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Chapter 85 Reconstructing the brain from immortal cell lines
D . M . tiach and J . R . Sladek, J r . (Eds.) Progress i n Brain Research, Vol. 18 t 1988 Elcevier Science Publishers B.V. (Biomedical Divirion)
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CHAPTER 85
Reconstructing the brain from immortal cell lines Ron McKay, Kristen Frederiksen, Parm-Jit Jat and Dan Levy Departments of Brain and Cognitive Science and Biology, E25-435, Massachusetts Institute of Technology, Cambridge, M A 02139, (1.S. A .
To study the molecular mechanisms of brain development we have generated clonal cell lines which can differentiate in tissue culture. The clonal cell lines were obtained by infecting primary rat central nervous system (CNS) cells with defective retroviruses carrying both a conditional oncogene and a dominant selectable marker. The dominant selectable marker conferred resistance to the cytotoxic effects of the antibiotic G418. Cell colonies resistant to the antibiotic G418 also carried an activated oncogene introduced by the retrovirus. Three different oncogenes were introduced into primary brain cells, v-myc, neu and SV40 large T antigen. We chose to use a temperature-sensitive derivative of SV40 T antigen because previous work suggested that cells could differentiate when an oncogene was inactivated (Holtzer et al., 1975; Fiszman and Fuchs, 1975). CNS precursor cell lines were established with all three retroviruses. These cell lines express functions characteristic of cells from different brain regions. The ability to generate region-specific cell lines which can differentiate has important implications for CNS transplantation experiments.
Immortalization of CNS precursors The nature of precursor-product relationships in brain development has assumed a central position in many current studies of the mammalian CNS. Experiments in invertebrates and vertebrates suggest that neurons and non-neuronal cells are derived from multipotential precursors (Ready et al., 1976; Lawrence and Green, 1979; Tomlinson and Ready, 1986; Turner and Cepko, 1987). We have shown that the monoclonal antibody Rat 401 re-
cognized CNS precursor cells in the rat (Frederiksen and McKay, 1988). Primary cell cultures from the differentiating cerebellum were infected with recombinant retroviruses carrying the tsA58 variant of SV40 large T antigen. A retrovirusimmortalized cerebellar cell line, STl5A, expresses the Rat 401 gene when grown at 33°C. When these cells are grown at 39°C T antigen is inactivated, the cells lose Rat 401 expression and gain glial fibrillary acidic protein (GFAP) expression. GFAP is an intermediate Qlament protein characteristic of astrocytes in vivo. This result suggests that STlSA cells can mimic an early step in glial fate. In this context it is interesting to ask if STl5A cells can acquire neuronal characteristics. The operational definition of a neuron is a cell containing the 200 kDa neurofilament antigen. This antigen appears very early in the differentiation of postmitotic neurons from the cerebral and cerebellar cortices (N. Valtz and R.McKay, unpublished data). The STlSA cell line expresses the 200 kDa antigen at 33 and 39°C. In the N2 serum-free medium, ST15A cells adopt an elongated morphology very similar to primary cerebellar neurons. Southern blotting shows that the ST15 cell line contains a single integrated provirus proving that the cell line is a clone. Because cell-cell interaction is likely to be important in determining cell fate during CNS development we have-co-cultured ST15A cells with primary cerebellar neurons. To follow the ST15A cells in co-culture they were internally labeled by incubation with a succinimidyl ester of fluorescein (Bronner-Fraser, 1985). This label allowed the ST15A cells to be identified in co-culture for four days. In co-culture two classes of ST15A cells were
648
found; flat, polygonal, GFAP-positive cells and neurofilament-positive cells with the morphology of neurons. These data suggest that STl5A is a clonal cell which can be manipulated in culture to adopt either a neuronal or a glial fate.
Hippocampal cell lines Cell lines were also obtained by retrovirus infection of primary hippocampal cultures obtained from embryonic day 18 animals. One of these cell lines, HT4, has been shown by Southern blot analysis to be a clone. HT4 was immortalized by the tsA58 variant of SV40 large T antigen. When grown at 33"C, HT4 cells are negative for Rat 401 and GFAP but express the 200 kDa subunit of the neurofilament triplet. If the cells are grown at 39°C with basic fibroblast growth factor they adopt a neuronal morphology by extending neurofilament-positive neurites with clear growth cones. In clonal culture the HT4 cells are GFAP negative, but when they are grown in co-culture with primary cortical cells immortalized cells can express GFAP. Our interpretation of this result is that the HT4 cell is in a state which favors neuronal differentiation but it is not committed to this fate. The differences between STI5A and HT4 cells suggest that the tsA58 oncogene can immortalize cells in different biochemical states. Changes in antigenicity can be induced in both cell types by controlling oncogene activity and extracellular signals. These changes in cell state support a model of CNS precursor cells which are multipotential.
Transplanting immortal cell lines One point of general interest is the potential clinical use of transplanted cell lines. They offer advantages as they can be carefully analyzed and genetically manipulated. However, they also suffer from the potential disadvantage that their proliferation may be uncontrolled. To test the ability of T antigen-immortalized cells to survive in the adult brain, we have transplanted a T antigen-immortalized cell line into the brain of neonatal rats. In these experiments cell lines generated by a non-condi.tional T antigen was used. The cell lines were obtained from primary cultures of embryonic day 11 rat CNS cultures.
The primary cells were placed in culture and infected the following day. After two further days in culture, G418 selection was made. Several colonies were isolated which were resistant to this selection and these cell lines were stable in culture over many months. Immunohistochemical analysis showed the cells to be strongly vimentin positive, weakly positive for the Rat 401 antigen and negative for GFAP and neurofilaments. Two of these cell lines, Al-A1 and A3-A3, were injected into the CNS of postnatal rat pups. The cells were labeled with [3H]thymidine and the DNA intercalating dye Hoechst 33258. Cell suspensions in 50 pl of media were injected either into the brain stem or into the right cerebrum of animals two to four days after birth. It was clear that only a portion of the injected material remained in the CNS after the needle was withdrawn. To calculate the delivered dose, a known number of fluorescent beads was included with the cell suspension. The number of beads found in the CNS three to five days after injection was calculated by two methods. In one case the CNS cells were dissociated using methods we have developed which provide accurate measurements of total cell number (Frederiksen and McKay, 1988). The number of fluorescent beads was counted in different brain regions. A representative animal was injected in the brain stem but beads were found distributed throughout the brain: olfactory bulb, 173; cerebrum, 1548; cerebellum, 560; rostra1 brain stem, 501 1; caudal brain stem, 2027; spinal cord, 180; total, 9499. The number of beads was also measured by fluorescent microscopy of serial-sectioned fixed brain. The total number of beads in a representative animal was 8750 and the distribution was similar to that of the dissociated preparation. From these data we estimate that lo6 cells were delivered by injection of 50 p1 of a cell suspension of 2.5 x lo6 cells. A total of 32 animals was injected and analyzed as long as 173 days after injection. Fixed brains were sectioned and analyzed by autoradiography and fluorescence microscopy. Even after long periods many thymidine and Hoechst-labeled cells were seen. Transplanted cells were found deep in brain tissue. In no case was there any evidence of tumors. These results with transplanted cells show that it
649
is possible to place large numbers of immortalized cells into the mammalian brain without gross evidence of hyperplasia. We are now setting up further transplants with conditionally immortalized cell lines. These cell lines can differentiate in tissue culture, suggesting that they may differentiate in vivo. It is also important to understand that the core body temperature of a rat is 39"C, favoring the differentiation of conditionally immortalized cell lines. These results and strategies suggest that immortal, functionally competent cell lines may allow reconstruction of the nervous system in vivo.
Acknowledgements This work has been supported by the National Institutes of Health and the Rita Allen Foundation.
References Bronner-Fraser, M. (1985) Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion. J . Cell. Biol., 101: 601-611. Fiszman, M. and Fuchs, P. (1975) Temperature sensitive expression of differentiation in myoblasts. Nature, 254: 429 - 431. Frederiksen, K. and McKay, R. (1988) Proliferation and differentiation of neuroepithelial stem cells. J . Neurosci., 8: 1144-1151. Holtzer, H.. Briehl, J., Yeoh, G., Meganathan, R. and Kaji, A. (1975) Effects of oncogenic virus on muscle differentiation. Proc. Nail. Acad. Sci., 72: 405 1 - 4055. Lawrence, P.A. and Green, S.M. (1979) Cell lineage in the developing retina of Drosophila. Dev. Biol., 71: 142- 152. Ready, D.F., Hanson, T.E. and Benzer, S. (1976) Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol., 53: 217-240. Tomlinson, A. and Ready, D.F. (1986) Sevenless, a cell specific homeotic mutation of the Drosophila retina. Science, 231: 400 - 402. Turner, D. and Cepko, C . (1987) A common progenitor for neurons and glia persists in rat retina late in development. Nature. 328: 131 - 136.
647
CHAPTER 85
Reconstructing the brain from immortal cell lines Ron McKay, Kristen Frederiksen, Parm-Jit Jat and Dan Levy Departments of Brain and Cognitive Science and Biology, E25-435, Massachusetts Institute of Technology, Cambridge, M A 02139, (1.S. A .
To study the molecular mechanisms of brain development we have generated clonal cell lines which can differentiate in tissue culture. The clonal cell lines were obtained by infecting primary rat central nervous system (CNS) cells with defective retroviruses carrying both a conditional oncogene and a dominant selectable marker. The dominant selectable marker conferred resistance to the cytotoxic effects of the antibiotic G418. Cell colonies resistant to the antibiotic G418 also carried an activated oncogene introduced by the retrovirus. Three different oncogenes were introduced into primary brain cells, v-myc, neu and SV40 large T antigen. We chose to use a temperature-sensitive derivative of SV40 T antigen because previous work suggested that cells could differentiate when an oncogene was inactivated (Holtzer et al., 1975; Fiszman and Fuchs, 1975). CNS precursor cell lines were established with all three retroviruses. These cell lines express functions characteristic of cells from different brain regions. The ability to generate region-specific cell lines which can differentiate has important implications for CNS transplantation experiments.
Immortalization of CNS precursors The nature of precursor-product relationships in brain development has assumed a central position in many current studies of the mammalian CNS. Experiments in invertebrates and vertebrates suggest that neurons and non-neuronal cells are derived from multipotential precursors (Ready et al., 1976; Lawrence and Green, 1979; Tomlinson and Ready, 1986; Turner and Cepko, 1987). We have shown that the monoclonal antibody Rat 401 re-
cognized CNS precursor cells in the rat (Frederiksen and McKay, 1988). Primary cell cultures from the differentiating cerebellum were infected with recombinant retroviruses carrying the tsA58 variant of SV40 large T antigen. A retrovirusimmortalized cerebellar cell line, STl5A, expresses the Rat 401 gene when grown at 33°C. When these cells are grown at 39°C T antigen is inactivated, the cells lose Rat 401 expression and gain glial fibrillary acidic protein (GFAP) expression. GFAP is an intermediate Qlament protein characteristic of astrocytes in vivo. This result suggests that STlSA cells can mimic an early step in glial fate. In this context it is interesting to ask if STl5A cells can acquire neuronal characteristics. The operational definition of a neuron is a cell containing the 200 kDa neurofilament antigen. This antigen appears very early in the differentiation of postmitotic neurons from the cerebral and cerebellar cortices (N. Valtz and R.McKay, unpublished data). The STlSA cell line expresses the 200 kDa antigen at 33 and 39°C. In the N2 serum-free medium, ST15A cells adopt an elongated morphology very similar to primary cerebellar neurons. Southern blotting shows that the ST15 cell line contains a single integrated provirus proving that the cell line is a clone. Because cell-cell interaction is likely to be important in determining cell fate during CNS development we have-co-cultured ST15A cells with primary cerebellar neurons. To follow the ST15A cells in co-culture they were internally labeled by incubation with a succinimidyl ester of fluorescein (Bronner-Fraser, 1985). This label allowed the ST15A cells to be identified in co-culture for four days. In co-culture two classes of ST15A cells were
648
found; flat, polygonal, GFAP-positive cells and neurofilament-positive cells with the morphology of neurons. These data suggest that STl5A is a clonal cell which can be manipulated in culture to adopt either a neuronal or a glial fate.
Hippocampal cell lines Cell lines were also obtained by retrovirus infection of primary hippocampal cultures obtained from embryonic day 18 animals. One of these cell lines, HT4, has been shown by Southern blot analysis to be a clone. HT4 was immortalized by the tsA58 variant of SV40 large T antigen. When grown at 33"C, HT4 cells are negative for Rat 401 and GFAP but express the 200 kDa subunit of the neurofilament triplet. If the cells are grown at 39°C with basic fibroblast growth factor they adopt a neuronal morphology by extending neurofilament-positive neurites with clear growth cones. In clonal culture the HT4 cells are GFAP negative, but when they are grown in co-culture with primary cortical cells immortalized cells can express GFAP. Our interpretation of this result is that the HT4 cell is in a state which favors neuronal differentiation but it is not committed to this fate. The differences between STI5A and HT4 cells suggest that the tsA58 oncogene can immortalize cells in different biochemical states. Changes in antigenicity can be induced in both cell types by controlling oncogene activity and extracellular signals. These changes in cell state support a model of CNS precursor cells which are multipotential.
Transplanting immortal cell lines One point of general interest is the potential clinical use of transplanted cell lines. They offer advantages as they can be carefully analyzed and genetically manipulated. However, they also suffer from the potential disadvantage that their proliferation may be uncontrolled. To test the ability of T antigen-immortalized cells to survive in the adult brain, we have transplanted a T antigen-immortalized cell line into the brain of neonatal rats. In these experiments cell lines generated by a non-condi.tional T antigen was used. The cell lines were obtained from primary cultures of embryonic day 11 rat CNS cultures.
The primary cells were placed in culture and infected the following day. After two further days in culture, G418 selection was made. Several colonies were isolated which were resistant to this selection and these cell lines were stable in culture over many months. Immunohistochemical analysis showed the cells to be strongly vimentin positive, weakly positive for the Rat 401 antigen and negative for GFAP and neurofilaments. Two of these cell lines, Al-A1 and A3-A3, were injected into the CNS of postnatal rat pups. The cells were labeled with [3H]thymidine and the DNA intercalating dye Hoechst 33258. Cell suspensions in 50 pl of media were injected either into the brain stem or into the right cerebrum of animals two to four days after birth. It was clear that only a portion of the injected material remained in the CNS after the needle was withdrawn. To calculate the delivered dose, a known number of fluorescent beads was included with the cell suspension. The number of beads found in the CNS three to five days after injection was calculated by two methods. In one case the CNS cells were dissociated using methods we have developed which provide accurate measurements of total cell number (Frederiksen and McKay, 1988). The number of fluorescent beads was counted in different brain regions. A representative animal was injected in the brain stem but beads were found distributed throughout the brain: olfactory bulb, 173; cerebrum, 1548; cerebellum, 560; rostra1 brain stem, 501 1; caudal brain stem, 2027; spinal cord, 180; total, 9499. The number of beads was also measured by fluorescent microscopy of serial-sectioned fixed brain. The total number of beads in a representative animal was 8750 and the distribution was similar to that of the dissociated preparation. From these data we estimate that lo6 cells were delivered by injection of 50 p1 of a cell suspension of 2.5 x lo6 cells. A total of 32 animals was injected and analyzed as long as 173 days after injection. Fixed brains were sectioned and analyzed by autoradiography and fluorescence microscopy. Even after long periods many thymidine and Hoechst-labeled cells were seen. Transplanted cells were found deep in brain tissue. In no case was there any evidence of tumors. These results with transplanted cells show that it
649
is possible to place large numbers of immortalized cells into the mammalian brain without gross evidence of hyperplasia. We are now setting up further transplants with conditionally immortalized cell lines. These cell lines can differentiate in tissue culture, suggesting that they may differentiate in vivo. It is also important to understand that the core body temperature of a rat is 39"C, favoring the differentiation of conditionally immortalized cell lines. These results and strategies suggest that immortal, functionally competent cell lines may allow reconstruction of the nervous system in vivo.
Acknowledgements This work has been supported by the National Institutes of Health and the Rita Allen Foundation.
References Bronner-Fraser, M. (1985) Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion. J . Cell. Biol., 101: 601-611. Fiszman, M. and Fuchs, P. (1975) Temperature sensitive expression of differentiation in myoblasts. Nature, 254: 429 - 431. Frederiksen, K. and McKay, R. (1988) Proliferation and differentiation of neuroepithelial stem cells. J . Neurosci., 8: 1144-1151. Holtzer, H.. Briehl, J., Yeoh, G., Meganathan, R. and Kaji, A. (1975) Effects of oncogenic virus on muscle differentiation. Proc. Nail. Acad. Sci., 72: 405 1 - 4055. Lawrence, P.A. and Green, S.M. (1979) Cell lineage in the developing retina of Drosophila. Dev. Biol., 71: 142- 152. Ready, D.F., Hanson, T.E. and Benzer, S. (1976) Development of the Drosophila retina, a neurocrystalline lattice. Dev. Biol., 53: 217-240. Tomlinson, A. and Ready, D.F. (1986) Sevenless, a cell specific homeotic mutation of the Drosophila retina. Science, 231: 400 - 402. Turner, D. and Cepko, C . (1987) A common progenitor for neurons and glia persists in rat retina late in development. Nature. 328: 131 - 136.