Cytokines regulate the cellular phenotype of developing neural lineage species

The patterns and mechanisms of action of inductive signals that orchestrate neural lineage commitment and differentiation in the mammalian brain are incompletely understood. To examine these developmental issues, we have utilized several culture systems including conditionally immortalized cell lines, subventricular zone progenitor cells and primary neuronal cultures. A neural stem and progenitor cell line (MK31) was established from murine embryonic hippocampus by retroviral transduction of temperature-sensitive alleles of the simian virus 40 large tumor antigen. At the non-permissive temperature for antigen expression (39°C) in serum-free media, the neural stem cells give rise to a series of increasingly mature neuronal progenitor and differentiated cellular forms under the influence of a subset of hematolymphopoietic cytokines (interleukins 5, 7, 9 and 11), when individually co-applied with transforming growth factor α, after pretreatment with basic fibroblast growth factor. These cellular forms elaborated a series of progressively more mature neurofilament proteins, a sequential pattern of ligand-gated channels, and inward currents and generation of action potentials with mature physiological properties. Because the factors regulating the development of central nervous system astrocytes have been so difficult to define, we have chosen to focus, in this manuscript, on the elaboration of this cell type. At 39°C, application of a subfamily of bone morphogenetic proteins of the transforming growth factor β superfamily of growth factors sanctioned the selective expression of astrocytic progenitor cells and mature astrocytes, as defined by sequential elaboration of the Yb subunit of glutathione-S-transferase and glial fibrillary acidic protein. These lineage-specific cytokine inductive relationships were verified using subventricular zone neural progenitor cells generated by the application of epidermal growth factor, alone or in combination with basic fibroblast growth factor, to dissociated cellular cultures derived from early embryonic murine brain, a normal non-transformed developmental population. Finally, application of a different series of cytokines from five distinct factor classes (basic fibroblast growth factor, platelet-derived growth factor-AA, insulin-like growth factor 1, neurotrophin 3 and representative gp130 receptor subunit-related ligands) caused the elaboration of oligodendroglial progenitor species and post-mitotic oligodendrocytes, defined by progressive morphological maturation and the expression of increasingly adbanced oligodendroglial and oligodendrocyte lineage markers. In addition, seven different gp130-associated neuropoietic (ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin-M) and hematopoietic (interleukins 6, 11, 12, granulocyte-colony stimulating factor) cytokines exhibited differential trophic effects on oligodendroglial lineage maturation and factor class interactions. Examination of the expression of hematolymphopoietic cytokines and their receptors in brain and neural cultures has confirmed that these epigenetic signals are present at the appropriate development times to mediate their neurotrophic actions. These cytokines signal through alternate receptor subunit motifs distinct from those of the traditional neurotrophins. The bone morphogenetic protein ligand, in particular, exhibit a complex spatiotemporal pattern of transcript expression that suggests a broad spectrum of developmental roles for these transforming growth factor β subclass factors. To examine the cellular action of the bone morphogenetic proteins on astroglial lineage elaboration in greater detail, we utilized several complementary developmental systems. When primary neuronal cultures from multiple brain regions of mid-gestational (embryonic day 15) fetuses in serum-free media were exposed to the same combination of bone morphogenetic proteins that sanctioned astroglial lineage elaboration from neural stem and progenitor cells, they exhibited significant suppression of neuronal viability. By contrast, application of the same factors to late embryonic day 17-18 neuronal cultures resulted in a regional and factor-specific potentiation of cellular survival and differentiation. The neurotrophic effects of the bone morphogenetic proteins appear to be indirect and mediated by stimulation of non-neuronal cells. Further, application of the bone morphogenetic proteins to purified O-2A progenitor cells, derived from early postnatal brain and from a clonal progenitor cell line resulted in the selective induction of type II astrocytes, suggesting that these transforming growth factor β subclass factors are acting directly on these bipotent astrocytic/oligodendroglial progenitor cells. These diverse experimental observations suggest that a single central nervous system neural stem cell can give rise to all three major cellular elements of the mammalian brain. Cytokines from the three major growth factor superfamilies (neurotrophins, hemopoietins and transforming growth factor β-related factors) exhibit a differential pattern of neurotrophic actions on distinct central nervous system lineage species during sequential developmental stages. These observations suggest that a complex hierarchy of interacting epigenetic signals is required for central nervous system neurogenesis.