Astrocyte Differentiation Research Articles

WNT signaling represses astrogliogenesis via Ngn2-dependent direct suppression of astrocyte gene expression.

Neural progenitor cells (NPCs) are sequentially specified into neurons and glia during the development of central nervous system. WNT/β-catenin signaling is known to regulate the balance between the proliferation and differentiation of NPCs during neurogenesis. However, the function of WNT/β-catenin signaling during gliogenesis remains poorly defined. Here, we report that activation of WNT/β-catenin signaling disrupts astrogliogenesis in the developing spinal cord. Conversely, inhibition of WNT/β-catenin signaling leads to precocious astrogliogenesis. Further analysis reveals that activation of WNT/β-catenin pathway results in a dramatic increase of neurogenin 2 (Ngn2) expression in transgenic mice, and knockdown of Ngn2 expression in neural precursor cells can reverse the inhibitory effect of WNT/β-catenin on astrocytic differentiation. Moreover, Ngn2 can directly bind to the promoters of several astrocyte specific genes and suppress their expression independent of STATs activity. Together, our studies provide the first in vivo evidence that WNT/β-catenin signaling inhibits early astrogliogenesis via an Ngn2-dependent transcriptional repression mechanism.

Deficiency of Fhl2 leads to delayed neuronal cell migration and premature astrocyte differentiation

The four and a half LIM domains protein 2 (Fhl2) is an adaptor protein capable of mediating protein-protein interactions. Here, we report for the first time phenotypic changes in the brain of Fhl2-deficient mice. We showed that Fhl2 is expressed in neural stem cells, precursors and mature cells of neuronal lineage. Moreover, Fhl2 deficiency leads to delayed neuroblast migration in vivo, premature astroglial differentiation of neural stem cells (NSCs) in vitro, and a gliosis-like accumulation of glial fibrillary acidic protein (GFAP)-positive astrocytes in vivo that substantially increases with age. Collectively, Fhl2-deficiency in the brain interrupts the maintenance and the balanced differentiation of adult NSCs, resulting in preferentially glial differentiation and early exhaustion of the NSC pool required for adult neurogenesis.

Notch Signaling Mediates Astrocyte Abnormality in Spinal Muscular Atrophy Model Systems

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of spinal motor neurons and muscle atrophy. The disease is mainly caused by low level of the survival motor neuron (SMN) protein, which is coded by two genes, namely SMN1 and SMN2, but leads to selective spinal motor neuron degeneration when SMN1 gene is deleted or mutated. Previous reports have shown that SMN-protein-deficient astrocytes are abnormally abundant in the spinal cords of SMA model mice. However, the mechanism of the SMN- deficient astrocyte abnormality remains unclear. The purpose of this study is to identify the cellular signaling pathways associated with the SMN-deficient astrocyte abnormality and propose a candidate therapy tool that modulates signaling. In the present study, we found that the astrocyte density was increased around the central canal of the spinal cord in a mouse SMA model and we identified the dysregulation of Notch signaling which is a known mechanism that regulates astrocyte differentiation and proliferation, in the spinal cord in both early and late stages of SMA pathogenesis. Moreover, pharmacological inhibition of Notch signaling improved the motor functional deficits in SMA model mice. These findings indicate that dysregulated Notch signaling may be an underlying cause of SMA pathology.

Foxg1 Antagonizes Neocortical Stem Cell Progression to Astrogenesis.

Neocortical astrogenesis follows neuronogenesis and precedes oligogenesis. Among key factors dictating its temporal articulation, there are progression rates of pallial stem cells (SCs) towards astroglial lineages as well as activation rates of astrocyte differentiation programs in response to extrinsic gliogenic cues. In this study, we showed that high Foxg1 SC expression antagonizes astrocyte generation, while stimulating SC self-renewal and committing SCs to neuronogenesis. We found that mechanisms underlying this activity are mainly cell autonomous and highly pleiotropic. They include a concerted downregulation of 4 key effectors channeling neural SCs to astroglial fates, as well as defective activation of core molecular machineries implementing astroglial differentiation programs. Next, we found that SC Foxg1 levels specifically decline during the neuronogenic-to-gliogenic transition, pointing to a pivotal Foxg1 role in temporal modulation of astrogenesis. Finally, we showed that Foxg1 inhibits astrogenesis from human neocortical precursors, suggesting that this is an evolutionarily ancient trait.

Proteomic analyses reveal misregulation of LIN28 expression and delayed timing of glial differentiation in human iPS cells with MECP2 loss-of-function.

Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation.

Resistin-Inhibited Neural Stem Cell-Derived Astrocyte Differentiation Contributes to Permeability Destruction of the Blood-Brain Barrier.

Neuroinflammation is an important part of the development of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's and amyotrophic lateral sclerosis. Inflammatory factors destroy the balance of the microenvironment, which results in changes in neural stem cell differentiation and proliferation behaviour. However, the mechanism underlying inflammatory factor-induced NSC behavioural changes is not clear. Resistin is a proinflammatory and adipogenic factor and is involved in several human pathology processes. The neural stem cell microenvironment changes when the concentration of resistin in the brain during an inflammatory response disease increases. In the present study, we explored the effect and mechanism of resistin on the proliferation and differentiation of neural stem cells. We found that intracerebroventricular injection of resistin induced a decrease in GFAP-positive cells in mice by influencing NSC differentiation. Resistin significantly decreased TEER and increased permeability in an in vitro blood-brain barrier model, which is consistent with the results of an HBMEC-astrocyte coculture system. Resistin-inhibited astrocyte differentiation is mediated through TLR4 on neural stem cells. To our knowledge, this is the first study reporting the effect of resistin on neural stem cells. Our findings shed light on resistin-involved neural stem cell degeneration mechanisms.

Novel Chiral 1,3,4‐Oxadiazole Derivatives Inducing Astrocyte Differentiation of Rat Fetal Neural Stem Cells

Novel Chiral 1,3,4‐Oxadiazole Derivatives Inducing Astrocyte Differentiation of Rat Fetal Neural Stem Cells

Astrocytes migrate from human neural stem cell grafts and functionally integrate into the injured rat spinal cord

Neural stem cells (NSCs) can differentiate into both neurons and glia after transplantation into spinal cord injury (SCI) sites. The neuronal component of stem cell grafts has the potential to form functional synaptic relays across the lesion site. The glial component may reform a blood-spinal cord barrier, support neuronal function, and contribute to remyelination. We performed a long-term, 1.5-year time course study focused on astrocyte migration, differentiation, integration, and safety following human NSC transplantation into C5 hemisection sites in immunodeficient rats. NSCs that adopted a neuronal fate did not migrate from the lesion site. In contrast, transplanted cells that adopted astrocyte fates exhibited long distance migration from the lesion site through host white matter in rostrocaudal directions. These cells migrated slowly at a mean rate of 2–3 mm/month, divided as they migrated, and gradually differentiated into astrocytes. After 1.5 years, human astrocytes migrated nine spinal cord segments, caudally to the mid-thoracic level, and rostrally into the brainstem. The migrated human astrocytes joined the endogenous population of astrocytes in the host spinal cord, extended perivascular endfeet towards host pericytes and endothelium, formed interspecies and intraspecies perivascular astrocytic networks connected by gap junctions, and expressed glutamate transporter proteins in perisynaptic processes, suggesting structural and functional integration. No adverse consequences of this extended glial migration were detected. Adjacent to the lesion site, chronic host astrocytic upregulation was significantly attenuated by NSC grafts. Thus, human astrocytes can migrate long distances from sites of SCI and safely integrate into the host central nervous system. Significance statementNeural stem cell (NSC) grafts are a candidate therapy for spinal cord injury (SCI). Here we report an 18-month study of astrocyte survival and migration from sites of SCI in immunodeficient rats. NSC grafts significantly attenuate host astrocyte reactivity at the lesion/host interface. Intra-graft astrocytes integrate into the host blood-spinal cord barrier (BSCB) and widely express glutamate transporter proteins characteristic of neurotransmitter regulation. Notably, astrocytic components of NSC grafts exhibit gradual yet extensive migration after implantation into the mid-cervical injury site; neurons do not migrate at all. This extensive astrocyte migration is not detectably associated with adverse outcomes anatomically or behaviorally.

Acetylcholine suppresses the increase of glia fibrillary acidic protein expression via acetylcholine receptors in cAMP-induced astrocytic differentiation of rat C6 glioma cells

Astrocytes, the most common glial cells in the central nervous system, maintain neuronal functions and have roles in neurological diseases. Acetylcholine (ACh) is one of the most essential neurotransmitters, and ACh receptor (AChR) ligands were recently reported to influence astrocyte functions. However, the functions of ACh, the only endogenous agonist of AChR, in astrocytogenesis and in the expression of astrocytic marker genes have not been known. We previously demonstrated that the inhibition of acetylcholine esterase (AChE) suppressed the differentiation of rat glioma C6 cells, an astrocyte differentiation model, and we observed a suppressive effect of ACh agonists on astrocyte differentiation. Our present study revealed that in the cAMP-induced differentiation of C6 cells, an AChR antagonist alleviated the expression of glia fibrillary acidic protein (GFAP) that had been suppressed by dichlorvos (DDVP), an organophosphate and an AChE inhibitor. Our findings also demonstrated a direct effect of ACh on the GFAP expression, and that muscarinic AChR is involved in the suppressive effect of ACh on the GFAP expression in differentiation-induced C6 cells. This is the first report indicating that ACh the only endogenous agonist for AChRs functions as a mediator of astrocyte differentiation.

A1 polarized IPSCs-derived astrocytes with the PSEN1EΔ9 mutation show deregulated inflammatory dynamics

Event Abstract Back to Event A1 polarized IPSCs-derived astrocytes with the PSEN1EΔ9 mutation show deregulated inflammatory dynamics Sofia Ferreira1, Gonçalo F. Garcia1, Sara Pinto1, Filipa Moreira1, Minna Oksanen2, Jari Koistinaho2, Adelaide Fernandes1, 3 and Dora Brites1, 3* 1 Research Institute for Medicines (iMed.ULisboa), Portugal 2 A.I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences, University of Eastern Finland, Finland 3 Faculdade de Farmácia, Universidade de Lisboa, Department of Biochemistry and Human Biology, Portugal Astrocytes with an A1 phenotype were identified in the prefrontal cortex of Alzheimer’s disease (AD) patients, and revealed to occur upon stimulation with specific inflammatory molecules released by activated microglia in an in vitro murine model (1). Differentiation of astrocytes from iPSCs generated from AD patients may overcome species differences and allow a better understanding of early alterations in AD pathogenesis. So, we investigated the phenotypes of astrocytes differentiated from AD-iPSCs, before and after A1-stimulation, focusing on the dynamics of inflammatory-associated mediators/miRNAs. iPSCs from patients with PSEN1ΔE9 mutation and controls were obtained under the JPND-MADGIC-project (2), and astroglial differentiation was performed as previously described (3). Cells were treated with TNF-α+IL-1α+C1q to induce A1 polarization (1) and small extracellular vesicles (sEVs) isolated by ultracentrifugation. Astrocyte characterization was accomplished by GFAP- and S100B-immunostaining, and detection of released sAPPβ by WB. Expression of pro-/anti-inflammatory markers and associated miRNAs was determined by qRT-PCR. Although almost 100% of astrocytes derived from iPSCs stained positive for S100B, less than 80% of AD ones were positive for GFAP. A1-activation of AD astrocytes further decreased GFAP+ cell number and led to increased sAPPβ secretion. No differences were observed in TNF-α and IL-10 gene expression between non-polarized control and AD astrocytes. However, A1-polarization elicited downregulation of TNF-α and IL-10 in control cells, in contrast with their marked upregulation in AD astrocytes. Further, AD non-polarized cells showed upregulated miR-21 and miR-125b vs. control astrocytes, while a marked downregulation occurred upon A1 polarization. Most attractively, these miRNAs were shown to be decreased in sEVs of AD non-polarized cells, but increased in AD astrocyte media, as well as in AD sEVs and media after A1 induction. Our data support the existence of deregulated inflammatory dynamics in iPSCs-derived astrocytes with the PSEN1EΔ9 mutation, and unveil miR-21 and miR-125b as potential biomarkers for AD. Acknowledgements Supported by EU-JPND research (grant agreement Nº643417 to DB) and by Fundação para a Ciência e a Tecnologia (JPco-fuND/0003/2015 to DB; UID/DTP/04138/2013 to iMed.ULIsboa and SFRH/BD/128738/2017 to GG). References 1- Liddelow SA. et al., Neurotoxic reactive astrocytes are induced by activated microglia, Nature. 2017; 541:481-487 2- http://www.cellmadgic.eu/p1 3- Oksanen M. et al., SEN1 Mutant iPSC-derived model reveals severe astrocyte pathology in Alzheimer's disease, Stem Cell Reports. 2017; 9:1885-1897. Keywords: Alzheimer’s disease, Astrocyte pathology, Inflammatory markers, Inflammatory-associated miRNAs, iPSC = induced pluripotent stem cell Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Glia / Neuroinflammation Citation: Ferreira S, Garcia GF, Pinto S, Moreira F, Oksanen M, Koistinaho J, Fernandes A and Brites D (2019). A1 polarized IPSCs-derived astrocytes with the PSEN1EΔ9 mutation show deregulated inflammatory dynamics. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00025 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. Dora Brites, Research Institute for Medicines (iMed.ULisboa), Lisboa, Portugal, dbrites@ff.ulisboa.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sofia Ferreira Gonçalo F Garcia Sara Pinto Filipa Moreira Minna Oksanen Jari Koistinaho Adelaide Fernandes Dora Brites Google Sofia Ferreira Gonçalo F Garcia Sara Pinto Filipa Moreira Minna Oksanen Jari Koistinaho Adelaide Fernandes Dora Brites Google Scholar Sofia Ferreira Gonçalo F Garcia Sara Pinto Filipa Moreira Minna Oksanen Jari Koistinaho Adelaide Fernandes Dora Brites PubMed Sofia Ferreira Gonçalo F Garcia Sara Pinto Filipa Moreira Minna Oksanen Jari Koistinaho Adelaide Fernandes Dora Brites Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Generating Human iPSC-Derived Astrocytes with Chemically Defined Medium for In Vitro Disease Modeling.

To better understand and model neurological, in particular neurodegenerative diseases, human induced pluripotent stem cells (hiPSCs) offer a great source for generation of neural cells. We provide a protocol for the differentiation of hiPSc-derived astrocytes in vitro. This protocol not only is chemically defined, that is, it does not use serum, but also allows for the expansion of astrocyte progenitor cells and mature astrocytes. Large batches of hiPSc-derived astrocytes can be stored and used for defined in vitro disease models.

Hypoxia epigenetically bestows astrocytic differentiation potential on human pluripotent cell-derived neural stem/precursor cells

The central nervous system (CNS) is composed of three major cell types, neurons, astrocytes, and oligodendrocytes, which differentiate from common multipotent neural stem/precursor cells (NS/PCs). However, NS/PCs do not have this multipotentiality from the beginning: neurons are generated first and astrocytes are later during CNS development. This developmental progression is observed in vitro by using human (h) NS/PCs derived from pluripotent cells, such as embryonic- and induced pluripotent-stem cells (ES/iPSCs), however, in contrast to rodent's pluripotent cells, they require quite long time to obtain astrocytic differentiation potential. Here, we show that hypoxia confers astrocytic differentiation potential on hNS/PCs through epigenetic alteration for gene regulation. Furthermore, we found that these molecular mechanisms can be applied to functional analysis of patient' iPSC-derived astrocytes. In this review, we summarize recent findings that address molecular mechanisms of epigenetic and transcription factor-mediated regulation that specify NS/PC fate and the development of potential therapeutic strategies for treating astrocyte-mediated neurological disorders.

Inhibition of astrocytic differentiation of transplanted neural stem cells by chondroitin sulfate methacrylate hydrogels for the repair of injured spinal cord.

Neural stem cell (NSC) transplantation exerts a therapeutic effect on spinal cord injury (SCI) but is limited to an unregulated differentiation pattern by which NSCs preferentially differentiate into astrocytes, with relatively few neurons. It is well established that the increased NSC-derived astrocytes exhibit aberrant axonal sprouting associated with allodynia-like symptoms of the forepaws. Some strategies have been used to overcome this issue, such as regulation of major pathways, ex vivo gene transfer, and genetic overexpression. However, lack of efficiency, viral vector safety issues and the risk of tumorigenesis have hindered the clinical application of these treatments. Here, we show that astrocytic differentiation of NSCs in vitro and in vivo can be inhibited by encapsulation of cells in a three-dimensional chondroitin sulfate methacrylate (CSMA) hydrogel. When CSMA hydrogels were used to transplant NSCs, the combinatory implant promoted functional recovery and attenuated the hypersensitivity responses of the forepaws. Further analysis showed that transplantation of NSCs within CSMA hydrogels reduced injured cavity areas and promoted neurogenesis rather than fibroglial formation after graft implantation. Furthermore, the treatment prevented allodynia-related CGRP/GAP43-positive nociception due to fibers sprouting into inappropriate lamina regions. Taken together, these findings show that CSMA/NSCs combined transplantation helps prevent adverse side effects of NSCs treatment and promotes recovery of SCI.

EGFRvIII Is Expressed in Cellular Areas of Tumor in a Subset of Glioblastoma.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific cell surface antigen often expressed in glioblastoma and has drawn much attention as a possible therapeutic target. We performed immunohistochemistry on histology sections of surgical specimens taken from 67 cases with glioblastoma, isocitrate dehydrogenase-wild type, and evaluated the morphological characteristics and distribution of the EGFRvIII-positive tumor cells. We then evaluated the localization of EGFRvIII-expression within the tumor and peritumoral areas. EGFRvIII immunopositivity was detected in 15 specimens taken from 13 patients, including two recurrent specimens taken from the same patient at relapse. Immunofluorescence staining demonstrated that EGFRvIII-positive cells were present in cells positive for glial fibrillary acidic protein (GFAP), and some showed astrocytic differentiation with multiple fine processes and others did not shown. The EGFRvIII-positive cells were located in cellular areas of the tumor, but not in the invading zone. In the two recurrent cases, EGFRvIII-positive cells were markedly decreased in one case and retained in the other. With regard to overall survival, univariate analysis indicated that EGFRvIII-expression in patients with glioblastoma was not significantly associated with a favorable outcome. Double-labeling immunofluorescence staining of EGFRvIII and GFAP showed that processes of large, well differentiated, GFAP-positive glia extend to and surround less differentiated, EGFRvIII-positive glial cells in cellular areas of tumor. However, in the tumor periphery, EGFRvIII-positive tumor cells were not observed. This finding suggests that EGFRvIII is involved in tumor proliferation, but that invading glioma cells lose their EGFRvIII expression.

SU31 - ESTABLISHING A BLOOD-BRAIN BARRIER MODEL FOR THE ANALYSIS OF ALZHEIMER'S DISEASE USING PATIENT DERIVED INDUCED PLURIPOTENT STEM CELLS

Background Alzheimer's Disease (AD) is the most common form of dementia. One characteristic of AD is the accumulation of amyloid-β (Aβ) peptides in different regions of the brain caused by the sequential cleavage of the transmembrane amyloid precursor protein. These extracellular amyloid plaques are a result of a dysregulated Blood-Brain Barrier (BBB) and emerge either due to an augmented production or a decreased excretion through brain barriers. The BBB with its several transporter systems shows age dependent expression levels of unspecific Aβ transporters. Recently, 19 loci including ABCA7, an ABC transporter responsible for Aβ efflux at the BBB, were discovered showing genome-wide significant association with the sporadic, late-onset form of AD (LOAD). Together with other research groups we identified several mutations in ABCA7 in AD patients, which is why an analysis of mutated ABCA7 in AD-related BBB models should be considered. We established patient-specific pluripotent stem cells from AD patients and healthy donors to differentiate endothelial cells and other related BBB cells for the investigation of disease mechanisms related to ABCA7 in AD patients. Methods Episomal vectors were used for the generation of induced Pluripotent Stem (iPS) cell lines from AD-specific B-lymphoblastoid cell lines (B-LCLs) and control B-LCLs of healthy donors. Alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers verified pluripotency. Successfully established lines were applied for the generation of BBB cells and neighboring cells. Endothelial cells and astrocytes were characterized by transcript analysis and flow cytometry. The differentiation process was analyzed for the altered expression of ABCA7. Results AD-specific iPS cells were successfully generated from B-LCLs. We could show alkaline phosphatase activity and the nuclear localization of pluripotency-related transcription factors including OCT4, SOX2, and NANOG. Transcript analysis and western blot analysis verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The efficient differentiation of high quality endothelial cells was shown by the expression of endothelial marker genes including von Willebrand factor. Astrocyte-specific markers including EAAT1 demonstrated efficient astrocyte differentiation. Discussion Human iPS cells provide a powerful tool for the analysis of AD-associated and patient-specific mutations in ABCA7. We successfully established endothelial cells and astrocytes representing suitable to study disease mechanisms of the BBB. Accordingly, this approach is an excellent opportunity to analyze pathogenic phenotypes of AD in vitro towards the identification of potential therapeutic targets.

Identification and epigenetic analysis of divergent long non-coding RNAs in multilineage differentiation of human Neural Progenitor Cells

ABSTRACTLong non-coding RNAs have emerged as an important regulatory layer in biological systems. Of the various types of lncRNAs, one class (designated as divergent RNAs/XH), which is in head-to-head overlap with the coding genes, has emerged as a critical biotype that regulates development and cellular differentiation. This work aimed to analyze previously published data on differential expression, epigenetic and network analysis in order to demonstrate the association of divergent lncRNAs, a specific biotype with the differentiation of human neural progenitor cells (hNPCs). We have analyzed various available RNAseq databases that address the neuronal and astrocytic differentiation of hNPCs and identified differentially expressed lncRNAs (DELs) during cell-fate determination. Key DELs identified from the databases were experimentally verified by us in our in-vitro hNPC differentiation system. We also analyzed the change in promoter activity using ChIP-seq datasets of the histone markers H3K4me3 (activation) and H3K27me3 (inactivation) of these DELs. Additionally, we explored the change in the euchromatinization state of DELs (by analyzing DNase-seq data) during lineage-specific differentiation of hNPCs and performed their network analysis. We were able to identify differences between neuronal and astrocytic differentiation of hNPCs at the level of divergent DELs epigenetic markers, DNAase hypersensitive sites and gene expression network. Divergent lncRNAs are more involved in neuronal rather than astrocytic differentiation, while the sense downstream lncRNA biotype appears to be more involved in astrocytic differentiation. By studying the lncRNA involvement of distinct biotypes, we have been able to indicate the preferential role of a particular biotype during lineage-specific differentiation.

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.

Conditioned Media From Glial Cells Promote a Neural-Like Connexin Expression in Human Adipose-Derived Mesenchymal Stem Cells.

The expression of neuronal and glial connexins (Cxs) has been evaluated in adipose-derived mesenchymal stem cells (ASCs) whose neural differentiation was promoted by a conditioned medium (CM) obtained from cultures of olfactory ensheathing cells (OECs) or Schwann cells (SCs). By immunocytochemistry and flow cytometer analysis it was found that Cx43 was already considerably expressed in naïve ASCs and further increased after 24 h and 7 days from CM exposition. Cx32 and Cx36 were significantly improved in conditioned cultures compared to control ASCs, whereas a decreased expression was noticed in the absence of CM treatments. Cx47 was virtually absent in any conditions. Altogether, high basal levels and induced increases of Cx43 expression suggest a potential attitude of ASCs toward an astrocyte differentiation, whereas the lack of Cx47 would indicate a poor propensity of ASCs to become oligodendrocytes. CM-evoked Cx32 and Cx36 increases showed that a neuronal- or a SC-like differentiation can be promoted by using this strategy. Results further confirm that environmental cues can favor an ASC neural differentiation, either as neuronal or glial elements. Of note, the use of glial products present in CM rather than the addition of chemical agents to achieve such differentiation would resemble “more physiological” conditions of differentiation. As a conclusion, the overexpression of typical neural Cxs would indicate the potential capability of neural-like ASCs to interact with neighboring neural cells and microenvironment.

3D Microfibrous Scaffolds Selectively Promotes Proliferation and Glial Differentiation of Adult Neural Stem Cells: A Platform to Tune Cellular Behavior in Neural Tissue Engineering.

Biomaterials are essential for the development of innovative biomedical and therapeutic applications. Biomaterials-based scaffolds can influence directed cell differentiation to improve cell-based strategies. Using a novel microfluidics approach, poly (ε-caprolactone) (PCL), is used to fabricate microfibers with varying diameters (3-40µm) and topographies (straight and wavy). Multipotent adult rat hippocampal stem/progenitor cells (AHPCs) are cultured on 3D aligned PCL microfibrous scaffolds to investigate their ability to differentiate into neurons, astrocytes, and oligodendrocytes. The results indicate that the PCL microfibers significantly enhance proliferation of the AHPCs compared to control, 2D planar substrates. While the AHPCs maintained their multipotent differentiation capacity when cultured on the PCL scaffolds, there is a significant and dramatic increase in immunolabeling for astrocyte and oligodendrocyte differentiation when compared with growth on planar surfaces. Our results show a 3.5-fold increase in proliferation and 23.4-fold increase in astrocyte differentiation for cells on microfibers. Transplantation of neural stem/progenitor cells within a PCL microfiber scaffold may provide important biological and topographic cues that facilitate the survival, selective differentiation, and integration of transplanted cells to improve therapeutic strategies.

Effects of cyclic AMP on the differentiation and bioenergetics of rat C6 glioma cells

Introduction: Elevation in the level of intracellular cAMP is known to induce astrocytic differentiation of C6 glioma cells by unknown mechanisms.Methods: Therefore, cytoskeletal protein genes (phalloidin) fluorescents to investigate morphological changes, cell proliferation assay, MTT assay, flow cytometry, western blotting, in-cell western, immune-cytochemical (protein expression and localization), and oxygen electrodes (oxygen consumption rate) after a treatment with 0.25 mM dbcAMP were conducted.Results: Undifferentiated cells (media without dbcAMP) showed a flat polygonal appearance, whereas those cultured in the presence of 0.25 mM dbcAMP exhibited a more differentiated astrocytic morphology. They had more numerous neurite-like thin processes. The cell proliferation of differentiated c6 glioma reduced at day 2 and then started to increase at day 3 till day 5 compared to undifferentiated c6 glioma cells. In terms of flow-cytometry data, dbcAMP had no apoptotic effect on the C6 glioma cells. There was an increase in the protein expression GFAP (specific marker for astrocytes). There was no significant effect between undifferentiated and 5-day differentiation regarding their response to glucose 10 mM. In addition, there were no significant effects of glucose on the basal of 5-day differentiation of C6 glioma cells. However, there was a significant correlation between the concentration of glucose and inhibition of the basal oxygen consumption. Finally, glucose 10 mM did not stimulate NAD (P)H levels of C6 glioma cells.Conclusion: The above results showed that cAMP induce C6 glioma cells differentiation without affecting its bioenergetics. Therefore cAMP is considered to be the best differentiating agent.