Isolation and characterization of GFAP-positive porcine neural stem/progenitor cells derived from a GFAP-CreERT2 transgenic piglet.
ABSTRACT: BACKGROUND:The porcine brain is gyrencephalic with similar gray and white matter composition and size more comparable to the human rather than the rodent brain; however, there is lack of information about neural progenitor cells derived from this model. RESULTS:Here, we isolated GFAP-positive porcine neural stem cells (NSCs) from the brain explant of a transgenic piglet, with expression of CreERT2 under the control of the GFAP promoter (pGFAP-CreERT2). The isolated pGFAP-CreERT2 NSCs showed self-renewal and expression of representative NSC markers such as Nestin and Sox2. Pharmacological inhibition studies revealed that Notch1 signaling is necessary to maintain NSC identity, whereas serum treatment induced cell differentiation into reactive astrocytes and neurons. CONCLUSIONS:Collectively, these results indicate that GFAP promoter-driven porcine CreERT2 NSCs would be a useful tool to study neurogenesis of the porcine adult central nervous system and furthers our understanding of its potential clinical application in the future. ?.
Project description:PTEN-induced putative kinase 1 (PINK1) is a Parkinson's disease (PD) gene. We examined miRNAs regulated by PINK1 during brain development and neural stem cell (NSC) differentiation, and found that lvels of miRNAs related to tumors and inflammation were different between 1-day-old-wild type (WT) and PINK1-knockout (KO) mouse brains. Notably, levels of miR-326, miR-330 and miR-3099, which are related to astroglioma, increased during brain development and NSC differentiation, and were significantly reduced in the absence of PINK1. Interestingly, in the presence of ciliary neurotrophic factor (CNTF), which pushes differentiation of NSCs into astrocytes, miR-326, miR-330, and miR-3099 levels in KO NSCs were also lower than those in WT NSCs. Furthermore, mimics of all three miRNAs increased expression of the astrocytic marker glial fibrillary acidic protein (GFAP) during differentiation of KO NSCs, but inhibitors of these miRNAs decreased GFAP expression in WT NSCs. Moreover, these miRNAs increased the translational efficacy of GFAP through the 3'-UTR of GFAP mRNA. Taken together, these results suggest that PINK1 deficiency reduce expression levels of miR-326, miR-330 and miR-3099, which may regulate GFAP expression during NSC differentiation and brain development.
Project description:Since the original isolation of neural stem cells (NSCs) in the adult mammalian brain, further work has revealed a heterogeneity in the NSC pool. Our previous work characterized a distinct, Oct4 expressing, NSC population in the periventricular region, through development and into adulthood. We hypothesized that this population is upstream in lineage to the more abundant, well documented, GFAP expressing NSC. Herein, we show that Oct4 expressing NSCs give rise to neurons, astrocytes and oligodendrocytes throughout the developing brain. Further, transgenic inducible mouse models demonstrate that the rare Oct4 expressing NSCs undergo asymmetric divisions to give rise to GFAP expressing NSCs in naïve and injured brains. This lineage relationship between distinct NSC pools contributes significantly to an understanding of neural development, the NSC lineage in vivo and has implications for neural repair.
Project description:Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.
Project description:Neurosphere formation is commonly used as a surrogate for neural stem cell (NSC) function but the relationship between neurosphere-initiating cells (NICs) and NSCs remains unclear. We prospectively identified, and isolated by flow cytometry, adult mouse lateral ventricle subventricular zone (SVZ) NICs as Glast(mid)EGFR(high)PlexinB2(high)CD24(-/low)O4/PSA-NCAM(-/low)Ter119/CD45(-) (GEPCOT) cells. They were highly mitotic and short-lived in vivo based on fate-mapping with Ascl1(CreERT2) and Dlx1(CreERT2). In contrast, pre-GEPCOT cells were quiescent, expressed higher Glast, and lower EGFR and PlexinB2. Pre-GEPCOT cells could not form neurospheres but expressed the stem cell markers Slc1a3-CreER(T), GFAP-CreER(T2), Sox2(CreERT2), and Gli1(CreERT2) and were long-lived in vivo. While GEPCOT NICs were ablated by temozolomide, pre-GEPCOT cells survived and repopulated the SVZ. Conditional deletion of the Bmi-1 polycomb protein depleted pre-GEPCOT and GEPCOT cells, though pre-GEPCOT cells were more dependent upon Bmi-1 for Cdkn2a (p16(Ink4a)) repression. Our data distinguish quiescent NSCs from NICs and make it possible to study their properties in vivo.DOI: http://dx.doi.org/10.7554/eLife.02669.001.
Project description:Adult forebrain definitive neural stem cells (NSCs) comprise a subpopulation of GFAP-expressing subependymal cells that arise from embryonic fibroblast growth factor (FGF)-dependent NSCs that are first isolated from the developing brain at E8.5. Embryonic FGF-dependent NSCs are derived from leukemia inhibitory factor (LIF)-responsive, Oct4-expressing primitive NSCs (pNSCs) that are first isolated at E5.5. We report the presence of a rare population of pNCSs in the periventricular region of the adult forebrain. Adult-derived pNSCs (AdpNSCs) are GFAP(-), LIF-responsive stem cells that display pNSC properties, including Oct4 expression and the ability to integrate into the inner cell mass of blastocysts. AdpNSCs generate self-renewing, multipotent colonies that give rise to definitive GFAP(+) NSCs in vitro and repopulate the subependyma after the ablation of GFAP(+) NSCs in vivo. These data support the hypothesis that a rare population of pNSCs is present in the adult brain and is upstream of the GFAP(+) NSCs.
Project description:This study is to determine the impact of QKI deletion on transcriptomes of mouse NSC and PM-NSC and to analyze the transcriptomic profiles of Nestin-CreERT2 PtenLoxP/LoxP p53LoxP/LoxP QKILoxP/LoxP (QPP) mouse glioblastoma and to determine which subtypes these tumors belong to Overall design: NSCs of different genetic background (Nestin-CreERT2 QKI+/+, Nestin-CreERT2 QKILoxP/LoxP, Nestin-CreERT2 PtenLoxP/LoxP p53LoxP/LoxP and Nestin-CreERT2 PtenLoxP/LoxP p53LoxP/LoxP QKILoxP/LoxP) were isolated from postnatal day one SVZs and then treated with either EtOH or 4OHT for two days. Nestin-CreERT2 PtenLoxP/LoxP p53LoxP/LoxP QKILoxP/LoxP (QPP) mice were treated with tamoxifen at day8-10, and mouse brain tumors were collected. The total RNAs of NSCs, PM-NSCs and brain tumors were extracted by Rnasey kit and sent for sequencing with llumina HiSeq2000 Sequencer.
Project description:Cellular metabolism plays a crucial role in controlling the proliferation, differentiation, and quiescence of neural stem cells (NSCs). The metabolic transition from aerobic glycolysis to oxidative phosphorylation has been regarded as a hallmark of neuronal differentiation. Understanding what triggers metabolism reprogramming and how glucose metabolism directs NSC differentiation may provide new insight into the regenerative potential of the brain. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is an endogenous inhibitor of glycolysis and is highly expressed in mature neurons. However, its function in embryonic NSCs has not yet been explored. In this study, we aimed to investigate the precise roles of TIGAR in NSCs and the possible involvement of metabolic reprogramming in the TIGAR regulatory network. We observed that TIGAR is significantly increased during brain development as neural differentiation proceeds, especially at the peak of NSC differentiation (E14.5-E16.5). In cultured NSCs, knockdown of TIGAR reduced the expression of microtubule-associated protein 2 (MAP2), neuron-specific class III beta-tubulin (Tuj1), glial fibrillary acidic protein (GFAP), Ngn1, and NeuroD1, and enhanced the expression of REST, suggesting that TIGAR is an important regulator of NSC differentiation. Furthermore, TIGAR enhanced the expression of lactate dehydrogenase B (LDHB) and the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) markers, peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1α), nuclear respiratory factor (NRF1), and MitoNEET during NSC differentiation. TIGAR can decrease lactate production and accelerate oxygen consumption and ATP generation to maintain a high rate of OXPHOS in differentiated NSCs. Interestingly, knockdown of TIGAR decreased the level of acetyl-CoA and H3K9 acetylation at the promoters of Ngn1, Neurod1, and Gfap. Acetate, a precursor of acetyl-CoA, increased the level of H3K9 acetylation and rescued the effect of TIGAR deficiency on NSC differentiation. Together, our data demonstrated that TIGAR promotes metabolic reprogramming and regulates NSC differentiation through an epigenetic mechanism.
Project description:Hypoxic-ischemic brain damage (HIBD) is the major recognized perinatal cause of neurological morbidity in full-term new borns. Neural stem cells (NSCs) have been extensively studied because of their clinical applications in treating neuro degenerative diseases and brain injuries, including HIBD, while microRNAs (miRNAs) are deemed critical regulators of the proliferation and differentiation of NSCs. However, the role of let-7f in NSC differentiation remains unknown. Our study aims to investigate the role of let-7f in the differentiation of NSCs and brain development in rats and hence to explore the therapeutic potential of let-7f in the treatment of HIBD. The quantitative real-time polymerase chain reaction (qRT-PCR) was applied to assess the expressions of let-7f, and western blot was performed to detect GFAP, Tuj1 and Nestin in rat brains at postnatal day 1, 8 and 14 (n=12 per time point). The NSCs isolated from the brains of rat fetuses at gestational day 15 were transduced with lenti virus expressing let-7f or let-7f inhibitor so as to observe altered expressions of let-7f, GFAP, Tuj1 and Nestin. A gradually-increasing expression of let-7f was detected by qRT-PCR in rat brain tissues during postnatal brain development. Increased levels of GFAP and Tuj1, while a decreased level of Nestin, were detected by western blot in let-7f-overexpressing NSCs. In contrast, the cells expressing the let-7f inhibitor exhibited lower levels of GFAP and Tuj1, while a higher level of Nestin, compared with control cells. Therefore, let-7f is involved in brain development and promotes the differentiation of NSCs in rats.
Project description:We describe the labeling of adult neural stem cells (aNSCs) in the mouse and human dentate gyrus (DG) by the combinatorial expression of glial fibrillary acidic protein (GFAP) and Prominin1, as revealed by immunohistochemistry. Split-Cre-based genetic fate mapping of these double-positive cells in the adult murine DG reveals their NSC identity, as they are self-renewing and contribute to neurogenesis over several months. Their progeny reacts to stimuli such as voluntary exercise with increased neurogenesis. Prominin1+/GFAP+ cells also exist in the adult human DG, the only region in the human brain for which adult neurogenesis has been consistently reported. Our data, together with previous evidence of such double-positive NSCs in the developing murine brain and in neurogenic regions of vertebrates with widespread neurogenesis, suggest that Prominin1- and GFAP-expressing cells are NSCs in a wide range of species in development and adulthood.
Project description:There are currently no effective treatments for moderate-to-severe traumatic brain injuries (TBIs). The paracrine functions of undifferentiated neural stem cells (NSCs) are believed to play a significant role in stimulating the repair and regeneration of injured brain tissue. We therefore hypothesized that fibroblast growth factor (FGF2) enriching chondroitin sulfate glycosaminoglycan (CS-GAG) matrices can maintain the undifferentiated state of neural stem cells (NSCs) and facilitate brain tissue repair subacutely post-TBI. Rats subjected to a controlled cortical impactor (CCI) induced TBI were intraparenchymally injected with CS-GAG matrices alone or with CS-GAG matrices containing PKH26GL labeled allogeneic NSCs. Nissl staining of brain tissue 4 weeks post-TBI demonstrated the significantly enhanced (p < 0.05) tissue protection in CS-GAG treated animals when compared to TBI only control, and NSC only treated animals. CS-GAG-NSC treated animals demonstrated significantly enhanced (p < 0.05) FGF2 retention, and maintenance of PKH26GL labeled NSCs as indicated by enhanced Sox1+ and Ki67+ cell presence over other differentiated cell types. Lastly, all treatment groups and sham controls exhibited a significantly (p < 0.05) attenuated GFAP+ reactive astrocyte presence in the lesion site when compared to TBI only controls.