,1 ml of R P M I 1640 + 15% FBS were s e e d e d onto r o u n d glass coverslips of 10 m m in d i a m e t e r p l a c e d in Falcon plastic dishes. Within 24 h after seeding, the coverslips carrying confluent cells (referred to as the m o t h e r culture) were washed with a fresh m e d i u m ( R P M I 1640 + 15% FBS) and transferred to the center of 6 coverslips placed in a new plastic dish containing 4.5 ml of a 1:1 mixture of C M and the above intensified R P M I 1640 ( M C M ) . F o r control, the fresh m e d i u m was used in place of CM. H a l f volume of culture m e d i u m was changed every 3 days. Cells migrated from the m o t h e r culture onto the surrounding coverslips. T h e y were used for immunofluorescence staining and a u t o r a d i o g r a p h y studies. F o r examining the proliferating activities, the cells ( 1 - 1 0 x 104) were s u s p e n d e d in 2 ml of M C M or fresh medium and i m m e d i a t e l y seeded into 24 well plastic plates (Coaster). The cells were cultured for 72 h and a d d e d with 0.5 k~Ci/ml of tritiated thymidine ([3H]TdR, spec. act. 47 Ci/mmol) for a n o t h e r 24 h until cell harvest. The amounts of [3H]TdR i n c o r p o r a t e d into the cells were expressed as cpm/culture. All cells were cultured at 37 °C in a humidified a t m o s p h e r e of 5% CO 2 in air. F o r a u t o r a d i o g r a p h i c studies, coverslips carrying migrated cells from 5 - 2 1 day cultures were first treated for 24 h with 1 ~tCi/ml [3H]TdR in M C M . The cells were then fixed with acid alcohol at - 2 0 °C for 10 rain, incubated with 0.1 ml of rabbit anti G F A P serum (1:40, supplied by Dr. S.U. Kim) and 0.1 ml of r h o d a m i n e - c o n j u g a t e d I g G fraction of goat against rabbit l g G (1:40) each for 20 rain at r o o m t e m p e r a ture before dipping in Sakura N R - H 2 emulsion. RESULTS AND DISCUSSION The effects of C M on cultured brain cells from rat newborns ( 1 - 3 days old) were r e p e a t e d l y tested. W h e n 1 x 105 brain cells were cultured for 96 h in CM, those cells were greatly stimulated to incorporate [3H]TdR into the D N A (Fig. 1A). The increased D N A synthesis of the brain cells by C M was always o b s e r v e d after the lag phase of 24 h. W e then tested organ specificity of the growth stimulating activity of CM. The heart muscle, skeletal muscle, kidney, liver, thymic fibroblasts, skin, spinal cord, dorsal root ganglia, spleen and brain cells were p r e p a r e d from one litter of rat fetuses at 17th gestation day and cul-
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dorssl heart root ganglia
muscle kidney
skin
liver
thymus
l
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Fig. 1. Effects of CM on [3H]TdR incorporation into rat cells. A: new-born brain cells (1 x 105) cultured in 2 ml of either MCM or control medium. Values between MCM and the control in each experiment were statistically significant (P < 0.01). Each column indicates mean of 4 cultures (+ S.E.M,) Dotted column, control; open column. MCM. B: Cells from various organs of fetuses on gestation day 17 cultured with or without CM. Only brain and spleen cells showed high [3H]TdR incorporation by stimulation with CM (P < 0.01). For cultures of brain, spleen and liver cells; 1 × 105 cells/culture. For cultures of spinal cord, dorsal root ganglia, heart muscle, skeletal muscle, kidney, skin and thymic fibroblast cells; 1 × 104 cells/culture. Each column indicates means of 3 cultures. Dotted column, control: open column, MCM. tured with or without CM. The growth p r o m o t i n g activities of C M were specific for the brain and spleen cells with 3 times of proliferation but not for cells from the o t h e r organs (Fig. 1B). In many cases, cells from other organs except for l y m p h o i d and brain were adversely affected with CM. This may be due to
Fig. 2. Cellular morphology of newborn rat brain cells responding to CM. A: cells cultured in control medium for 5 days. B and (~: cells cultured in CM for 5 days. D - F : intracellular changes occurred in CM cultures of 7-21 days. Arrows in A and B indicate the. edgc of ~ round glass coverslip in a plastic dish of 60 mm in diameter. Ba: dendritic cells. Bb: cells with long processes. C: spindle type cells. D: flat cells in a cluster. E: astrocytes with a characteristic intracellular fibrous morphology (arrows), F: many astroeytes inc~rpor~ting [3H]TdR (heavy grains in the nuclei) were shown to be stained with anti G F A P antibody.
307 nutritional exhaustion of CM and/or inhibitors produced by myoid cells. Unresponsiveness of spinal cord and dorsal root ganglia cells seems to be caused by the maturity of those cells at the 17th gestation day, while the cerebral hemispheres and cerebellum are composed mainly of undifferentiated cells. CM also induced morphological changes in rat brain cells. The characteristic changes were as follows (Fig. 2); when the mother culture coverslips were transferred into the MCM, the first visible changes occurred as early as 8-16 h after transfer with appearance of round cells piling up on the mother culture. Those cells contained many large granules and resembled the immature macrophage-granulocyte lineage. Also, the cells started to migrate from the mother culture to the secondary coverslip. In controls, most of the cells remained on the mother culture (Fig. 2A). The cells first attaching to the secondary coverslips appeared to be dendritic (Fig. 2Ba) or spindle shaped cells (Fig. 2C, different field of 2B). Those adherent cells gradually transformed their morphology and another two cell types became prominent: cells connected with each other by long processes (Fig. 2Bb) and flat cells forming clusters (Fig. 2D). Those cells finally changed their intracellular morphology: they developed characteristic intracellular fibrous structures (Fig. 2E, arrows). We followed those morphological changes until day 21 after culture start. In order to investigate target cell specificity, cells migrated onto the secondary coverslip were treated with [3H]TdR and anti-GFAP antibodies. All anti-GFAP-positive cells incorporated [3H]TdR in the nuclei (shown as heavy grains) in response to CM (Fig. 2F, cells harvested on day 12). This tendency was also observed at two other different time points, cells harvested on day 5 and 21 (data not shown). These results suggested that target cells of CM were a glioblast-astrocyte lineage. However, mature astrocytes gradually ceased appreciable cell proliferation in MCM after morphological changes. Taken together, these results strongly indicate that CM stimulates exclusively the growth of glioblasts REFERENCES 1 Barclay, A.-N. and Ward, H.A., Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain recognized by monoclonal antibody MR( OX2. Eur. J. Biochem., 129 (1982) 447-458.
rather than that of mature astrocytes. Our present studies seemed to indicate another possible link between the thymus and CNS 16. However, the exact roles of thymic myoid cells in vivo remain unclear. A putative 'glial cell growth-promoting cytokine' produced by thymic myoid cells might be involved in generation of astrocytes in the CNS and etiology of some autoimmune disease 19. CNS also contains cells with myogenic potential. Generation of astrocytes and striated muscle cells in optic nerve cell cultures 2° may suggest a close relation between myoid cells and astrocytes in their development. In fact, our preliminary results indicated that cells with myoid potential from newborn rat brain cells stimulated the growth of glial cells. In this regard, various glial cell growth and/or maturation factors have been reported in cell extracts of brain 9-]2,~5, pituitary 2, submaxillary gland 13 and rat astrocytoma C6 m. Also, a platelet-derived growth factor (P-DGF)6 and an epidermal growth factor (EGF) 1] have been reported to affect the growth of cultured astrocytes. Besides those factors, a 'lymphokine' named GSF has been reported in culture supernatant of T and B lymphocytes stimulated with mitogens-~, 4. As for glial cell stimulating activity, CM has quite similar potency to lymphocyte-derived GSF. However, our preliminary results indicated that CM contains an IL 1, CSF and lectins beyond a glial cell growth-promoting activity. We are currently investigating whether an astrocyte differentiating activity in CM is carried by one of the above characterized molecular entities or other uncharacterized entities. ACKNOWLEDGEMENTS We thank Drs. Eiko Okada-Kobayasjai, Seung U. Kim and Kamesaburo Yoshino for helpful support. Also, we appreciate the excellent technical assistance of Ms. Aiko Sato. This work was supported by Grant no. 58570369 from the Ministry of Education of Japan.
2 Brockes, J.P., Lemke, G.E. and Balzer, D.R. Jr., Purification and preliminary characterization of a glial growth factor from the bovine pituitary, J. Biol. Chem., 255 (1980) 8374-8377. 3 Fontana, A., Dubs, R., Merchant, R., Balsiger, S. and Grob, P.J., Glia cell stimulating factor (GSF): a new lym-
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