Astroglial differentiation of fibronectin­positive human "glia-like" cells.

OBJECTIVES: Fibronectin (Fn) is a glycoprotein of extracellular matrix produced by a variety of mesenchymal and neoplastic cell types. BACKGROUND: In adult brain tissue, Fn is restricted to blood vessels. However, adult human brain cultures are almost entirely comprised of flat or spindle­shaped Fn-positive cells usually referred to as "glia-like" cells. Since Fn is primarily present in fibroblasts, these cultures may be considered to be of non-glial origin. METHODS: Cells gained by long-term culturing of adult human brain tissue derived from brain biopsies obtained from 12 patients with non-malignant diagnoses were examined by immunofluorescence methods. RESULTS: Primary cultures contained GFAP-/Vim+/Fn+ "glia-like" cells (95-98 %) and GFAP+/Vim+/Fn- astrocytes (0.1 %) which disappeared by passage number 3. The formation of cell processes and enlargement of cell bodies was observed in 9 of 12 cultures with decreased cell growth during passages 12 to 17. It is remarkable that during this period, all "glia-like" cells became GFAP+/Vim+/Fn+. CONCLUSION: Herein, we confirm our previously published hypothesis about the origin of adult human "glia-like" cells, which we consider to be precursor cells scattered through the brain cortex and subcortical white matter. Cultures were comprised entirely of GFAP-/Fn+ "glia-like" cells and showed morphological and immunochemical astroglial differentiation with spontaneously decelerated growth during prolonged passaging. We propose that the adult human brain tissue contains a "dormant population" of undefined glial precursor cells. Under culture, these cells show to have a high proliferative capacity and different stages of cell dedifferentiation (Fig. 2, Ref. 21).

Rapid and Efficient Differentiation of Rodent Neural Stem Cells into Oligodendrocyte Progenitor Cells.

Oligodendrocyte progenitor cells (OPCs) may have beneficial effects in cell replacement therapy of neurodegenerative disease owing to their unique capability to differentiate into myelinogenic oligodendrocytes (OLs) in response to extrinsic signals. Therefore, it is of significance to establish an effective differentiation methodology to generate highly pure OPCs and OLs from some easily accessible stem cell sources. To achieve this goal, in this study, we present a rapid and efficient protocol for oligodendroglial lineage differentiation from mouse neural stem cells (NSCs), rat NSCs, or mouse embryonic stem cell-derived neuroepithelial stem cells. In a defined culture medium containing Smoothened Agonist, basic fibroblast growth factor, and platelet-derived growth factor-AA, OPCs could be generated from the above stem cells over a time course of 4-6 days, achieving a cell purity as high as ∼90%. In particular, these derived OPCs showed high expandability and could further differentiate into myelin basic protein-positive OLs within 3 days or alternatively into glial fibrillary acidic protein-positive astrocytes within 7 days. Furthermore, transplantation of rodent NSC-derived OPCs into injured spinal cord indicated that it is a feasible strategy to treat spinal cord injury. Our results suggest a differentiation strategy for robust production of OPCs and OLs from rodent stem cells, which could provide an abundant OPC source for spinal cord injury.

Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin.

Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.

Suppressor of fused controls cerebellar neuronal differentiation in a manner modulated by GLI3 repressor and Fgf15.

BACKGROUND: Deficiency of Suppressor of Fused (SuFu), an intracellular mediator of Hedgehog signaling, in the murine mid-hindbrain disrupts cerebellar morphogenesis and cell differentiation in a manner that is rescued by constitutive expression of GLI3 transcriptional repressor (GLI3R). Here, we determined SuFu functions in cerebellar radial precursors following the stage of mid-hindbrain specification using a Blbp-Cre transgene. RESULTS: SuFu-deficient cerebella were severely dysplastic, and characterized by laminar disorganization, and delayed differentiation of ventricular zone-derived precursors. In vitro analysis of cerebellar precursors isolated from control and mutant mice demonstrated an increased proportion of radial glial precursors vs. Tuj1-positive neurons in mutant cultures. Abnormal cell differentiation in SuFu-deficient precursors was rescued by a constitutively expressed GLI3R knock-in allele, albeit with variable penetrance. Using RNA expression analysis in control and SuFu-deficient cerebellar anlage, we identified up-regulation of Fgf15 in mutant tissue. Strikingly, exogenous hFGF19, a mFGF15 ortholog, inhibited neuronal differentiation in cultures of wild-type cerebellar precursors. Moreover, siRNA-mediated knockdown of Fgf15 in SuFu-deficient cerebellar precursors rescued their delayed differentiation to neurons. CONCLUSIONS: Together, our results show that SuFu promotes cerebellar radial precursor differentiation to neurons. SuFu function is mediated in part by GLI3R and down-regulation of Fgf15 expression. Developmental Dynamics 247:156-169, 2018. © 2017 Wiley Periodicals, Inc.

A transcriptional network controlling glial development in the Drosophila visual system.

In the nervous system, glial cells need to be specified from a set of progenitor cells. In the developing Drosophila eye, perineurial glia proliferate and differentiate as wrapping glia in response to a neuronal signal conveyed by the FGF receptor pathway. To unravel the underlying transcriptional network we silenced all genes encoding predicted DNA-binding proteins in glial cells using RNAi. Dref and other factors of the TATA box-binding protein-related factor 2 (TRF2) complex were previously predicted to be involved in cellular metabolism and cell growth. Silencing of these genes impaired early glia proliferation and subsequent differentiation. Dref controls proliferation via activation of the Pdm3 transcription factor, whereas glial differentiation is regulated via Dref and the homeodomain protein Cut. Cut expression is controlled independently of Dref by FGF receptor activity. Loss- and gain-of-function studies show that Cut is required for glial differentiation and is sufficient to instruct the formation of membrane protrusions, a hallmark of wrapping glial morphology. Our work discloses a network of transcriptional regulators controlling the progression of a naïve perineurial glia towards the fully differentiated wrapping glia.

Development of the intrinsic and extrinsic innervation of the gut.

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract.

A synthetic BMP-2 mimicking peptide induces glioblastoma stem cell differentiation.

BACKGROUND: Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments. METHODS: The BMP-2 mimicking peptide, named GBMP1a, was synthesized on solid-phase by Fmoc chemistry. Structural characterization and prediction of receptor binding were obtained by Circular Dicroism (CD) and NRM analyses. Activation of BMP signalling was evaluated by a luciferase reporter assay and western blot. Pro-differentiating effects of GBMP1a were verified by immunostaining and neurosphere assay in primary glioblastoma cultures. RESULTS: CD and NMR showed that GBMP1a correctly folds into expected tridimensional structures and predicted its binding to BMPR-IA to the same epitope as in the native complex. Reporter analysis disclosed that GBMP1a is able to activate BMP signalling in GBM cells. Moreover, BMP-signalling activation was specifically dependent on smad1/5/8 phosphorylation. Finally, we confirmed that GBMP1a treatment is sufficient to enhance osteogenic differentiation of Mesenchymal Stem Cells and to induce astroglial differentiation of glioma stem cells (GSCs) in vitro. CONCLUSIONS: GBMP1a was demonstrated to be a good inducer of GSC differentiation, thus being considered a potential anti-cancer tool to be further developed for GBM treatment. GENERAL SIGNIFICANCE: These data highlight the role of BMP-mimicking peptides as potential anti-cancer agents against GBM and stimulate the further development of GBMP1a-based structures in order to enhance its stability and activity.

Secretome profiling of cytokines and growth factors reveals that neuro-glial differentiation is associated with the down-regulation of Chemokine Ligand 2 (MCP-1/CCL2) in amniotic fluid derived-mesenchymal progenitor cells.

Secreted cytokines and growth factors play a key role in the modulation of stem cell proliferation, differentiation and survival. To investigate the interplay between the changes in their expression levels, we used the newly characterized human amniotic fluid derived-mesenchymal progenitor MePR-2B cell line differentiated to a neuro-glial phenotype and exploited the very high sensitivity and versatility of magnetic beads-based immunoassays. We found that a sub-set of proteins, including the cytokines IL-6, TNFα, IL-15, IFNγ, IL-8, IL-1ra, MCP-1/CCL2, RANTES and the growth factor PDGFbb, underwent a significant down-regulation following neuro-glial differentiation, whereas the expression levels of IL-12 p70, IL-5, IL-7, bFGF, VEGF and G-CSF were increased. The role of MCP-1/CCL2, previously identified as a regulator of neural progenitor stem cell differentiation, has been further investigated at transcriptional level, revealing that both the chemokine and its receptor are co-expressed in MePR-2B cells and that are regulated upon differentiation, suggesting the presence of an autocrine and paracrine loop in differentiating cells. Moreover, we demonstrated that exogenous CCL2 is capable to affect neuro-glial differentiation in MePR-2B cells, thus providing novel evidences for the potential involvement of chemokine-mediated signaling in progenitor/stem cells differentiation processes and fate specification.

The transcription factor hairy/E(spl)-related 2 induces proliferation of neural progenitors and regulates neurogenesis and gliogenesis.

The study of molecular regulation in neural development provides information to understand how diverse neural cells are generated. It also helps to establish therapeutic strategies for the treatment of neural degenerative disorders and brain tumors. The Hairy/E(spl) family members are potential targets of Notch signaling, which is fundamental to neural cell maintenance, cell fate decisions, and compartment boundary formation. In this study, we isolated a zebrafish homolog of Hairy/E(spl), her2, and showed that this gene is expressed in neural progenitor cells and in the developing nervous system. The expression of her2 required Notch activation, as revealed by a Notch-defective mutant and a chemical inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The endogenous expression of Her2 was altered by both overexpression and morpholino-knockdown approaches, and the results demonstrated that Her2 was both necessary and sufficient to promote the proliferation of neural progenitors by inhibiting the transcription of the cell cycle inhibitors cdkn1a, cdkn1ba, and cdkn1bb. Her2 knockdown caused premature neuronal differentiation, which indicates that Her2 is essential for inhibiting neuronal differentiation. At a later stage of neural development, Her2 could induce glial differentiation. The overexpression of Her2 constructs lacking the bHLH or WRPW domain phenocopied the effect of the morpholino knockdown, demonstrating the essential function of these two domains and further confirming the knockdown specificity. In conclusion, our data reveal that Her2 promotes progenitor proliferation and maintains progenitor characteristics by inhibiting neuronal differentiation. Together, these two mechanisms ensure the proper development of the neural progenitor cell pool.

Minocycline mitigates the gliogenic effects of proinflammatory cytokines on neural stem cells.

Mobilizing endogenous neural stem cells (NSCs) in the adult brain is designed to enhance the brain's regenerative capacity after cerebral lesions, e.g., as a result of stroke. Cerebral ischemia elicits neuroinflammatory processes affecting NSCs in multiple ways, the precise mechanisms of which currently remain elusive. An inhibitory effect of minocycline on microglia activation, a hallmark of postischemic neuroinflammation, has already been demonstrated in clinical trials, showing minocycline to be safe and potentially effective in ischemic stroke. Here we investigate the direct effects of minocycline and of proinflammatory cytokines on the differentiation potential of NSCs in vitro and in vivo. Primary fetal rat NSCs were treated with minocycline plus a combination of the proinflammatory cytokines tumor necrosis factor-α, interleukin 1ß, and interleukin 6. The differentiation fate of NSCs was assessed immunocytochemically. To investigate the effects of minocycline and inflammation in vivo, minocycline or lipopolysaccharides were injected intraperitoneally into adult rats, with subsequent immunohistochemistry. Minocycline alone did not affect the differentiation potential of NSCs in vivo or in vitro. In contrast, proinflammatory cytokines accelerated the differentiation of NSCs, promoting an astrocytic fate while inhibiting neurogenesis in vitro and in vivo. It is interesting to note that minocycline counteracted this cytokine-induced rapid astrocytic differentiation and restored the neurogenic and oligodendrogliogenic potential of NSCs. Data suggest that minocycline antagonizes the rapid glial differentiation induced by proinflammatory cytokines following cerebral ischemia but without having a direct effect on the differentiation potential of NSCs. Thus, minocycline constitutes a promising drug for stroke research, counteracting the detrimental effects of postischemic neuroinflammation in multiple ways.

Secreted type of amyloid precursor protein induces glial differentiation by stimulating the BMP/Smad signaling pathway.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases leading to dementia. Although cytotoxicity of amyloid ß peptides has been intensively studied within pathophysiology of AD, the physiological function of amyloid precursor protein (APP) still remains unclarified. We have shown previously that secreted APPα (sAPPα) is associated with glial differentiation of neural stem cells. To elucidate specific mechanisms underlying sAPPα-induced gliogenesis, we examined the potential involvement of bone morphogenic proteins (BMPs). BMPs are one of the factors involved in glial differentiation of neural progenitor cells. When expressions of BMP-2, -4, and -7 were examined, upregulation of BMP-4 expression was solely observed as a result of treatment with sAPPα in a time and dose-dependent manner. Furthermore, the treatment of sAPPα promoted phosphorylation of Smad1/5/8, a downstream signaling mediator of BMP receptors. Interestingly, N-terminal domain of APP (1-205) was sufficient to elevate BMP4 expression, resulting in an increase of glial fibrillary acidic protein (GFAP) expression and phosphorylation of Smad1/5/8. However, the application of APP neutralizing antibody and anti-BMP4 antibody significantly suppressed expression of BMP-4 as well as phosphorylation of Smad1/5/8. Thus, our results indicate that sAPPα-induced gliogenesis is in part mediated by the BMP-4 signaling pathway. We also observed upregulation of BMP-4 and phosphorylation of Smad1/5/8 in APP transgenic mice. It is imperative to unravel the mechanisms underlying the role of BMP-4 during APPα-induced glial differentiation in hope of providing novel prevention or treatment for AD.

Embryonic Stem Cells Lacking DNA Methyltransferases Differentiate into Neural Stem Cells that Are Defective in Self-Renewal.

Background and Objectives: DNA methyltransferases (Dnmts) play an important role in regulating DNA methylation during early developmental processes and cellular differentiation. In this study, we aimed to investigate the role of Dnmts in neural differentiation of embryonic stem cells (ESCs) and in maintenance of the resulting neural stem cells (NSCs). Methods and Results: We used three types of Dnmt knockout (KO) ESCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO), to investigate the role of Dnmts in neural differentiation of ESCs. All three types of Dnmt KO ESCs could form neural rosette and differentiate into NSCs in vitro. Interestingly, however, after passage three, Dnmt KO ESC-derived NSCs could not maintain their self-renewal and differentiated into neurons and glial cells. Conclusions: Taken together, the data suggested that, although deficiency of Dnmts had no effect on the differentiation of ESCs into NSCs, the latter had defective maintenance, thereby indicating that Dnmts are crucial for self-renewal of NSCs.

Axonal ion homeostasis and glial differentiation.

The brain is the ultimate control unit of the body. It conducts accurate, fast and reproducible calculations to control motor actions affecting mating, foraging and flight or fight decisions. Therefore, during evolution, better and more efficient brains have emerged. However, even simple brains are complex organs. They are formed by glial cells and neurons that establish highly intricate networks to enable information collection, processing and eventually, a precise motor control. Here, we review and connect some well-established and some hidden pieces of information to set the focus on ion homeostasis as a driving force in glial differentiation promoting signalling speed and accuracy.

A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan.

The molecular diversity of glia in the human hippocampus and their temporal dynamics over the lifespan remain largely unknown. Here, we performed single-nucleus RNA sequencing to generate a transcriptome atlas of the human hippocampus across the postnatal lifespan. Detailed analyses of astrocytes, oligodendrocyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease relevance. We further characterized spatiotemporal heterogeneity of GFAP-enriched astrocyte subpopulations in the hippocampal formation using immunohistology. Leveraging glial subpopulation classifications as a reference map, we revealed the diversity of glia differentiated from human pluripotent stem cells and identified dysregulated genes and pathological processes in specific glial subpopulations in Alzheimer's disease (AD). Together, our study significantly extends our understanding of human glial diversity, population dynamics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem-cell-based glial differentiation.

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis.

The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed.

Embryonic neural stem/progenitor cells as model to characterize dystrophin and dystrophin-associated proteins expression during neuronal or astrocytic differentiation.

Neural stem/progenitor cells (NSPC) are multipotent cells that renew themselves and could differentiate into neurons and macro glia (astrocytes and oligodendrocytes) of the nervous system during embryonic development. Duchenne muscular dystrophy is a severe type of muscular dystrophy caused by mutations in the dmd gene, and one-third of patients cursed with neuro-cognitive impairments. In this data article, we take advantage of the differentiation capacity of NSPC as a model to increase our knowledge in the neuronal and/or astrocytic differentiation and to evaluate the expression of dystrophins and dystrophin-associated proteins. We showed the characterization of undifferentiated and neuron and/or astrocyte differentiated NSPC. In addition, we evaluated the expression and subcellular localization of dystrophins and ß-dystroglycan in undifferentiated NSPC and differentiated to neurons and astrocytes.•Primary culture of NSPC was characterized by the expression of multipotent markers nestin and Sox2.•Neuronal or astrocytic differentiation of NSPC was performed by basic fibroblast growth factor (FGF2) withdrawal, histamine or ciliary neurotrophic factor (CNTF) treatment, and expression of ßIII-tubulin or glial fibrillary acidic protein (GFAP) as differentiation markers for neurons or astrocytes was evaluated.•This study will contribute to the understanding of dystrophins and dystrophin-associated proteins expression and function during neuronal or astrocytic differentiation of NSPC.

Optogenetically Controlled TrkA Activity Improves the Regenerative Capacity of Hair-Follicle-Derived Stem Cells to Differentiate into Neurons and Glia.

Hair-follicle-derived stem cells (HSCs) originating from the bulge region of the mouse vibrissa hair follicle are able to differentiate into neuronal and glial lineage cells. The tropomyosin receptor kinase A (TrkA) receptor that is expressed on these cells plays key roles in mediating the survival and differentiation of neural progenitors as well as in the regulation of the growth and regeneration of different neural systems. In this study, the OptoTrkA system is introduced, which is able to stimulate TrkA activity via blue-light illumination in HSCs. This allows to determine whether TrkA signaling is capable of influencing the proliferation, migration, and neural differentiation of these somatic stem cells. It is found that OptoTrkA is able to activate downstream molecules such as ERK and AKT with blue-light illumination, and subsequently able to terminate this kinase activity in the dark. HSCs with OptoTrkA activity show an increased ability for proliferation and migration and also exhibited accelerated neuronal and glial cell differentiation. These findings suggest that the precise control of TrkA activity using optogenetic tools is a viable strategy for the regeneration of neurons from HSCs, and also provides a novel insight into the clinical application of optogenetic tools in cell-transplantation therapy.

Neural crest derived stem cells from dental pulp and tooth-associated stem cells for peripheral nerve regeneration.

The peripheral nerve injuries, representing some of the most common types of traumatic lesions affecting the nervous system, are highly invalidating for the patients besides being a huge social burden. Although peripheral nervous system owns a higher regenerative capacity than does central nervous system, mostly depending on Schwann cells intervention in injury repair, several factors determine the extent of functional outcome after healing. Based on the injury type, different therapeutic approaches have been investigated so far. Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries, however these approaches own limitations, such as scarce donor nerve availability and donor site morbidity. Cell based therapies might provide a suitable tool for peripheral nerve regeneration, in fact, the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade. Dental pulp is a promising cell source for regenerative medicine, because of the ease of isolation procedures, stem cell proliferation and multipotency abilities, which are due to the embryological origin from neural crest. In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models, highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.

Characterization of the expression of dystrophins and dystrophin-associated proteins during embryonic neural stem/progenitor cell differentiation.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene. Dystrophin is required for the organization of a complex consisting of dystroglycans, sarcoglycans, dystrobrevins and syntrophins, known as the dystrophin-associated proteins complex (DAPC). In addition to muscle degeneration, cognitive impairment has been reported in DMD patients. To characterize a suitable model for studying the embryonic cerebral functions of dystrophin, we analyzed the expression patterns of dystrophins/DAPC in undifferentiated and differentiated embryonic neural stem/progenitor cells (NSPC). We found that NSPC express mRNAs for dystrophins Dp427, Dp140, Dp71 and Dp40; ß-dystroglycan; α- and ß-dystrobrevin; α1-, ß1-, ß2- and γ2-syntrophin; and ß-, γ-, δ- and ε-sarcoglycan. Some of these were differentially regulated during neuronal or astrocytic differentiation. Interestingly, the protein expression levels of Dp140, ß-dystroglycan and α2-dystrobrevin were also differentially regulated. Additionally, we found that proliferating NSPC and differentiated neurons and astrocytes show immuno-positive staining for dystrophins and ß-dystroglycan. Our results show that dystrophins and DAPC components are expressed and regulated during the neuronal or astrocytic differentiation of NSPC, suggesting that these proteins may have different roles in the brain development.

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 (3-40 µm) 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.