Reconstituting
animals
from
immortal
precursors
Ron McKay Massachusetts Several
institute
recent
cells retain
experiments
cells in the
animal. in
In addition, suggest that
approaches the ordered
Current
the the
immortal
Opinion
Several groups have recently made use of retroviral transduction of an oncogene to immortalize cells from the developing brain [3-91. Two strategies have been used. In the first, cell lines have been derived from transgenic animals expressing oncogenes under the control of celltype-specific promoters. Two recent examples show the successful application of this approach [ 10,111. In both cases cell lines were derived from tumors that occurred in transgenic mice as a result of the expression of the oncogene in restricted cell types by a cell-specific promoter. The second strategy uses somatic cell fusion of a tumor cell with a primary cell to introduce the oncogene into the cell type of choice. This procedure is based on the initial methods for generating somatic cell hybrids that were applied with such success to hybridomas. Cell lines from the nemous system have also been derived by cell fusion techniques [ 12,131, and cell lines derived in this
in vivo.
the The
582
Current
to of
lead
that generate
1992, 2:582-585
way are used as model systems for neuronal properties [ 14,15*]. As Han et al. [ 15.1 show, these hybrid cells can generate structures that have many of the biochemical and ultrastructural features of synapses. Although many hybrid cell lines have useful properties, the phenotype of the hybrid cell is often a complex product of nuclear and cytoplasmic factors in the interacting cell types. This is particularly important because the differentiated state is not fixed and genes characteristic of a new differentiated state can be induced in terminally differentiated cells. In the above experiments both the transgenic tumor cell lines and the primaty tumor cell develop into fully transformed cells. While these methods are clearly generating interesting cell lines, the rest of this review will be concerned with cell lines that are not fully transformed. These are generated by introducing single oncogenes into primary cells. The lack of transformation is compatible with a more extensive differentiation than had previously been thought possible. precursors
of the nervous
system
The many molecularly distinct neurons in the adult brain are derived from multipotential precursors. The processes regulating the differentiation of these multipotential stem cells to multiple fates are of great intrinsic interest and are also relevant to understanding neurodegenerative diseases. In the central nervous system, the differentiation of neurons is a precisely timed process in which large numbers of neuronal precursors are present transiently during development and are absent or much reduced in number in the adult brain [7]. The neuronal precursor cell is recognized by its expression of nestin, an intermediate filament protein that is specifically expressed in this cell type [ 161. Nestin expression is first seen in the neuroepithelium following gastrulation, and nestin-positive cells are abundant in regions of the brain before neurons differentiate. Nestin-positive cells can divide in vitro and when growth conditions are altered they differentiate into neurons [ 171. The transient abundance of central nervous system neuroepithelial
light chain; ts-temperature
Biology
ability results
cell lines may
Abbreviations MLC-myosin
and muscle
of their precursor
the mechanisms
Immortal
methods
USA
of cell types in mammals.
in Neurobiology
It is clear to virologists that viruses interact in specific ways with different host cells. A good example is the avian erythroblastosis virus, which contains two immortalizing genes, v-erbA and v-erbB. These interact in a very elegant way with erythroblasts at a specific stage of their differentiation [ 1,2]. There are many other examples of a specific interaction between an oncogene and a particular cell type. It is also clear, however, that some oncogenes act on widely different cell types. Recent work has generated cell lines that represent precursor states found in vivo using retrovimlly transduced oncogenes to immor talize cells. Immortal precursor cells differentiate in interesting ways both in culture and after grafting into a host. The ability to systematically generate immortal precursor cells will be a powerful tool in the analysis of mammalian development. The application of cell lines to outstanding issues in this field will form the central topic of this brief review.
neural
cells retain
use of precursor
for understanding distribution
Introduction
Immortalization
that immortal
and spatial properties
vitro and, after transplantation,
experiments
to powerful
Cambridge, Massachusetts,
have shown
the specific temporal
differentiate these
of Technology,
sensitive.
Ltd ISSN 0959-4388
Reconstituting
stem cells makes them dillicult to identify and characterize. From a theoretical and practical viewpoint it is clearly interesting to ask whether nestin-positive precursor cells can be captured by oncogene-mediated immortalization. Nestin-positive cell lines can be generated by infecting primary cells in culture with retroviruses transducing oncogenes. The retroviral technology allows different immortalizing oncogenes to be expressed from the same promoter/enhancer elements. Thus, the immortalizing efftciency of different oncogenes can be directly compared. When three different oncogenes, SV40 T-antigen, v-myc and neu were used to infect cells derived from the developing cerebellum, all three were capable of establishing nestin-positive cell lines [ 181. The neu immortaized lines grew slowly relative to the T-antigen and myc immortalized cells. These results suggest that although the nestin-positive state is transient in zlivo, it is cell autonomous, and that if a cell is induced to proliferate by the expression of an oncogene it can be maintained in the nestin-positive condition. A more demanding question is whether these immortal cells are still capable of differentiating. Experiments in vitro support the view that differentiation can occur. Nestin-positive cerebellar cell lines immortalized with either T-antigen or v-myc can undergo partial differentiation in culture [ 181. The T-antigen-derived cells were immortalized with a temperature sensitive (ts) allele of the T-antigen (tsA58). The permissive temperature for tsA58 is 33°C and the non-permissive temperature 39°C. Simply shifting the cells to 39°C is sufficient to drive the cells through the early steps of neuronal differentiation [ 191. Surprisingly, if the culture medium contains horse serum then these same cells will differentiate in vitro into muscle [20]. In this respect the immortal cells behave similarly to a particular cerebellar tumor cell, the medulloblastoma. Results show that nestin-positive cerebellar cells can differentiate in vitro to a limited extent, The oligodendrocyte precursor is one of the best characterized precursor states in the central nervous system. It can be readily identified in the optic nerve by the specific expression of a glycolipid recognized by the man oclonal antibody A2B5. A2B5-positive cell lines can be derived from the optic nerve by T-antigen immortalization, and these cells can be induced to differentiate, losing A2B5 expression and gaining the expression of genes characteristic of differentiated oligodendrocytes [3]. It is interesting to note that although there are nestin-positive cells in the developing optic nerve, the cell line, tsUl95, which has been most extensively characterized as an A2B5-positive oligodendrocyte precursor, is nestin negative. The results show that nestin expression is not a default state for an immortalized central nervous system cell. They also show that T-antigen can immortalize different types of precursor cells. Neuronal precursor cells immortalized by v-myc can also differentiate in vitro. The best example is a cell line derived from the neural crest. In this case the cells used for immortalization were derived from the developing adrenal medulla, which differentiate into sympathetic neurons [5]. At present, the differentiation of this cell
animals
from
immortal
precursors
McKay
line in vitro is inefficient, but this may simply be a consequence of multiple differentiation factors being required. This cell line is important as it provides a system that allows the factors regulating neuronal differentiation to be defined. Muscle precursor
lines retain positional
value
In the embryo very few cells are transiently precursors, but oncogenes can immortalize precursor states of interest in both neuronal and glial systems. A temperaturesensitive oncogene offers a simple strategy for inducing differentiation, and one of the best examples is the differentiation of muscle cells from immortal cells [20]. This strategy has recently been used to immortalize precursor cells from different muscle types in the mouse [21*-l. The primary cells were obtained from a transgenic iine in which enhancer elements from the myosin light chain (MIC) promoter were linked to the E. coli P-galactosidase gene. It was observed that in these mice the E. coligene was expressed in differentiated intercostal muscle cells in a gradient that increased loo-fold as cells from increasing caudal muscles were tested. This gradient was also found when cells from different intercostal muscles were placed in primary culture. In contrast, the acetylcholine receptor gene showed no gradient of expression. Cells from different adult muscles were placed in culture and immortalized with a retrovirus transducing the tsA58 allele of SV40 T-antigen. The cell lines derived were myoblasts. These precursors do not express P-galactosidase, but when the temperature is raised the T-antigen is rapidly degraded and the cells can be induced to differentiate into myotubes. The MLC enhancer/promoter is activated by differentiation and the level of fi-galactosidase activity reflects the location of the muscle from which the cell lines were originally generated. The conclusion is that positional values acquired by the cells during development are retained through long periods of culture after immortalization. The implication is that this family of cell lines will be important for understanding the cell autonomous mechanisms that specify these positional values. The availability of cell lines that retain a memory of their axial location opens up new approaches for studying this key feature of embryonic patterning. Chimeric
brains
The review has so fdr been concerned with in z&To differentiation paradigms, but I will now turn to the differentiation of immortal cells in viz)o.If transplanted precursor cells differentiate in vivo then animals could be generated that carry immortal cells integrated into specific host tissues. Experiments from two laboratories suggest that chimeric animals of this kind can be generated. Both groups have generated cell lines from the developing brain that differentiate into neurons and glia when transplanted back into the brain of newborn rats [ 22,23**,24**]. In both cases cells were placed into a host site at a developmental stage when neurons were being generated in large numbers. Although the two groups differed in the
583
584
Neuronal
and glial cell biology
labelling methods they used to follow and analyze the transplanted cells, they both reached the same general conclusion. Immortal precursors integrate and differentiate in the host with no sign of tumor formation. In one set of experiments, the immortal cells were placed in two sites in the developing brain, the original site that was the source of the immortal cells and a second site [23-l. Neurons were derived from the transplanted cells at both sites, and were characteristic of the transplant site rather than the site of origin of the transplanted cells. The main conclusion of this experiment is that immortal stem cells can respond to local signals in different regions of the developing brain and become recruited to local fates. It is possible that the immortal cells are unusual in this regard as they may have lost their regional specification in prolonged culture, but the experiments on muscle precursor lines show that this is not necessarily the case. Both transplant studies show that established cell lines can be grown for long periods in culture and still retain the ability to differentiate in response to signals present in vivo. This result needs to be extended to obtain quantitative measures of differentiation, but it already holds the promise for the development of powerful new assays for the molecular mechanisms underlying brain development. The main point to stress here is that the immortal cells give rise to neurons that are present in the adult brain, and that these neurons appear to be integrated into the circuitry of the brain. The main objection to the use of cell lines is that they do not represent the cell types found in viva. While this may be true in some circumstances, the two studies discussed here suggest that under appropriate conditions immortal precursor cells can differentiate into neurons that are closely related to the neuronal types normally found in rho. Of mice and man mammalian
new methods
in
genetics
The potential importance of the ability to reconstruct animals from immortal cell lines encourages a re-examination of the simple question: what is a cell line? The concept of a cell line is closely related to the ideas of senescence and death. It is widely accepted that pnmat-y embryonic libroblasts show senescence, and many cultured cells of different kinds will not divide indefinitely in culture. It may also be the case that cell death is a common phenomenon in vivo [ 251. The accepted view is that cells are mortal, i.e. they undergo a finite number of divisions and then die. The idea that cell senescence/death is a cell autonomous property of most cell types is not uniformly supported by experiments in vitro, however. For example, myoblasts can be maintained in culture for very long periods by the appropriate growth factors. In addition defined cytokines can cause a great expansion of lymphocytes. The large scale expansion of primary cells is also used routinely in skin culture treatments for burn victims. Embryonic stem cells are used in mouse genetics because these cells can be cultured indefinitely without immortalization [2628]. In all of these cases there is good evidence that the cells retain their ‘primary’ nature even though they have been kept for long periods in
culture. In the case of embryonic stem cells, completely normal animals can be derived from cultured cells. The production of a transgenic mouse in which the tsA58 allele of SV40 T-antigen is driven from the H-2Kb promoter should allow a new strategy for immortalizing cells from the developing mouse [29]. This promoter is induced in the presence of y-interferon. Culture data on skin fibroblasts and thymocytes suggest that the presence of the inducing agent and a non-permissive temperature for the &A58 allele are required for the growth of some cell lines. In principle, this transgenic mouse strain may permit the derivation of many cell lines in a double conditional manner, dependent on temperature and y-interferon. The most common view of cell lines is that they require activated oncogenes for continued growth, but the above examples show that there is not a sharp boundary between a primary cell and a transformed cell. In view of these recent experiments, it seems reasonable to adopt a very flexible view of what a cell line is and how a cell line relates to cell types found in vizm. Perspective It has been known since the pioneering aggregation chimera studies of Mintz and colleagues that teratocarcinema cells contribute to many tissues of the adult mouse [30]. Aggregation chimeras obtained by introducing embryonic stem cells into blastulae have been used to generate new strains of mice carrying mutations that were introduced into the embryonic stem cells in culture. It is possible to mutate both alleles of a gene in culture allowing recessive phenotypes to be analyzed [31,32]. The homologous recombination mechanisms that allow directed mutation in embryonic stem cells are found in all cells; indeed, homologous recombination in cultured mammalian cells was first observed in somatic cells. These techniques permit powerful analyses of gene function in transgenic mice, but the same homologous recombination techniques may be applied to immortalized precursor cells of any species. After transplantation into a host, the cells will integrate and differentiate. The phenotypes of the mutations can then be assessed in chimeric animals. This approach to genetics may have important applications. There are some problems in basic science and clinical medicine that would be best addressed in mammalian species other than rodents. For example, much of our knowledge of integrated cellular functions in neurobiology comes from studies of the visual system in carnivores and primates. The potential for genetic analysis in chimeric animals and tissue engineering in man will ensure that these new approaches are actively explored. References Papers of view, have . of .. of 1.
and recommended
particular interest, published been highlighted as: special interest outstanding interest
GANDRIIUJN
reading
within the annual period of re-
0, Jrrfmc P, PAIN C, DESBOIS B, MADJARJJ, MOSCO~ICI MG, MOSCO~I C, SAMARUTJ: Expression of v-
Reconstituting
erbA, an Altered Nuclear Hormone Receptor, is Sufficient to Transform Erythrocytic CeUs In Vitro. Cell 1989, 58:115-121. 2.
PAIN B, WOODS CM, PE-YROL S, Moscovlc~ as a Hematopoietic Present in Normal and Controls Their
3.
ALMAZXN G, MCKAYRDG: An Oligodendrocyte Precursor CelI Line from Rat Optic Nerve. Bruin Res 1992, 579:234-245.
4.
BARTLETTPF, REm HH, BAIIEY KA, BERNAKD0: ImmortaIiza-
tion of Mouse Neural Precursor Cells by the c-myc gene. Pm Natl Acad Sci lJsA 1988, 85:3255-3259. 5.
BIRREN SJ, ANI)ERSON DJ: A v-myc
adrenal Progenitor tiation is Initiated 4:189-201. 6.
7.
Onco-
ImmortaUzed SympathoCeU Line in which Neuronal Differenby FGF and not NGF. Neuron 1990,
E\w> C, BORDE I, MAR~N P, GAUNA E, PRFMONT J, GROS F: Immortalization of BipotentiaI and Plastic Glio-Neuronal Precursor Cells. Pra Nutl Acad Sci USA 1990, 87:3062-3066 FREDERIKSONK, MCKAY R: Proliferation
of Rat NeuroepitheUaI
and Differentiation Cells In Vivo. .I Neurosci
Precursor
1988, 8:1144-1151. 8.
&DIES C, LENDAI~LLJ, MCKAY RDG: Differentiation
erogeneity in T-Antigen from Mouse Cerebellum.
and HetImmortalized Precursor CeU Lines J Neurmci Res 1991, 30:601415.
from
immortal
precursors
McKay
19.
of CDC2 HAVESTE, VAL~L NLM, MCKAY RDG: Downregulation upon Terminal Differentiation of Neurons. New Biologist 1991, 3~259-269.
20.
VALXZN, NOREGAARDT, HAVES T, Liu S, MCKAY R: An Embryonic Origin for MeduUoblastoma. Neu) Biologist 1991, 3:364-371.
21. ..
D~NOGHUE MJ, MORRIS-VALERO R, JOHNSON YR, MERLIEJP, SANES JR: Mammalian Muscle Cells Bear a Cell-Autonomous Heritable Memory of Their Rostro-CaudaI Position. Cell 1992,
SAEZJ, FUCKINGERT, RAIN% M, KUNG H.J,
C, MOSCOVICIMG, JURDIC P, et al.: EGF-R Growth Factor: the c-erbB Product is Chicken Erythrocytic Progenitor Cells Self-Renewal. Cell 1991, 65:3746.
animals
6967-78. Cell lines ate derived from adult muscle that differentiate
into myotubes. The donor for the cell lines was a mouse that carried a transgene expressed in a caudo-rostra1 gradient. The cell lines express this gene to the same relative extent a? the cells of origin. 22.
MCKAY RDG, CUNNINGHAMMG, VALTZN, HAYEST: Embryonic
Mechanisms Regulating the Number and Type of Cells in the Mammalian Central Nervous System. Cold Spring Hur6 hjmp Quant Biol 1990, 55:291-301. 23.
KENFRANZPJ, CLINNINGHA~IMG, MCKAY RDG: Region-Specific Differentiation of the Hippocampai Stem CeU Line HiB5 upon Implantation into the Developing Mammalian Brain. Cell 1991, 66:713_729. Demonstrates that immortal cells integrate and differentiate following transplantation into the hippocampus and cerebellum of a newborn rat. ..
24. ..
SNV~EREY, DEITCHERDL, WALSH C, ARNOU)-ALDEAS, HARTWEIG Neural CeU Lines can Engraft and EA, CEPKO CL: Multipotent
9.
RXXW EF, SNYDER EY, CEIJK~ CL: Establishment and Characterization of Multipotent Neural CeU Lines Using Retrovirus-Mediated Oncogene Transfer. .I Nczrrohiol 1989. 2 1:356-375.
Participate in the Development of the Mouse Cerebellum. Cell 1992, 68:33-51. Demonstrates that immortal cells integrate and differentiate following transplantation into the cerebellum of a newborn rat.
10.
HAMMANG JP, BAETGI: FE, BEHIRINCERRR, BIUNSTERRL, PAL~IITER Retinal Neurons Derived from RD, MESSINGA: Immortalized SV40 T-Antigen-Induced Tumors in Transgenic Mice. Neuro,~ 1990, 4x775-782.
25.
WF MC: Social Controls Nuture 1992, 356:397-400.
26.
BRADLEYA, EVANS MJ, KAUFMAN MII, ROBERTSONE: Formation of Germ-Line Chimeras from Embryo-Derived Teratocarcinoma CeU Lines. Nature 1984. 309:255256.
11.
12.
M~ll.0~ PL, WINDLE JJ, GOLDSMITH PC, PAD~IA CA, Ro~w?la JI., WEINERRI: Immortalization of Hypothalamic GnRH Neurons by Genetically Targeted Tumorigenesis. Neuron 1990, 5:1-IO. GWXNE I& SWN W. CHAIAXONITI~ A, BKEAKUEU) X, MINNA HG, NIKE~EHG M: Neuronal Properties of Hybrid Neuroblastoma x Sympathetic Ganglion Cells. Proc Null Acad Sci IISA 1975, 72:49234927.
J, Coon
13.
14.
Krr~ WA, NIRENHEWM: A Neuroblastoma CeU Line with Morphine Receptors. tkx 1974, 69:3474-3477.
x GIioma
Nutl Aad
Hybrid Sci USA
BLIJSZTAJNJK, VENT~JRINIA, JACKSON DA, L!& HJ, WAINEK
27.
PWAIONNOLI VE, ROSSANTJ: Effects
ronment on Proliferation Carcinoma Cells. Cuncer 28.
on CeU Survival and CeU Death.
ROI$EKTSON E,
of the Embryonic Enviand Differentiation of EmbryonaI Surrs 1983, 2:165-172.
BRAZIER A,
KIIEHN M,
EvxNb
Line Transmission of Genes Introduced PluripotentiaI Cells by RetroviraI Vectors. 323:445-448. .
M; Germ into Cultured Nuture 1986,
JAM’PS, NOBLE MD, ATALIOTISP, TANAKA Y, YANNOUTSo5 N, LARSENL, KIOLISSISD: Direct Derivation of Conditionally Immortal CeU Lines from an H-2Kb-tsA58 Transgenic Mouse. PYOC Nat1 Acud Sci USA 1991, 88:50965100. Describes a potentially general strategy for obtaining immortal cell lines.
29. .
BH: Acetylcholine Synthesis and Release is Enhanced by Dibutryl c-AMP in a Neuronal CeU Line Derived From Mouse Septum. J Nettrrxci 1992, 12:793-799.
30.
lli\N H-Q, NIC~~OISRA, RI!HIN MR, BAH~~K M, GKEENC;ARDP: Induction of Formation of Presynaptic Terminals in Neuroblastoma Cells by Synapsin IIb. Nature 1991. 349:697-700. Describes the genetic modilication of synapse formation in a cell line.
DEWT:Y MJ, MARTIN DW, MARTIN GR, MINTZ B: Mosaic Mice with Teratocarcinoma-Derived Mutant Cells Deficient in Hypoxanthine Phosphoribosyltransferase. Proc N&l Acud Sci USA 1977, 74:5564-556X.
31.
Hksn P, RAMI~ZSOLISR, KRIIM~ALIFR, BRADIEYA: Introduction of a Subtle Mutation into the Hox-2.6 Locus in Embryonic Stem Cells. Nutt!re 1991, 350:243-246.
32.
VAIANCI~~SV, SMITHIES0: Testing
15. .
16.
~NDAHL IJ, ZIMMERMANL, MCKAY RDG:
Express a New 60:1585-1595. 17.
Intermediate
CNS Stem Cells Protein. Ccl/ 1990,
CATTANEO E, MCKAY R: Proliferation
Neuronal Stem Cells Regulated Nature 199Q, 347~762-765. 18.
Filament
and Differentiation of by Nerve Growth Factor.
FREDERIKSEN K, JAT PS, VAL?“% N, LEVYD, MCKAY R: Immmtab ization of Precursor Cells from the MammaIian CNS. Neuroll 1988, 1:43%448.
an ‘in-out’ Targeting Procedure for Making Subtle Genomic Modifications in Mouse Embryonic Stem Cells. ,1fol CeN Biol 1991, 11:1402-1408.
R McKay, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
585
animals
from
immortal
precursors
Ron McKay Massachusetts Several
institute
recent
cells retain
experiments
cells in the
animal. in
In addition, suggest that
approaches the ordered
Current
the the
immortal
Opinion
Several groups have recently made use of retroviral transduction of an oncogene to immortalize cells from the developing brain [3-91. Two strategies have been used. In the first, cell lines have been derived from transgenic animals expressing oncogenes under the control of celltype-specific promoters. Two recent examples show the successful application of this approach [ 10,111. In both cases cell lines were derived from tumors that occurred in transgenic mice as a result of the expression of the oncogene in restricted cell types by a cell-specific promoter. The second strategy uses somatic cell fusion of a tumor cell with a primary cell to introduce the oncogene into the cell type of choice. This procedure is based on the initial methods for generating somatic cell hybrids that were applied with such success to hybridomas. Cell lines from the nemous system have also been derived by cell fusion techniques [ 12,131, and cell lines derived in this
in vivo.
the The
582
Current
to of
lead
that generate
1992, 2:582-585
way are used as model systems for neuronal properties [ 14,15*]. As Han et al. [ 15.1 show, these hybrid cells can generate structures that have many of the biochemical and ultrastructural features of synapses. Although many hybrid cell lines have useful properties, the phenotype of the hybrid cell is often a complex product of nuclear and cytoplasmic factors in the interacting cell types. This is particularly important because the differentiated state is not fixed and genes characteristic of a new differentiated state can be induced in terminally differentiated cells. In the above experiments both the transgenic tumor cell lines and the primaty tumor cell develop into fully transformed cells. While these methods are clearly generating interesting cell lines, the rest of this review will be concerned with cell lines that are not fully transformed. These are generated by introducing single oncogenes into primary cells. The lack of transformation is compatible with a more extensive differentiation than had previously been thought possible. precursors
of the nervous
system
The many molecularly distinct neurons in the adult brain are derived from multipotential precursors. The processes regulating the differentiation of these multipotential stem cells to multiple fates are of great intrinsic interest and are also relevant to understanding neurodegenerative diseases. In the central nervous system, the differentiation of neurons is a precisely timed process in which large numbers of neuronal precursors are present transiently during development and are absent or much reduced in number in the adult brain [7]. The neuronal precursor cell is recognized by its expression of nestin, an intermediate filament protein that is specifically expressed in this cell type [ 161. Nestin expression is first seen in the neuroepithelium following gastrulation, and nestin-positive cells are abundant in regions of the brain before neurons differentiate. Nestin-positive cells can divide in vitro and when growth conditions are altered they differentiate into neurons [ 171. The transient abundance of central nervous system neuroepithelial
light chain; ts-temperature
Biology
ability results
cell lines may
Abbreviations MLC-myosin
and muscle
of their precursor
the mechanisms
Immortal
methods
USA
of cell types in mammals.
in Neurobiology
It is clear to virologists that viruses interact in specific ways with different host cells. A good example is the avian erythroblastosis virus, which contains two immortalizing genes, v-erbA and v-erbB. These interact in a very elegant way with erythroblasts at a specific stage of their differentiation [ 1,2]. There are many other examples of a specific interaction between an oncogene and a particular cell type. It is also clear, however, that some oncogenes act on widely different cell types. Recent work has generated cell lines that represent precursor states found in vivo using retrovimlly transduced oncogenes to immor talize cells. Immortal precursor cells differentiate in interesting ways both in culture and after grafting into a host. The ability to systematically generate immortal precursor cells will be a powerful tool in the analysis of mammalian development. The application of cell lines to outstanding issues in this field will form the central topic of this brief review.
neural
cells retain
use of precursor
for understanding distribution
Introduction
Immortalization
that immortal
and spatial properties
vitro and, after transplantation,
experiments
to powerful
Cambridge, Massachusetts,
have shown
the specific temporal
differentiate these
of Technology,
sensitive.
Ltd ISSN 0959-4388
Reconstituting
stem cells makes them dillicult to identify and characterize. From a theoretical and practical viewpoint it is clearly interesting to ask whether nestin-positive precursor cells can be captured by oncogene-mediated immortalization. Nestin-positive cell lines can be generated by infecting primary cells in culture with retroviruses transducing oncogenes. The retroviral technology allows different immortalizing oncogenes to be expressed from the same promoter/enhancer elements. Thus, the immortalizing efftciency of different oncogenes can be directly compared. When three different oncogenes, SV40 T-antigen, v-myc and neu were used to infect cells derived from the developing cerebellum, all three were capable of establishing nestin-positive cell lines [ 181. The neu immortaized lines grew slowly relative to the T-antigen and myc immortalized cells. These results suggest that although the nestin-positive state is transient in zlivo, it is cell autonomous, and that if a cell is induced to proliferate by the expression of an oncogene it can be maintained in the nestin-positive condition. A more demanding question is whether these immortal cells are still capable of differentiating. Experiments in vitro support the view that differentiation can occur. Nestin-positive cerebellar cell lines immortalized with either T-antigen or v-myc can undergo partial differentiation in culture [ 181. The T-antigen-derived cells were immortalized with a temperature sensitive (ts) allele of the T-antigen (tsA58). The permissive temperature for tsA58 is 33°C and the non-permissive temperature 39°C. Simply shifting the cells to 39°C is sufficient to drive the cells through the early steps of neuronal differentiation [ 191. Surprisingly, if the culture medium contains horse serum then these same cells will differentiate in vitro into muscle [20]. In this respect the immortal cells behave similarly to a particular cerebellar tumor cell, the medulloblastoma. Results show that nestin-positive cerebellar cells can differentiate in vitro to a limited extent, The oligodendrocyte precursor is one of the best characterized precursor states in the central nervous system. It can be readily identified in the optic nerve by the specific expression of a glycolipid recognized by the man oclonal antibody A2B5. A2B5-positive cell lines can be derived from the optic nerve by T-antigen immortalization, and these cells can be induced to differentiate, losing A2B5 expression and gaining the expression of genes characteristic of differentiated oligodendrocytes [3]. It is interesting to note that although there are nestin-positive cells in the developing optic nerve, the cell line, tsUl95, which has been most extensively characterized as an A2B5-positive oligodendrocyte precursor, is nestin negative. The results show that nestin expression is not a default state for an immortalized central nervous system cell. They also show that T-antigen can immortalize different types of precursor cells. Neuronal precursor cells immortalized by v-myc can also differentiate in vitro. The best example is a cell line derived from the neural crest. In this case the cells used for immortalization were derived from the developing adrenal medulla, which differentiate into sympathetic neurons [5]. At present, the differentiation of this cell
animals
from
immortal
precursors
McKay
line in vitro is inefficient, but this may simply be a consequence of multiple differentiation factors being required. This cell line is important as it provides a system that allows the factors regulating neuronal differentiation to be defined. Muscle precursor
lines retain positional
value
In the embryo very few cells are transiently precursors, but oncogenes can immortalize precursor states of interest in both neuronal and glial systems. A temperaturesensitive oncogene offers a simple strategy for inducing differentiation, and one of the best examples is the differentiation of muscle cells from immortal cells [20]. This strategy has recently been used to immortalize precursor cells from different muscle types in the mouse [21*-l. The primary cells were obtained from a transgenic iine in which enhancer elements from the myosin light chain (MIC) promoter were linked to the E. coli P-galactosidase gene. It was observed that in these mice the E. coligene was expressed in differentiated intercostal muscle cells in a gradient that increased loo-fold as cells from increasing caudal muscles were tested. This gradient was also found when cells from different intercostal muscles were placed in primary culture. In contrast, the acetylcholine receptor gene showed no gradient of expression. Cells from different adult muscles were placed in culture and immortalized with a retrovirus transducing the tsA58 allele of SV40 T-antigen. The cell lines derived were myoblasts. These precursors do not express P-galactosidase, but when the temperature is raised the T-antigen is rapidly degraded and the cells can be induced to differentiate into myotubes. The MLC enhancer/promoter is activated by differentiation and the level of fi-galactosidase activity reflects the location of the muscle from which the cell lines were originally generated. The conclusion is that positional values acquired by the cells during development are retained through long periods of culture after immortalization. The implication is that this family of cell lines will be important for understanding the cell autonomous mechanisms that specify these positional values. The availability of cell lines that retain a memory of their axial location opens up new approaches for studying this key feature of embryonic patterning. Chimeric
brains
The review has so fdr been concerned with in z&To differentiation paradigms, but I will now turn to the differentiation of immortal cells in viz)o.If transplanted precursor cells differentiate in vivo then animals could be generated that carry immortal cells integrated into specific host tissues. Experiments from two laboratories suggest that chimeric animals of this kind can be generated. Both groups have generated cell lines from the developing brain that differentiate into neurons and glia when transplanted back into the brain of newborn rats [ 22,23**,24**]. In both cases cells were placed into a host site at a developmental stage when neurons were being generated in large numbers. Although the two groups differed in the
583
584
Neuronal
and glial cell biology
labelling methods they used to follow and analyze the transplanted cells, they both reached the same general conclusion. Immortal precursors integrate and differentiate in the host with no sign of tumor formation. In one set of experiments, the immortal cells were placed in two sites in the developing brain, the original site that was the source of the immortal cells and a second site [23-l. Neurons were derived from the transplanted cells at both sites, and were characteristic of the transplant site rather than the site of origin of the transplanted cells. The main conclusion of this experiment is that immortal stem cells can respond to local signals in different regions of the developing brain and become recruited to local fates. It is possible that the immortal cells are unusual in this regard as they may have lost their regional specification in prolonged culture, but the experiments on muscle precursor lines show that this is not necessarily the case. Both transplant studies show that established cell lines can be grown for long periods in culture and still retain the ability to differentiate in response to signals present in vivo. This result needs to be extended to obtain quantitative measures of differentiation, but it already holds the promise for the development of powerful new assays for the molecular mechanisms underlying brain development. The main point to stress here is that the immortal cells give rise to neurons that are present in the adult brain, and that these neurons appear to be integrated into the circuitry of the brain. The main objection to the use of cell lines is that they do not represent the cell types found in viva. While this may be true in some circumstances, the two studies discussed here suggest that under appropriate conditions immortal precursor cells can differentiate into neurons that are closely related to the neuronal types normally found in rho. Of mice and man mammalian
new methods
in
genetics
The potential importance of the ability to reconstruct animals from immortal cell lines encourages a re-examination of the simple question: what is a cell line? The concept of a cell line is closely related to the ideas of senescence and death. It is widely accepted that pnmat-y embryonic libroblasts show senescence, and many cultured cells of different kinds will not divide indefinitely in culture. It may also be the case that cell death is a common phenomenon in vivo [ 251. The accepted view is that cells are mortal, i.e. they undergo a finite number of divisions and then die. The idea that cell senescence/death is a cell autonomous property of most cell types is not uniformly supported by experiments in vitro, however. For example, myoblasts can be maintained in culture for very long periods by the appropriate growth factors. In addition defined cytokines can cause a great expansion of lymphocytes. The large scale expansion of primary cells is also used routinely in skin culture treatments for burn victims. Embryonic stem cells are used in mouse genetics because these cells can be cultured indefinitely without immortalization [2628]. In all of these cases there is good evidence that the cells retain their ‘primary’ nature even though they have been kept for long periods in
culture. In the case of embryonic stem cells, completely normal animals can be derived from cultured cells. The production of a transgenic mouse in which the tsA58 allele of SV40 T-antigen is driven from the H-2Kb promoter should allow a new strategy for immortalizing cells from the developing mouse [29]. This promoter is induced in the presence of y-interferon. Culture data on skin fibroblasts and thymocytes suggest that the presence of the inducing agent and a non-permissive temperature for the &A58 allele are required for the growth of some cell lines. In principle, this transgenic mouse strain may permit the derivation of many cell lines in a double conditional manner, dependent on temperature and y-interferon. The most common view of cell lines is that they require activated oncogenes for continued growth, but the above examples show that there is not a sharp boundary between a primary cell and a transformed cell. In view of these recent experiments, it seems reasonable to adopt a very flexible view of what a cell line is and how a cell line relates to cell types found in vizm. Perspective It has been known since the pioneering aggregation chimera studies of Mintz and colleagues that teratocarcinema cells contribute to many tissues of the adult mouse [30]. Aggregation chimeras obtained by introducing embryonic stem cells into blastulae have been used to generate new strains of mice carrying mutations that were introduced into the embryonic stem cells in culture. It is possible to mutate both alleles of a gene in culture allowing recessive phenotypes to be analyzed [31,32]. The homologous recombination mechanisms that allow directed mutation in embryonic stem cells are found in all cells; indeed, homologous recombination in cultured mammalian cells was first observed in somatic cells. These techniques permit powerful analyses of gene function in transgenic mice, but the same homologous recombination techniques may be applied to immortalized precursor cells of any species. After transplantation into a host, the cells will integrate and differentiate. The phenotypes of the mutations can then be assessed in chimeric animals. This approach to genetics may have important applications. There are some problems in basic science and clinical medicine that would be best addressed in mammalian species other than rodents. For example, much of our knowledge of integrated cellular functions in neurobiology comes from studies of the visual system in carnivores and primates. The potential for genetic analysis in chimeric animals and tissue engineering in man will ensure that these new approaches are actively explored. References Papers of view, have . of .. of 1.
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R McKay, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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