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We have been studying the differing characteristics of oligodendrocyte-type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O-2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.

Cellular and humoral immune mechanisms have been implicated in the pathogenesis of human and experimental demyelinating diseases of the CNS. How these interact in the complex sequence of events that culminates in phagocytosis of myelin by macrophages has yet to be resolved. The relationship between leakage of the blood-brain barrier and demyelination, the reason why recurrent inflammatory demyelination occurs--seemingly in the absence of an antigen-specific immune response--and the lack of effective remyelination all require explanation if a coherent account of immunologically mediated demyelination is to be achieved. One approach to these problems is to study in vitro the developmental and cellular biology of oligodendrocytes--the glial cells responsible for the synthesis and maintenance of CNS myelin. This provides experimental opportunities not offered by more direct investigation of the intact nervous system, but carries the clear disadvantage that observations made in vitro cannot necessarily be extrapolated to humans.

Bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve, are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. Previous studies have shown that O-2A progenitors exposed to platelet-derived growth factor (PDGF) divide as migratory bipolar cells a limited number of times, with a cell cycle time of 18 hr, before clonally related progenitors differentiate into nondividing oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but instead differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. We now have found that O-2A progenitors can be induced to undergo continuous self-renewal in the absence of oligodendrocytic differentiation by exposure to a combination of PDGF and basic fibroblast growth factor (bFGF). With the exception of the inhibition of differentiation, the O-2A progenitors exposed to PDGF and bFGF behaved similarly to those exposed to PDGF alone. In contrast, progenitors exposed to basic bFGF alone were multipolar, had a cell-cycle length of 45 hr, showed little migratory behavior, underwent premature oligodendrocytic differentiation, and did not cease division upon expression of oligodendrocyte marker antigens. Thus, inhibition of differentiation required the presence of both mitogens. Our results demonstrate that PDGF and bFGF act on O-2A progenitors as both inducers of division and as regulators of differentiation that modulate multiple aspects of O-2A progenitor development and, additionally, reveal a previously unrecognized means of regulating self-renewal processes, wherein cooperation between growth factors promotes continuous division in the absence of differentiation.

Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve. These progenitors are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. When exposed to platelet-derived growth factor (PDGF), O-2A progenitors divide a limited number of times before clonally related cells differentiate into oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. O-2A progenitors can become immortalized through at least two different mechanisms. First, when O-2A progenitors are exposed to a combination of PDGF and basic fibroblast growth factor (bFGF) these cells undergo continuous self-renewal in the absence of differentiation. In contrast, the application of bFGF alone is associated with premature oligodendrocytic differentiation of dividing O-2A lineage cells. Thus, cooperation between growth factors can modulate O-2A progenitor self-renewal in a defined chemical environment by eliciting a novel programme of division and differentiation which cannot be predicted from the effects of either factor examined in isolation. A further mechanism which allows prolonged self-renewal in the O-2A lineage is the generation of a stem cell. O-2A progenitors isolated from optic nerves of perinatal rats also have the capacity to give rise to a population of cells called O-2Aadult progenitors, which differ from their perinatal counterparts in many characteristics. Most importantly, O-2Aadult progenitors have a slow cell cycle, divide and differentiate asymmetrically and appear to have the capacity for prolonged self-renewal. Thus, immortalization in this lineage can also be achieved by the generation of a cell with stem cell-like characteristics from a rapidly dividing progenitor population.

By studying the response of a well-defined progenitor cell to two well-defined mitogens, we have been able to provide a dramatic example of the complex relationships which can exist between the control of cell division and the control of differentiation. In previous studies we have described the development of the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell, a glial progenitor cell isolated from the rat optic nerve. Although originally described as a bipotential cell, we have recently identified a new differentiation pathway in this lineage. We have found that O-2Aperinatal progenitors, with properties appropriate for early development, give rise to O-2Aadult progenitors, which have stem cell-like properties more appropriate to the physiological needs of adult animals. Our studies thus indicate that the population of O-2Aperinatal progenitors is tripotential, and also suggests a possible developmental origin for self-renewing stem cells. Moreover, the properties of O-2Aadult progenitor cells may provide a cellular biological basis for understanding the failure of remyelination in multiple sclerosis. The division of both O-2Aperinatal and O-2Aadult progenitors is stimulated by type-1 astrocytes (which are themselves derived from a separate glial lineage) but this cell-cell interaction promotes different programs of differentiation in the two progenitor populations. The effects of type-1 astrocytes on perinatal and adult progenitors appears to be mediated by platelet-derived growth factor (PDGF), and this mitogen will also induce different programs of differentiation in the two progenitor populations. Moreover, the patterns of differentiation promoted by PDGF are different from those promoted by fibroblast growth factor (FGF), demonstrating that the modulation of division can be distinguished from the modulation of differentiation.

The central nervous system of individuals with multiple sclerosis contains lesions specifically characterized by breakdown of myelin sheaths associated with a general failure of repair of demyelinating damage. The cause of myelin breakdown is unknown. Although immune mechanisms have been implicated in this breakdown, no convincing demonstrations of specific immune reaction against myelin have yet been provided in multiple sclerosis patients. Similarly, the cellular biological mechanisms which underlie the failure of myelin repair are unknown. We have found that (i) oligodendrocytes, the cells that produce myelin sheaths in the central nervous system, and (ii) oligodendrocyte/type-2 astrocyte (O/2A) progenitor cells derived from optic nerves of adult rats bind and activate complement in the absence of antibody in vitro, leading to destruction of these cells. Susceptibility to antibody-independent lysis by complement was a cell-type-specific trait of oligodendrocytes and adult O/2A progenitors and was not shared by perinatal O/2A progenitors, type-2 astrocytes, type-1 astrocytes, meningeal cells, or Schwann cells. We suggest that the susceptibility of oligodendrocytes and adult O/2A progenitor cells to complement-induced lysis, combined with other specific properties of adult O/2A progenitors, are consistent with--and may be a contributing factor--both in the generation of demyelinating lesions in multiple sclerosis and also in the failure of these lesions to be successfully repaired in adult multiple sclerosis patients.