Bipotential Glial Progenitor Cell Research Articles

A2B5 Expression in Central Nervous System and Gliomas.

A2B5 IgM recognizes c-series gangliosides with three sialic acids. The aim of this review was to focus on A2B5 expression in the central nervous system and gliomas. In brain development, A2B5+ cells are recorded in areas containing multipotent neural stem cells (NSC). In adults, A2B5+ cells persist in neurogenic areas and in white matter where it identifies oligodendrocyte precursor cells (OPCs) but also cells with NSC properties. Although the expression of A2B5 has been widely studied in culture, where it characterizes bipotential glial progenitor cells, its expression in vivo is less characterized mainly because of technical issues. A new interest was given to the NSCs and OPCs since the discovery of cancer stem cells (CSC) in gliomas. Among other cell surface molecules, A2B5 has been identified as an accurate marker to identify glioma CSCs. We and others have shown that all types of gliomas express A2B5, and that only A2B5+ cells, and not A2B5- cells, can generate a tumor after orthotopic implantation in immunocompromised animals. Moreover, A2B5 epitope expression is positively correlated with stemness and tumor growth. This review highlights that A2B5 is an attractive target to tackle glioma CSCs, and a better characterization of its expression in the developing and adult CNS will benefit to a better understanding of gliomagenesis.

Low-Field Magnetic Stimulation Accelerates the Differentiation of Oligodendrocyte Precursor Cells via Non-canonical TGF-β Signaling Pathways.

Demyelination and oligodendrocyte loss are characteristic changes in demyelinating disorders. Low-field magnetic stimulation (LFMS) is a novel transcranial neuromodulation technology that has shown promising therapeutic potential for a variety of neuropsychiatric conditions. The cellular and molecular mechanisms of magnetic stimulation remain unclear. Previous studies mainly focused on the effects of magnetic stimulation on neuronal cells. Here we aimed to examine the effects of a gamma frequency LFMS on the glial progenitor cells. We used rat central glia-4 (CG4) cell line as an in vitro model. CG4 is a bipotential glial progenitor cell line that can differentiate into either oligodendrocyte or type 2-astrocyte. The cells cultured in a defined differentiation media were exposed to a 40-Hz LFMS 20min daily for five consecutive days. We found that LFMS transiently elevated the level of TGF-β1 in the culture media in the first 24h after the treatment. In correlation with the TGF-β1 levels, the percentage of cells possessing complex branches and expressing the late oligodendrocyte progenitor marker O4 was increased, indicating the accelerated differentiation of CG4 cells towards oligodendrocyte in LFMS-treated cultures. LFMS increased phosphorylation of Akt and Erk1/2 proteins, but not SMAD2/3. TGF-β1 receptor I specific inhibitor LY 364947 partially suppressed the effects of LFMS on differentiation and on levels of pAkt and pErk1/2, indicating that LFMS enhances the differentiation of oligodendrocyte progenitor cells via activation of non-canonical TGF-β-Akt and TGF-β-Erk1/2 pathways but not the canonical SMAD pathway. The data from this study reveal a novel mechanism of magnetic stimulation as a potential therapy for demyelination disorders.

Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation.

Huntington's disease (HD) is characterized by hypomyelination and neuronal loss. To assess the basis for myelin loss in HD, we generated bipotential glial progenitor cells (GPCs) from human embryonic stem cells (hESCs) derived from mutant Huntingtin (mHTT) embryos or normal controls and performed RNA sequencing (RNA-seq) to assess mHTT-dependent changes in gene expression. In human GPCs (hGPCs) derived from 3 mHTT hESC lines, transcription factors associated with glial differentiation and myelin synthesis were sharply downregulated relative to normal hESC GPCs; NKX2.2, OLIG2, SOX10, MYRF, and their downstream targets were all suppressed. Accordingly, when mHTT hGPCs were transplanted into hypomyelinated shiverer mice, the resultant glial chimeras were hypomyelinated; this defect could be rescued by forced expression of SOX10 and MYRF by mHTT hGPCs. The mHTT hGPCs also manifested impaired astrocytic differentiation and developed abnormal fiber architecture. White matter involution in HD is thus a product of the cell-autonomous, mHTT-dependent suppression of glial differentiation.

Optogenetic control of cell differentiation in channelrhodopsin-2-expressing OS3, a bipotential glial progenitor cell line

Alterations in the intracellular ion environment have been identified as one of the signals playing a critical role in the control of cellular proliferation and differentiation; however, the mechanisms responsible for signal transduction remain unclear. Recent studies have reported that channelrhodopsin-2 (ChR2) is a rapidly gated blue light (BL)-sensitive cation channel suitable for the non-invasive control of ion influx. We herein examined the expression of differentiation-associated markers by photo-activation and its signal transduction in ChR2-expressing OS3 (OS3ChR2) cells, which are clonal bipotential glial progenitor cells. Increases were observed in intracellular Na+ and Ca2+ concentrations in OS3ChR2 cells with BL exposure. Alterations in the intracellular ion environment, particularly in Ca2+, led to increases in the expression of oligodendrocyte markers including galactocerebrosides (GalC) and decreases in that of astrocyte markers such as glial fibrillary acidic protein (GFAP). These alterations also triggered activation of the ERK1/2 signaling pathway, which is involved in cell survival, and PI3K/Akt/mTOR signaling pathway, which is involved in oligodendrocyte differentiation, characterized by GalC expression. Moreover, when photo-activated OS3ChR2 cells were injected into mice with lysophosphatidyl choline (LPC)-induced demyelination, deficits in motor function were reduced. Our results demonstrated that signal transduction by ChR2-expressing glial progenitor cells may be controlled through alterations induced in the intracellular ion environment by photo-activation and results in oligodendrocyte differentiation from glial progenitor cells. Our results also suggest that ChR2-expressing glial progenitor cells have potential as a useful tool for therapeutic approaches to brain and spinal cord disorders associated with oligodendrocyte dysfunctions.

The bipotential glial progenitor cell line can develop into both oligodendrocytes and astrocytes in the mouse forebrain

Oligodendrocyte-type 2 astrocyte (O2A) progenitor cells in vivo might differentiate into oligodendrocytes. To examine the influence of the brain micro-environment on the differentiation, a bipotential glial cell line from the mouse cerebrum, designated OS3 cells, was implanted into the telencephalon of infant and adult mice. About a half of the OS3 cells injected into 1-week postnatal brain expressed galactocerebroside (GalC), and even myelin basic protein, which were not observed to be expressed in vitro. By contrast, in the brain over 6 months postnatally, many OS3 cells expressed glial fibrillary acidic protein, and did not express much GalC. These findings suggest that the differentiation of glial cells is controlled by stage specific factors in the brain.

A role for the acetylcholine receptor-inducing protein ARIA in oligodendrocyte development.

ARIA acetylcholine receptor-inducing activity protein, is a member of a family of ligands that includes the Neu differentiation factor, heregulin, and glial growth factor. These ligands all act through one or more receptor tyrosine kinases of approximately 185 kDa. In some conditions these ligands promote proliferation, whereas in others they induce differentiation. ARIA was originally isolated from chick brain on the basis of its ability to induce synthesis of nicotinic acetylcholine receptors in skeletal muscle. In this paper we show that ARIA is expressed in the subventricular zone of the rat brain and that it enhances the development of oligodendrocytes from bipotential (O2A) glial progenitor cells. We have also found that ARIA induces tyrosine phosphorylation of a 185-kDa protein in O2A progenitor cells. ARIA does not increase bromodeoxyuridine incorporation by oligodendrocytes but is mitogenic when added to Schwann cells in vitro. Thus, ARIA accelerates the formation of oligodendrocytes in vitro and is expressed where it could exercise the same influence in vivo.

PDGF increases the expression of Fos and Jun in newly formed oligodendrocytes that have become resistant to the mitogenic effect of PDGF.

Bipotential glial (O-2A) progenitor cells give rise to oligodendrocytes on a predictable schedule in the developing rat optic nerve. The loss of mitotic responsiveness to platelet-derived growth factor (PDGF) that is seen when O-2A progenitor cells initially differentiate into oligodendrocytes seems to reflect blocks or deficiencies downstream to some of the early intracellular signalling events induced by PDGF. Here we show that PDGF increases the expression of the Fos and Jun nuclear proteins in newly formed oligodendrocytes in vitro, suggesting that at least one intracellular signalling pathway to the nucleus is activated by PDGF in these cells even though they do not synthesize DNA in response to PDGF.

A rapid procedure for the preparation of oligodendrocyte-enriched cultures from rat spinal cord

Spinal cords and cerebra from 7-day-old rat pups were compared as tissue sources for the isolation of oligodendrocytes and for studies on the development of these cells in culture. After 1 day in culture the serum-containing medium was replaced by a chemically-defined medium, which contained a cocktail of hormones that stimulated oligodendrocyte development. The cultures were characterized with various immunocytochemical markers; monoclonal A2B5 for bipotential glial progenitor cells, anti-galactocerebroside (GC) serum for oligodendrocytes, and anti-glial fibrillary acidic protein (GFAP) serum for astrocytes. The number of positive cells was counted and expressed as a percentage of total cells. At 1 day in culture the cell cultures from spinal cord contained 30% GC + cells, increasing to 90% after 7 days in culture. In cultures derived from cerebra the percentage of GC + cells was always lower than in cultures from spinal cord. In cerebral cultures GFAP + cells increased from 15% at 1 day in culture to 30% at 7 days in culture, whereas it remained low in spinal cord cultures. The activity of the oligodendroglial marker enzyme 2′,3′-cyclic-nucleotide 3′-phosphodiesterase was followed during development in culture. The specific activity increased rapidly in both types of culture but was more than threefold higher in cultures derived from spinal cord. This procedure yields, within one week and without subculture, primary glial cultures from rat spinal cord, that are highly enriched in oligodendrocytes (≥ 90%; 3 · 10 5 oligodendrocytes per rat pup).

An Analysis of the Cell-Cell Interactions That Control the Proliferation and Differentiation of a Bipotential Glial Progenitor Cell in Culture

An Analysis of the Cell-Cell Interactions That Control the Proliferation and Differentiation of a Bipotential Glial Progenitor Cell in Culture

Neuronal influences on glial progenitor cell development

The role of cell-cell interactions in the development of bipotential glial progenitor cells in cultures of rat cerebellum and optic nerve was studied. In the cerebellar cultures, progenitor cells divide slowly and most of their progeny develop into additional progenitor cells. Progenitor cells isolated from postconfluent cultures of cerebellum, however, develop rapidly into oligodendrocytes when grown in a serum-free medium. Factors secreted or shed into the medium by young cerebellar interneurons stimulate optic nerve progenitor cells to divide and promote the survival of progenitor cells. These factors appear to alter the function of the internal clock that regulates the timing of oligodendrocyte differentiation. These results suggest that the neuronal microenvironment can influence the lineage decisions of multipotential glial progenitor cells.

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.

Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture.

We have been studying a population of bipotential glial progenitor cells in the perinatal rat optic nerve and brain in an attempt to understand how cells choose between alternative fates in the developing mammalian central nervous system (CNS). This cell population gives rise initially to oligodendrocytes and then to type-2 astrocytes, both of which apparently collaborate in sheathing axons in the CNS. In vitro studies suggest that oligodendrocyte differentiation is the constitutive pathway of development for the oligodendrocyte-type-2-astrocyte (O-2A) progenitor cell, whereas type-2 astrocyte differentiation depends on a specific inducing protein. This protein is present in the developing optic nerve when type-2 astrocytes are differentiating and can induce O-2A progenitor cells in vitro to express glial fibrillary acidic protein (GFAP), a marker of astrocyte differentiation. Here we show that the type-2-astrocyte-inducing protein is similar or identical to ciliary neutrotrophic factor (CNTF), which promotes the survival of some types of peripheral neurons in vitro, including ciliary ganglion neurons. This suggests that CNTF, in addition to its effect on neurons, may be responsible for triggering type-2 astrocyte differentiation in the developing CNS.

CNS neuronal cell line‐derived factors regulate gliogenesis in neonatal rat brain cultures

Recent studies suggest that heterotypic cell-cell interactions influence gliogenesis in the developing rat central nervous system. CNS neuron-derived factors have been hypothesized to exist, and several have been identified and partially characterized which affect the number of oligodendrocytes in vitro. In order to study further the role of neurons in gliogenesis, we have used serum-free culture conditions, the B104 CNS neuronal cell line as a source of soluble factors, and dissociated neonatal rat brain cells as a source of glial cells. We have analyzed the response of the glial cells to serum-free B104 conditioned medium using morphological, immunocytochemical, autoradiographic, and enzymatic methods. Dose-dependent increases in the number of morphologically identified oligodendrocytes occur in response to this conditioned medium. Galactocerebroside (GalC) is a specific marker for oligodendrocytes, and the A2B5 antigen marks bipotential glial progenitor cells and their progeny: immature oligodendrocytes and type 2 astrocytes. In the presence of conditioned medium, the number of cells expressing GalC and/or A2B5 antigen increases over time when measured at 4, 8, and 12 days in vitro. A significantly weaker effect is seen if serum is also present. Since the vast majority of A2B5-positive cells in conditioned medium treated cultures lack glial fibrillary acidic protein (GFA), indicative of type 2 astrocytes, they represent glial progenitors and immature oligodendrocytes. Double immunostaining combined with autoradiography suggests that the latter cell types are the target cells for the oligodendrocyte-promoting activity. In addition, the conditioned medium markedly increases 2',3' cyclic nucleotide 3'-phosphodiesterase (an oligodendrocyte marker) and to a lesser extent enhances glutamine synthetase activity (an astrocyte marker). Type 1 astrocytes are also more morphologically differentiated in this condition, and their percentage is decreased simultaneously. Conditioned medium from other donor neural cells either has no activity or is much less effective than B104 conditioned medium. The active factors are soluble, sensitive to both trypsin and 100 degrees C treatment for 20 min, and appear to be 30-100 kilodaltons by stirred cell ultrafiltration. In summary, we have identified a potent source of growth-stimulating factors that produce increased numbers of glial progenitor cells and oligodendrocytes; the same conditioned medium also appears to inhibit type 1 astrocyte proliferation.

Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture.

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.

Differentiation of a bipotential glial progenitor cell: what controls the timing and the choice of developmental pathway?

In the rat central nervous system (CNS) oligodendrocytes and type-2 astrocytes are thought to develop from a common precursor - the O-2A progenitor cell. Oligodendrocytes develop first and make myelin; type-2 astrocytes develop later and extend processes to nodes of Ranvier. The timing of differentiation of O-2A progenitor cells seems to depend on chemical signals secreted by another type of glial cell - the type-1 astrocyte. Type-1 astrocytes secrete platelet-derived growth factor (PDGF), which stimulates O-2A progenitor cell proliferation and drives the clock that controls the onset of oligodendrocyte differentiation, which is the constitutive pathway of progenitor cell development. Later, type-1 astrocytes are thought to secrete a CNTF-like protein that initiates type-2 astrocyte 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.

Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway.

A single bipotential glial progenitor cell of newborn rat optic nerve (the O-2A progenitor) characterized by its reactivity with antibodies to surface gangliosides (A2B5) and the presence of vimentin, can grow in microcultures in conditions which favor this progenitor's differentiation into oligodendrocytes. We selected at 8 days larger clones derived from such bipolar progenitors which had steadily proliferated on a layer of Type-1 astrocytes during the first week. Clonal growth and ratio of progenitor cells to oligodendrocytes was measured over the next two weeks by phase microscopy and double immunofluorescence labeling for the specific markers A2B5, GC (galactocerebroside, a surface marker for differentiated oligodendrocytes), O4 (a glycolipid marker of oligodendrocytes and some progenitors), GFAP (an astrocyte specific intermediate filament protein) and vimentin. Two types of clones were identified: type A clones (the majority), which were still slowly expanding at three weeks, and type B clones (the minority), which had stopped proliferating during the second week. In type A clones, some cells became GC positive multipolar oligodendrocytes, while other multipolar cells remained GC negative for days and expressed A2B5 and O4, but not GFAP or vimentin. Type B clones contained only GC positive, vimentin negative oligodendrocytes, which were generated during the second week and then decreased in number because of their restricted lifespan. Type B clones only developed in the presence of insulin or neurons. The number of GC negative cells in type A clones increased when insulin or neurons were deleted.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferating bipotential glial progenitor cells in adult rat optic nerve.

We have shown previously that the rat optic nerve contains three types of macroglial cells--oligodendrocytes and two types of astrocytes--which develop as two distinct lineages. Type-1 astrocytes develop from one type of precursor cell beginning at embryonic day 16 (E16), while oligodendrocytes and then type-2 astrocytes develop from a common, bipotential progenitor cell beginning at birth (E21) and postnatal days 7-10 (P7-10), respectively. Here we report that proliferating bipotential oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells are present in the adult rat optic nerve, raising the possibility that these cells are produced continually from self-renewing stem cells throughout life.

Differentiation of a bipotential glial progenitor cell in a single cell microculture.

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.

Effects of neonatal transection on glial cell development in the rat optic nerve: evidence that the oligodendrocyte-type 2 astrocyte cell lineage depends on axons for its survival.

We have previously provided evidence that the rat optic nerve contains three types of macroglial cells that develop as two distinct lineages: one lineage comprises type 1 astrocytes, which develop before birth, while the other comprises oligodendrocytes and type 2 astrocytes, which develop after birth from a common, bipotential glial progenitor cell. In the present study we have examined the influence of axons on the development of these two glial cell lineages by cutting the optic nerve at birth so that the retinal ganglion cell axons in the nerve degenerate. Using antibodies to distinguish the different types of glial cells in suspensions and semithin frozen sections of cut and uncut optic nerves, we show that neonatal transection results in a striking decrease in the total number of oligodendrocytes, type 2 astrocytes and their progenitor cells but has much less effect on the number of type 1 astrocytes. Since the [3H]thymidine labelling indices of oligodendrocytes and their progenitor cells were not significantly decreased in cut nerves, our results suggest that the progenitor cells and/or their progeny die in large numbers following neonatal nerve transection. We conclude that axons are required for the survival of cells of the oligodendrocyte-type 2 astrocyte lineage, at least during postnatal development.