The use of a SOX10 reporter toward ameliorating oligodendrocyte lineage differentiation from human induced pluripotent stem cells.

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells. Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, toward enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but also holds promise to accelerate research and translational work with iPSC-derived OLs.

Mapping Angiopoietin1 Expression in the Developing and Adult Brain.

Angiopoietin1 (Angpt1) is a secreted protein that activates the endothelial Tie2 receptor. Angpt1 plays a critical role in cardiac development and vascular remodeling in response to disease or injury and shows cell type-restricted expression in the lung, eye, and hematopoietic system. However, the expression of Angpt1 in the developing and adult brain is not known. Here, we employ Angpt1-GFP knock-in reporter mice and a systematic analysis of multiple single-cell RNA sequencing datasets to map the expression of Angpt1 during brain development and adulthood. In the developing brain, Angpt1 displays specific spatiotemporal patterns, with strong expression in cerebellar GABA interneuron progenitors and, to a lower level, in glial progenitor and astrocyte lineages. In the adult brain, on the other hand, we show that neurons are the main source of Angpt1 in the cerebrum, while in the cerebellum, expression is mostly restricted to astrocytes. Together, our data provide clarity on the cell types that express Angpt1 in the developing and adult brain and can be utilized to guide future studies, examining Angpt1 function in brain development, homeostasis, and pathological conditions.

Comparative Analysis of the Paracrine Action of Neuronal and Glial Progenitor Cells Derived from Induced Human Pluripotent Stem Cells.

We performed comparative analysis of paracrine activity of neuronal and glial progenitors derived from induced pluripotent stem cells under conditions of hypoxia modeled by addition of cobalt dichloride. Neuronal and glial progenitors produced neuroprotective and neurotrophic effects on SHSY-5Y neuroblastoma cells in co-culture during the post-hypoxic recovery and reduced the number of apoptotic and necrotic cells. Moreover, they produced a neurotrophic effect and promote the formation and growth of neurites in neuroblastoma cells. The paracrine effect of glial progenitors was more pronounced, which can be explained by more intensive expression and secretion of neurotrophic factors in these cells.

Migratory potential of transplanted glial progenitors as critical factor for successful translation of glia replacement therapy: The gap between mice and men.

Neurological disorders are a major threat to public health. Stem cell-based regenerative medicine is now a promising experimental paradigm for its treatment, as shown in pre-clinical animal studies. Initial attempts have been on the replacement of neuronal cells only, but glial progenitors (GPs) are now becoming strong alternative cellular therapeutic candidates to replace oligodendrocytes and astrocytes as knowledge accumulates about their important emerging role in various disease processes. There are many examples of successful therapeutic outcomes for transplanted GPs in small animal models, but clinical translation has proved to be challenging due to the 1,000-fold larger volume of the human brain compared to mice. Human GPs transplanted into the mouse brain migrate extensively and can induce global cell replacement, but a similar extent of migration in the human brain would only allow for local rather than global cell replacement. We review here the mechanisms that govern cell migration, which could potentially be exploited to enhance the migratory properties of GPs through cell engineering pre-transplantation. We furthermore discuss the (dis)advantages of the various cell delivery routes that are available, with particular emphasis on intra-arterial injection as the most suitable route for achieving global cell distribution in the larger brain. Now that therapeutic success has proven to be feasible in small animal models, future efforts will need to be directed to enhance global cell delivery and migration to make bench-to-bedside translation a reality.

Mechanisms of mouse neural precursor expansion after neonatal hypoxia-ischemia.

Neonatal hypoxia-ischemia (H-I) is the leading cause of brain damage resulting from birth complications. Studies in neonatal rats have shown that H-I acutely expands the numbers of neural precursors (NPs) within the subventricular zone (SVZ). The aim of these studies was to establish which NPs expand after H-I and to determine how leukemia inhibitory factor (LIF) insufficiency affects their response. During recovery from H-I, the number of Ki67(+) cells in the medial SVZ of the injured hemisphere increased. Similarly, the number and size of primary neurospheres produced from the injured SVZ increased approximately twofold versus controls, and, upon differentiation, more than twice as many neurospheres from the damaged brain were tripotential, suggesting an increase in neural stem cells (NSCs). However, multimarker flow cytometry for CD133/LeX/NG2/CD140a combined with EdU incorporation revealed that NSC frequency diminished after H-I, whereas that of two multipotential progenitors and three unique glial-restricted precursors expanded, attributable to changes in their proliferation. By quantitative PCR, interleukin-6, LIF, and CNTF mRNA increased but with significantly different time courses, with LIF expression correlating best with NP expansion. Therefore, we evaluated the NP response to H-I in LIF-haplodeficient mice. Flow cytometry revealed that one subset of multipotential and bipotential intermediate progenitors did not increase after H-I, whereas another subset was amplified. Altogether, our studies demonstrate that neonatal H-I alters the composition of the SVZ and that LIF is a key regulator for a subset of intermediate progenitors that expand during acute recovery from neonatal H-I.

Distinct progenitor behavior underlying neocortical gliogenesis related to tumorigenesis.

Radial glial progenitors (RGPs) give rise to the vast majority of neurons and glia in the neocortex. Although RGP behavior and progressive generation of neocortical neurons have been delineated, the exact process of neocortical gliogenesis remains elusive. Here, we report the precise progenitor behavior and gliogenesis program at single-cell resolution in the mouse neocortex. Fractions of dorsal RGPs transition from neurogenesis to gliogenesis progressively, producing astrocytes, oligodendrocytes, or both in well-defined propensities of ∼60%, 15%, and 25%, respectively, by fate-restricted "intermediate" precursor cells (IPCs). Although the total number of IPCs generated by individual RGPs appears stochastic, the output of individual IPCs exhibit clear patterns in number and subtype and form discrete local subclusters. Clonal loss of tumor suppressor Neurofibromatosis type 1 leads to excessive production of glia selectively, especially oligodendrocyte precursor cells. These results quantitatively delineate the cellular program of neocortical gliogenesis and suggest the cellular and lineage origin of primary brain tumor.

Immunological Characteristics and Properties of Glial Restricted Progenitors of Mice, Canine Primary Culture Suspensions, and Human QSV40 Immortalized Cell Lines for Prospective Therapies of Neurodegenerative Disorders.

Neurodegeneration can be defined as a process in which neuronal structures and functions undergo changes leading to reduced neuronal survival and increased cell death in the central nervous system (CNS). Neuronal degeneration in specific regions of the CNS is a hallmark of many neurodegenerative disorders, and there is reliable proof that neural stem cells bring therapeutic benefits in treatment of neurological lesions. However, effective therapy with neural stem cells is associated with their biological properties. The assessment of immunological properties and comprehensive studies on the biology of glial restricted progenitors (GRP) are necessary prior to the application of these cells in humans. This study provides an in vitro characterization of the QSV40 glial human cell line, as well as murine and canine primary culture suspensions of GRPs and their mature, astrocytic forms using flow cytometry and immunohistochemical staining. Cytokines and chemokines released by GRPs were assessed by Multiplex ELISA. Some immunological differences observed among species suggest the necessity of reconsidering the pre-clinical model, and that careful testing of immunomodulatory strategies is required before cell transplantation into the CNS can be undertaken.

Biodistribution of Glial Progenitors in a Three Dimensional-Printed Model of the Piglet Cerebral Ventricular System.

White matter damage persists in hypoxic-ischemic newborns even when treated with hypothermia. We have previously shown that intraventricular delivery of human glial progenitors (GPs) at the neonatal stage is capable of replacing abnormal host glia and rescuing the lifespan of dysmyelinated mice. However, such transplantation in the human brain poses significant challenges as related to high-volume ventricles and long cell migration distances. These challenges can only be studied in large animal model systems. In this study, we developed a three dimensional (3D)-printed model of the ventricular system sized to a newborn pig to investigate the parameters that can maximize a global biodistribution of injected GPs within the ventricular system, while minimizing outflow to the subarachnoid space. Bioluminescent imaging and magnetic resonance imaging were used to image the biodistribution of luciferase-transduced GPs in simple fluid containers and a custom-designed, 3D-printed model of the piglet ventricular system. Seven independent variables were investigated. The results demonstrated that a low volume (0.1 mL) of cell suspension is essential to keep cells within the ventricular system. If higher volumes (1 mL) are needed, a very slow infusion speed (0.01 mL/min) is necessary. Real-time magnetic resonance imaging demonstrated that superparamagnetic iron oxide (SPIO) labeling significantly alters the rheological properties of the GP suspension, such that, even at high speeds and high volumes, the outflow to the subarachnoid space is reduced. Several other factors, including GP species (human vs. mouse), type of catheter tip (end hole vs. side hole), catheter length (0.3 vs. 7.62 m), and cell concentration, had less effect on the overall distribution of GPs. We conclude that the use of a 3D-printed phantom model represents a robust, reproducible, and cost-saving alternative to in vivo large animal studies for determining optimal injection parameters.

In vitro transdifferentiation of human adipose tissue-derived stem cells to neural lineage cells - a stage-specific incidence.

The present Study investigated the intrinsic ability of adipose tissue-derived stem cells (ADSCs) and their neural transdifferentiation in a stage-specific manner. Woodbury's Chemical induction was implemented with modifications to achieve neural transdifferentiation. In Group I, ADSCs were preinduced with ß-mercaptoethanol (ß-ME) and later, with neural induction medium (NIM). In Group II, ADSCs were directly treated with NIM. In Group III, a DNA methyltransferase (DNMT) inhibitor 5-azacytidine was applied to understand whether transdifferentiation is controlled by epigenetic marks. Irrespective of the presence of (ß-ME), the differentiation protocol resulted in glial-lineage cells. Group III produced poorly -differentiated neural cells with neuron-specific enolase positivity. A neuroprogenitor stage (NPC) was identified at d 11 after induction only in Group I. In other groups, this stage was not morphologically distinct. We explored the stage-specific incidence NPC, by alternatively treating them with basic fibroblast growth factor (bFGF), and antioxidants to validate if different signalling could cause varied outcomes (Group IV). They differentiated into neurons, as defined by cell polarity and expression of specific proteins. Meanwhile, neuroprogenitors exposed to NIM (Group I) produced glial-lineage cells. Further refinement and study of the occurrence and terminal differentiation of neuroprogenitors would identify a promising source for neural tissue replacement.

Single-Cell Transcriptomics in Medulloblastoma Reveals Tumor-Initiating Progenitors and Oncogenic Cascades during Tumorigenesis and Relapse.

Progenitor heterogeneity and identities underlying tumor initiation and relapse in medulloblastomas remain elusive. Utilizing single-cell transcriptomic analysis, we demonstrated a developmental hierarchy of progenitor pools in Sonic Hedgehog (SHH) medulloblastomas, and identified OLIG2-expressing glial progenitors as transit-amplifying cells at the tumorigenic onset. Although OLIG2+ progenitors become quiescent stem-like cells in full-blown tumors, they are highly enriched in therapy-resistant and recurrent medulloblastomas. Depletion of mitotic Olig2+ progenitors or Olig2 ablation impeded tumor initiation. Genomic profiling revealed that OLIG2 modulates chromatin landscapes and activates oncogenic networks including HIPPO-YAP/TAZ and AURORA-A/MYCN pathways. Co-targeting these oncogenic pathways induced tumor growth arrest. Together, our results indicate that glial lineage-associated OLIG2+ progenitors are tumor-initiating cells during medulloblastoma tumorigenesis and relapse, suggesting OLIG2-driven oncogenic networks as potential therapeutic targets.

Function of B-Cell CLL/Lymphoma 11B in Glial Progenitor Proliferation and Oligodendrocyte Maturation.

B-cell CLL/lymphoma 11B (Bcl11b) - a C2H2 zinc finger transcriptional factor - is known to regulate neuronal differentiation and function in the development of the central nervous system (CNS). Although its expression is reduced during oligodendrocyte (OLG) differentiation, its biological role in OLGs remains unknown. In this study, we found that the downregulation of Bcl11b gene expression in glial progenitor cells (GPCs) by lentivirus-mediated gene knockdown (KD) causes a reduction in cell proliferation with inhibited expression of stemness-related genes, while increasing the expression of cell cyclin regulator p21. In contrast, OLG specific transcription factors (Olig1) and OLG cell markers, including myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), were upregulated in Bcl11b-KD GPCs. Chromatin immunoprecipitation (ChIP) analysis indicated that Bcl11b bound to the promoters of Olig1 and PLP, suggesting that Bcl11b could act as a repressor for Olig1 and PLP, similar to its action on p21. An increase in the number of GC+- or PLP+- OLGs derived from Bcl11b-KD GPCs or OLG precursor cells was also observed. Moreover, myelin basic protein (MBP) expression in OLGs derived from Bcl11b-KD GPCs was enhanced in hippocampal neuron co-cultures and in cerebellar brain-slice cultures. The in vivo study using a lysolecithin-induced demyelinating animal model also indicated that larger amounts of MBP+-OLGs and PLP+-OLGs derived from implanted Bcl11b-KD GPCs were present at the lesioned site of the white matter than in the scramble group. Taken together, our results provide insight into the functional role of Bcl11b in the negative regulation of GPC differentiation through the repression of OLG differentiation-associated genes.

Transplanted human glial-restricted progenitors can rescue the survival of dysmyelinated mice independent of the production of mature, compact myelin.

The therapeutic effect of glial progenitor transplantation in diseases of dysmyelination is currently attributed to the formation of new myelin. Using magnetic resonance imaging (MRI), we show that the therapeutic outcome in dysmyelinated shiverer mice is dependent on the extent of cell migration but not the presence of mature and compact myelin. Human or mouse glial restricted progenitors (GRPs) were transplanted into rag2-/- shiverer mouse neonates and followed for over one year. Mouse GRPs produced mature myelin as detected with multi-parametric MRI, but showed limited migration without extended animal lifespan. In sharp contrast, human GRPs migrated extensively and significantly increased animal survival, but production of mature myelin did not occur until 46weeks post-grafting. We conclude that human GRPs can extend the survival of transplanted shiverer mice prior to production of mature myelin, while mouse GRPs fail to extend animal survival despite the early presence of mature myelin. This paradox suggests that transplanted GRPs provide therapeutic benefits through biological processes other than the formation of mature myelin capable to foster rapid nerve conduction, challenging the current dogma of the primary role of myelination in regaining function of the central nervous system.

Neocortical Sox9+ radial glia generate glutamatergic neurons for all layers, but lack discernible evidence of early laminar fate restriction.

Glutamatergic neurons in the cerebral cortex are derived from embryonic neural stem cells known as radial glial progenitors (RGPs). Early RGPs, present at the onset of cortical neurogenesis, are classically thought to produce columnar clones of glutamatergic neurons spanning the cortical layers. Recently, however, it has been reported that a subset of early RGPs may undergo early commitment to upper layer neuron fates, thus bypassing genesis of deep layer neurons. However, the latter mode of early RGP differentiation was not confirmed in some other studies, and remains controversial. To further investigate the clonal output from early RGPs, we employed genetic lineage tracing driven by Sox9, a transcription factor gene that is expressed in all early RGPs. We found that early RGPs produced columnar clones spanning all cortical layers, with no evidence of significant laminar fate restriction. These data support the classic progressive restriction model of cortical neurogenesis, and suggest that early RGPs do not undergo early commitment to only upper or lower layer fates.

Cellular and subcellular imaging of motor protein-based behavior in embryonic rat brain.

Development of the cerebral cortex is a very dynamic process, involving a series of complex morphogenetic events. Following division of progenitor cells in the ventricular zone, neurons undergo a series of morphological changes and migrate outward toward the cortical plate, where they differentiate and integrate into functional circuits. Errors at several of stages during neurogenesis and migration cause a variety of severe cortical malformations. A number of disease genes encode factors associated with the cytoskeleton, which plays a crucial role throughout cortical development. Methods for regulating gene expression coupled with imaging of subcellular structures have provided important insight into the mechanisms governing normal and abnormal brain development. We describe here a series of protocols for imaging motor protein-dependent processes in real time in the developing rat brain.

Unmasking the responses of the stem cells and progenitors in the subventricular zone after neonatal and pediatric brain injuries.

There is great interest in the regenerative potential of the neural stem cells and progenitors that populate the subventricular zone (SVZ). However, a comprehensive understanding of SVZ cell responses to brain injuries has been hindered by the lack of sensitive approaches to study the cellular composition of this niche. Here we review progress being made in deciphering the cells of the SVZ gleaned from the use of a recently designed flow cytometry panel that allows SVZ cells to be parsed into multiple subsets of progenitors as well as putative stem cells. We review how this approach has begun to unmask both the heterogeneity of SVZ cells as well as the dynamic shifts in cell populations with neonatal and pediatric brain injuries. We also discuss how flow cytometric analyses also have begun to reveal how specific cytokines, such as Leukemia inhibitory factor are coordinating SVZ responses to injury.

Regulation of the neural stem cell compartment by extracellular matrix constituents.

Neural stem cells (NSCs) derive from the neuroepithelium of the neural tube, develop into radial glial cells, and recede at later developmental stages. In the adult, late descendants of these embryonic NSCs reside in discretely confined areas of the central nervous system, the stem cell niches. The best accepted canonical niches are the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus of the hippocampus. Stem cell niches provide a privileged environment to NSCs that supports self-renewal and maintenance of this cellular compartment. While numerous studies have highlighted the importance of transcription factors, morphogens, cytokines, and growth factors as intrinsic and extrinsic factors of stem cell regulation, less attention has been paid to the molecular micromilieu that characterizes the stem cell niches. In this chapter, we summarize increasing evidence that the extracellular matrix (ECM) of the stem cell environment is of crucial importance for the biology of this cellular compartment. A deeper understanding of the molecular composition of the ECM, the complementary receptors, and the signal transduction pathways engaged may prove highly relevant for harnessing NSCs in the context of biotechnological applications.

Low Concentration Microenvironments Enhance the Migration of Neonatal Cells of Glial Lineage.

Glial tumors have demonstrated abilities to sustain growth via recruitment of glial progenitor cells (GPCs), which is believed to be driven by chemotactic cues. Previous studies have illustrated that mouse GPCs of different genetic backgrounds are able to replicate the dispersion pattern seen in the human disease. How GPCs with genetic backgrounds transformed by tumor paracrine signaling respond to extracellular cues via migration is largely unexplored, and remains a limiting factor in utilizing GPCs as therapeutic targets. In this study, we utilized a microfluidic device to examine the chemotaxis of three genetically-altered mouse GPC populations towards tumor conditioned media, as well as towards three growth factors known to initiate the chemotaxis of cells excised from glial tumors: Hepatocyte Growth Factor (HGF), Platelet-Derived Growth Factor-BB (PDGF-BB), and Transforming Growth Factor-α (TGF-α). Our results illustrate that GPC types studied exhibited chemoattraction and chemorepulsion by different concentrations of the same ligand, as well as enhanced migration in the presence of ultra-low ligand concentrations within environments of high concentration gradient. These findings contribute towards our understanding of the causative and supportive roles that GPCs play in tumor growth and reoccurrence, and also point to GPCs as potential therapeutic targets for glioma treatment.

IN VITRO EFFECTS OF GLIA MATURATION FACTOR ON THE DEVELOPMENT AND DIFFERENTIATION OF CENTRAL MACROGLIAL CELLS / 解剖学报

Optic nerves were dissected from 7-day-old Sprague-Dawley rats, dissociated with collagenase and trypsin, and cultured on poly-L-lysine coated glass coverslips. Cultures were grown in the B-S medium (Bottenstein and Sato, 1979) containing 0.5% fetal calf serum(FCS). All of the coverslip cultures were divided into 4 groups: i. e. Ⅰ, "Ⅰ+GMF", Ⅱ, and "Ⅱ+GMF". Optic nerve glial cells were cocultured with glioblast monolayers in both group Ⅱ and group "Ⅱ+GMF". Glia maturation factor (GMF) was added to the medium in a concentration of 250 ng/ml for group "Ⅰ+GMF" and group "Ⅱ+GMF". No GMF was added to the medium in both group Ⅰ and group Ⅱ. The coverslip cultures were treated with indirect immunofluorescence to identify the cell types of optic nerve. Cells were double-labled with monoclonal gotibody A2B5 and anti-galactocerebroside(GC) monoclonal antibody, or A2B5 and anti-glisl fibrillary acidicprotein (GFAP) antibody. The bipotential glial progenitor cells (A2B5~+, GC~-; or A2B5~+, GFAP~-), mature oligodendrocytes (A2B5~-, GC~+), immature oligodendrocytes(A2B5~+, GC~+), type 1 astrocytes (A2BS~-, GFAP~+) and type 2 astrocytes (A2B5~+, CFAP~+) can be distinguished.After 5 days in culture, the number of cells in group "Ⅱ+GMF" showed a significant increase (P