Pluripotent stem cell‐derived radial glia‐like cells as stable intermediate for efficient generation of human oligodendrocytes

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor‐mediated expansion with pre‐exposure to the differentiation‐inducing agent retinoic acid and subsequent immunoisolation of CD133‐positive cells. This protocol yields an adherent and self‐renewing population of hindbrain/spinal cord radial glia (RG)‐like neural precursor cells (RGL‐NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL‐NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate‐determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL‐NPCs efficiently convert into NG2‐positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL‐NPCs expedite the generation of PSC‐derived oligodendrocytes with O4‐, 4860‐, and myelin basic protein (MBP)‐positive cells that already appear within 7 weeks following growth factor withdrawal‐induced differentiation. Thus, RGL‐NPCs may serve as robust tool for time‐efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. GLIA 2015;63:2152–2167     

Pax6 directly modulate Sox2 expression in the neural progenitor cells

Pax6 is a key regulator in the neuronal fate determination as well as the proliferation of neural stem cells, but the mechanisms are still unknown. Our study shows that Pax6 regulate the proliferation of neural progenitor cells of cortical subventricular zone, through direct modulation of the Sox2 expression during the late developmental stage in mice. We found a dramatic decrease in the number of Sox2+ neural progenitor cells in the subventricular zone of E18.5 Pax6(-/-) mice. We confirmed that Pax6 could bind to the Sox2 promoter by chromatin immunoprecipitation assay and activate Sox2 expression by a luciferase reporter gene assay. Moreover, neural progenitors isolated from the Pax6(-/-) embryos showed a decreased neurosphere formation as well as proliferation.     

Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro

Adult human and rodent brains contain neural stem and progenitor cells, and the presence of neural stem cells in the adult rodent spinal cord has also been described. Here, using electron microscopy, expression of neural precursor cell markers, and cell culture, we investigated whether neural precursor cells are also present in adult human spinal cord. In well-preserved nonpathological post-mortem human adult spinal cord, nestin, Sox2, GFAP, CD15, Nkx6.1, and PSA-NCAM were found to be expressed heterogeneously by cells located around the central canal. Ultrastructural analysis revealed the existence of immature cells close to the ependymal cells, which display characteristics of type B and C cells found in the adult rodent brain subventricular region, which are considered to be stem and progenitor cells, respectively. Completely dissociated spinal cord cells reproducibly formed Sox2(+) nestin(+) neurospheres containing proliferative precursor cells. On differentiation, these generate glial cells and gamma-aminobutyric acid (GABA)-ergic neurons. These results provide the first evidence for the existence in the adult human spinal cord of neural precursors with the potential to differentiate into neurons and glia. They represent a major interest for endogenous regeneration of spinal cord after trauma and in degenerative diseases.     

Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse

We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1-GFP-expressing population. Thus, the Sox1-GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1-GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1-GFP precursors were derived. On the other hand, Sox1-GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1-GFP cells possess the same capacity for neuronal differentiation in vivo.     

Gfap‐positive radial glial cells are an essential progenitor population for later‐born neurons and glia in the zebrafish spinal cord

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189     

Light‐controlled astrocytes promote human mesenchymal stem cells toward neuronal differentiation and improve the neurological deficit in stroke rats

Astrocytes are key components of the central nervous system (CNS) and release factors to support neural stem cell proliferation, differentiation, and migration. Adenosine 5'‐triphosphate (ATP) is one of the key factors released upon activation of astrocytes that regulates the neural stem cell's function. However, it is not clear whether ATP derived from the depolarized astrocytes plays a vital role in promoting the neuronal differentiation of mesenchymal stem cells (MSCs) in vitro and in vivo. Herein, for the first time, we co‐cultured MSCs with light‐stimulated‐channelrhodopsin‐2 (ChR2)‐astrocytes, and observed that the neuronal differentiation of MSCs was enhanced by expressing more neuronal markers, Tuj1 and NeuN. The ChR2‐astrocyte‐conditioned medium also stimulated MSCs differentiating into neuronal lineage cells by expressing more Tuj1 and Pax6, which was blocked by the P2X receptor antagonist, TNP‐ATP. Then we found that light‐depolarization of astrocytes significantly increased ATP accumulation in their bathing medium without impairing the cell membrane. We further found that ATP up‐regulated the Tuj1, Pax6, FZD8 and β‐catenin mRNA levels of MSCs, which could be reversed by application of TNP‐ATP. Together these in vitro data provided convergent evidence that ATP from light‐depolarized‐astrocytes activated the wnt/β‐catenin signaling of MSCs through binding to the P2X receptors, and promoted the neuronal differentiation of MSCs. Finally but importantly, our study also demonstrated in stroke rats that light‐controlled astrocytes stimulated endogenous ATP release into the ischemic area to influence the transplanted MSCs, resulting in promoting the MSCs towards neuronal differentiation and improvements of neurological deficit. GLIA 2013;62:106–121     

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ. © 2010 Wiley‐Liss, Inc.     

A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages

Glial fibrillary acidic protein (GFAP)-positive cells derived from the neurogenic areas of the brain can be stem/progenitor cells and give rise to new neurons in vitro and in vivo. We report here that a population of GFAP-positive cells derived from fetal human brain parenchyma coexpress markers of early neural and neuronal cells, and have neural progenitor cell characteristics. We used a monolayer culture system to expend and differentiate these cells. During the initial proliferative phase, all cells expressed GFAP, nestin and low levels of betaIII-tubulin. When these cells were cultured in serum and then basic fibroblast growth factor, they generated two distinct progenies: (i) betaIII-tubulin- and nestin-positive cells and (ii) GFAP- and nestin-positive cells. These cells, when subsequently cultured in serum-free media without growth factors, ceased to proliferate and differentiated into two major neural cell classes, neurons and glia. In the cells of neuronal lineage, nestin expression was down-regulated and betaIII-tubulin expression became robust. Cells of glial lineage differentiated by down-regulating nestin expression and up-regulating GFAP expression. These data suggest that populations of parenchymal brain cells, initially expressing both glial and neuronal markers, are capable of differentiating into single neuronal and glial lineages through asymmetric regulation of gene expression in these cells, rather than acquiring markers through differentiation.