Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.     

In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells

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.     

Proliferation and differentiation potential of rat forebrain oligodendroglial progenitors both in vitro and in vivo

We have followed the development of the O-2A progenitor cell from the neonatal rat forebrain, both in dissociated cell culture and in cryostat sections, using immunocytochemical techniques employing a panel of antibodies that recognise the cells at different stages of their development. This included the monoclonal antibody LB1, which binds to the surface ganglioside GD3 expressed on O-2A progenitor cells. In secondary cultures enriched for O-2A progenitors maintained in a serum-free chemically defined medium, a large proportion of the cells are primed to differentiate into oligodendroglia and go on to express the oligodendroglial specific surface glycolipid galactocerebroside (GC) and then the myelin proteins CNP and MBP. However, a significant proportion of immature bipolar GD3+ cells remained after 6 days in secondary culture. It appears that not all the O-2A progenitors in our cultures differentiate immediately and some cells remain in an undifferentiated state and divide to replenish progenitor numbers. We have also identified in our cultures a small apolar GD3- cell, which when isolated differentiated into a GD3+ bipolar O-2A progenitor cell. We have termed this cell type a preprogenitor. The differentiation of this cell type into O-2A progenitors may be the source of the immature GD3+ cells present at the later stages of our secondary cultures. The proliferative profile of the cultures was studied using 5'bromo-2-deoxyuridine (BrdU) incorporation as an index of mitosis. Only the immature, bipolar O-2A progenitors were seen to divide at any time in serum-free culture. Neither the more mature multipolar O-2A cells nor the oligodendroglia were seen to divide. The developmental profile of the O-2A cells in the rat forebrain in vivo showed a largely similar progression to that in culture, with a time lag of at least 6 days between GD3 expression and the onset of myelination. BrdU incorporation studies in vivo also showed that the GD3+ progenitor cell is mitotic whereas the GC(+)-expressing oligodendroglia is not. We have shown that there are several significant alterations in the timing of antigen expression in both O-2A progenitors and oligodendroglia in vitro compared to that seen in vivo.     

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.     

Development and regeneration in the central nervous system

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.     

The O-2Aadult progenitor cell: a glial stem cell of the adult central nervous system

Systematic comparison of the properties of oligodendrocytetype-2 astrocyte (O-2A) progenitor cells derived from optic nerves of perinatal and adult rats has revealed that these two populations differ in many fundamental properties. In particular, O-2A^perinatal progenitor cells are rapidly dividing cells capable of generating large numbers of oligodendrocytes over a relatively short time span. Oligodendrocyte differentiation generally occurs synchronously in all members of a clone, thus leading to elimination of that clone from the pool of dividing cells. However, some O-2A^perinatal progenitors are also capable of giving rise to O-2A^adult progenitors. These latter cells express many of the characteristics of stem cells of adult animals, including the capacity to undergo asymmetric division and differentiation. We suggest that precursors which function during early development give rise to terminally differentiated end-stage cells and to a second generation of precursors with properties more appropriate for later developmental stages. It is this second generation of precursors which express the properties of stem cells in adult animals, and we therefore further suggest that our work offers novel insights into the possible developmental origin of stem cells.     

FGF modulates the PDGF-driven pathway of oligodendrocyte development

PDGF promotes the growth of oligodendrocyte type-2 astrocyte (O-2A) glial progenitor cells and allows their timely differentiation into oligodendrocytes, the CNS myelin-forming cells. We demonstrate that basic FGF is a potent mitogen for brain O-2A progenitor cells, but blocks their differentiation into oligodendrocytes. Treatment with basic FGF also influences the level of expression of PDGF receptors on O-2A progenitor cells. These cells express only the alpha chain PDGF receptor, and the levels of PDGF alpha receptors decrease as the cells differentiate. In contrast, basic FGF maintains a high level of functionally responsive PDGF alpha receptors in O-2A progenitors. Thus basic FGF activates a signaling pathway that can positively regulate PDGF receptors in O-2A progenitor cells. In this way basic FGF or an FGF-like factor may modulate the production of myelin-forming cells in the CNS.     

The complex relationship between cell division and the control of differentiation in oligodendrocyte-type-2 astrocyte progenitor cells isolated from perinatal and adult rat optic nerves

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 relationship 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-2A^perinatal progenitors, with properties appropriate for early development, give rise to O-2A^adult 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-2A^perinatal progenitors is tripotential, and also suggests a possible developmental origin for self-renewing stem cells. Moreover, the properties of O-2A^adult progenitor cells may provide a cellular biological basis for understanding the failure of remyelination in multiple sclerosis.The division of both O-2A^perinatal and O-2A^adult 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.     

An inducer protein may control the timing of fate switching in a bipotential glial progenitor cell in rat optic nerve

In rat optic nerve, oligodendrocytes and type-2 astrocytes develop from a common (O-2A) progenitor cell. The first oligodendrocytes differentiate at birth, while the first type-2 astrocytes differentiate in the second postnatal week. We previously showed that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell. Here we provide evidence that the timing of type-2 astrocyte differentiation, by contrast, may depend on an inducing protein that appears late in the developing nerve. We show that extracts of 3- to 4-week-old, but not 1-week-old, rat optic nerve contain a protein (apparent Mr approximately 25,000) that induces O-2A progenitor cells in culture to express glial fibrillary acidic protein (GFAP), an astrocyte-specific marker in the rat central nervous system.     

Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell.

Optic nerves of neonatal rats contain a bipotential glial progenitor cell which can be induced by tissue culture conditions to differentiate into either an oligodendrocyte (the myelin-forming cell of the CNS) or a type 2 astrocyte (an astrocyte population found only in the myelinated tracts of the CNS). In our previous studies most oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells differentiated within 3 days in vitro with relatively little division of the progenitors or their differentiated progeny. We have now found that the O-2A progenitors are stimulated to divide in culture by purified populations of type 1 astrocytes, another glial cell-type found in the rat optic nerve. This cell-cell interaction appears to be mediated by a soluble factor(s) and results in the production of large numbers of both progenitor cells and oligodendrocytes. As type 1 astrocytes are the major glial cell-type in the optic nerve when oligodendrocytes first begin to be produced in large numbers in vivo, our results suggest that this astrocyte subpopulation may play an important role in expanding the oligodendrocyte population during normal development.     

PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.     

Identification of an adult-specific glial progenitor cell

We have found that glial progenitor cells isolated from the optic nerves of adult rats are fundamentally different from their counterparts in perinatal animals. In our studies on bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, we have seen that O-2Aadult progenitor cells can be distinguished from O-2Aperinatal progenitors by their morphology and antigenic phenotype, their much longer cell cycle time (65 h versus 18 h), slower rate of migration rate (4 microns h-1 versus 21 microns h-1), and their time course of differentiation into oligodendrocytes or type-2 astrocytes in vitro (less than or equal to 3 days versus greater than 5 days). At least some of the differences between O-2Aadult and O-2Aperinatal progenitor cells appear to be clearly related to the differing cellular requirements of the adult and perinatal central nervous system (CNS). The properties of the O-2Aadult progenitor cells may make these cells ideally suited for the needs of the adult CNS, where rapid exponential increases in the number of oligodendrocytes and O-2A progenitor cells would be inappropriate. However, the properties of the O-2Aadult progenitor cells are such that they may not be able to replace oligodendrocytes in sufficient numbers to repair extensive or recurrent damage in the adult brain, such as in patients suffering from the human demyelinating disease multiple sclerosis. Moreover, available information about other tissues suggests that the transition from perinatal to adult progenitor cell types may represent a developmental mechanism of general importance.     

A role for platelet-derived growth factor in normal gliogenesis in the central nervous system

The bipotential progenitor cells (O-2A progenitors) that produce oligodendrocytes and type-2 astrocytes in the developing rat optic nerve are induced to proliferate in culture by type-1 astrocytes. Here, we show that the astrocyte-derived mitogen is platelet-derived growth factor (PDGF). PDGF is a potent mitogen for O-2A progenitor cells in vitro. Mitogenic activity in astrocyte-conditioned medium comigrates with PDGF on a size-exclusion column, competes with PDGF for receptors, and is neutralized by antibodies to PDGF. PDGF dimers can be immunoprecipitated from astrocyte-conditioned medium, and mRNA encoding PDGF is present in rat brain throughout gliogenesis. We propose that astrocyte-derived PDGF is crucial for the control of myelination in the developing central nervous system.     

Repopulation of O-2A progenitor cells after irradiation of the adult rat optic nerve analyzed by an in vitro clonogenic assay.

In the central nervous system (CNS) O-2A (Oligodendrocyte type 2 Astrocyte) progenitor cells have been proposed as potential target cells, and their depletion by irradiation will cause demyelination. The extent and time course of repopulation of these glial stem cells were studied in the adult rat optic nerve after irradiation in vivo. The number of O-2A progenitor cells was measured quantitatively by an in vitro clonogenic assay. Although the CNS is typically a late-responding tissue, repopulation was initiated almost immediately after irradiation and after several weeks a plateau was reached that lasted up to 6 months. Single doses of 4-12 Gy of X rays caused a permanent reduction in the number of O-2A progenitor cells. An analysis of the colony size of O-2A progenitor cells showed a sustained reduction in the number of offspring of cells surviving a dose of 12 Gy. In addition, the colony size of unirradiated progenitors diminished with increasing age of the animals.     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

IGF-I synergizes with FGF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle

Secreted peptide growth factors are critical extracellular signals that interact to promote the proliferation, differentiation, and survival of progenitor cells in developing tissues. IGF-I signaling through the IGF type I receptor provides a mitogenic signal for numerous cell types, including stem and progenitor cells. We have utilized the O-2A oligodendrocyte progenitor to study the mechanism of IGF-I mitogenic actions since these progenitors respond to IGF-I in vitro, and gene targeting studies in mice have demonstrated that IGF-I is essential for normal oligodendrocyte development in vivo. The goal of this study was to elucidate the mechanism by which IGF-I promotes the proliferation of oligodendrocyte progenitors in the context of other mitogens critical for their proliferation. Results presented here show that IGF-I significantly amplified the actions of FGF-2 and PDGF to promote DNA synthesis in O-2A progenitors. Investigation of cell cycle kinetics revealed that IGF-I had no significant effect on the rate of cell cycle progression. Instead, IGF-I promoted increased recruitment of O-2A progenitors into the S phase of the cell cycle. These studies support a role for IGF-I as a cell cycle progression factor for progenitor cells.     

Evidence for nonlinear capacitance in biomembrane channel system.

The electrophysiological properties of voltage-dependent anion channels from mitochondrial membrane have been studied in a bilayer membrane system. It was observed that the probability of opening of the membrane channel depends on externally applied voltage and the plot is a bell-shaped curve symmetric around probability axis. A scheme of conformational energy levels under varying externally applied voltage was formulated. Assuming that the probability follows Boltzmann distribution, we arrive at an expression of change in energy containing a separate term identical to the energy of a capacitor. This fact indicates the possibility of existence of an added capacitance due to the channel protein. Further it was shown that the aforesaid channel capacitor could be a function of voltage leading to nonlinearity. We have offered a general method of calculating nonlinear capacitance from the experimental data on opening probability of a membrane channel. In case of voltage-dependent anion channel the voltage dependence of the capacitor has a power 0.786. The results have been interpreted in view of the structural organization of the channel protein in the membrane. Our hypothesis is that the phenomenon of capacitor behaviour is a general one for membrane channels.     

Identification of neural progenitors in the adult mammalian eye

We have shown that the embryonic mammalian retina contains neural progenitors which display stem cell properties in vitro. Here we report the characterization of neural progenitors isolated from the adult mammalian eye. These quiescent cells, located in the pigmented ciliary bodies, proliferate in the presence of FGF2 and express the neuroectodermal marker nestin. The proliferating cells give rise to neural spheres and are multipotential; they express cell type-specific markers corresponding to neurons and glia. In addition, neural progenitors can generate secondary neural spheres, thus displaying potential to self-renew. The ciliary body-derived neural progenitors display retina-specific properties; the undifferentiated cells express Chx10, a retinal progenitor marker, and upon differentiation express markers corresponding to specific retinal cell types. Therefore, the pigmented ciliary body in the adult mammalian eye harbors neural progenitors that display stem cell properties and have the capacity to give rise to retinal neurons in vitro.