Differentiation Of Mouse Neural Stem Cells Research Articles

Effects of overexpression of Hsp70 in neural stem cells on neurotoxicity and cognitive dysfunction in neonatal mice under sevoflurane exposure.

As one of the commonly used inhalation anesthetics in clinical practice, sevoflurane is currently widely applied in surgery for children and the elderly due to its safety and efficacy. However, the neurotoxicity and cognitive impairment induced by sevoflurane exposure cannot be ignored. A recombinant adenovirus with green fluorescent protein-labeled heat shock protein 70 (Hsp70) was constructed and used to infect neural stem cells (NSCs) separated from neonatal mice. Quantitative real-time PCR and Western blot assays were used to evaluate the expression of certain genes. 5‑Ethynyl‑2'‑deoxyuridine staining and cell counting kit assay were used to detect the proliferation and differentiation ability of NSCs. The Morris water maze experiment was used to test the cognitive abilities of mice. Adv-Hsp70 induced the overexpression of Hsp70 in mouse NSCs. Upregulation of Hsp70 promoted the proliferation ability and differentiation of mouse NSCs. NSCs that overexpressed Hsp70 attenuated sevoflurane-induced neurotoxicity and protected cognitive dysfunction in mice under sevoflurane exposure. In summary, our findings demonstrate the potential of overexpression of Hsp70 in NSCs against sevoflurane-induced impairments.

A causal association of ANKRD37 with human hippocampal volume.

Human hippocampal volume has been separately associated with single nucleotide polymorphisms (SNPs), DNA methylation and gene expression, but their causal relationships remain largely unknown. Here, we aimed at identifying the causal relationships of SNPs, DNA methylation, and gene expression that are associated with hippocampal volume by integrating cross-omics analyses with genome editing, overexpression and causality inference. Based on structural neuroimaging data and blood-derived genome, transcriptome and methylome data, we prioritized a possibly causal association across multiple molecular phenotypes: rs1053218 mutation leads to cg26741686 hypermethylation, thus leads to overactivation of the associated ANKRD37 gene expression in blood, a gene involving hypoxia, which may result in the reduction of human hippocampal volume. The possibly causal relationships from rs1053218 to cg26741686 methylation to ANKRD37 expression obtained from peripheral blood were replicated in human hippocampal tissue. To confirm causality, we performed CRISPR-based genome and epigenome-editing of rs1053218 homologous alleles and cg26741686 methylation in mouse neural stem cell differentiation models, and overexpressed ANKRD37 in mouse hippocampus. These in-vitro and in-vivo experiments confirmed that rs1053218 mutation caused cg26741686 hypermethylation and ANKRD37 overexpression, and cg26741686 hypermethylation favored ANKRD37 overexpression, and ANKRD37 overexpression reduced hippocampal volume. The pairwise relationships of rs1053218 with hippocampal volume, rs1053218 with cg26741686 methylation, cg26741686 methylation with ANKRD37 expression, and ANKRD37 expression with hippocampal volume could be replicated in an independent healthy young (n = 443) dataset and observed in elderly people (n = 194), and were more significant in patients with late-onset Alzheimer's disease (n = 76). This study revealed a novel causal molecular association mechanism of ANKRD37 with human hippocampal volume, which may facilitate the design of prevention and treatment strategies for hippocampal impairment.

Mouse Neural Stem Cell Differentiation and Human Adipose Mesenchymal Stem Cell Transdifferentiation Into Neuron- and Oligodendrocyte-like Cells With Myelination Potential.

Stem cell therapy is an interesting approach for neural repair, once it can improve and increase processes, like angiogenesis, neurogenesis, and synaptic plasticity. In this regard, adult neural stem cells (NSC) are studied for their mechanisms of proliferation, differentiation and functionality in neural repair. Here, we describe novel neural differentiation methods. NSC from adult mouse brains and human adipose-derived stem cells (hADSC) were isolated and characterized regarding their neural differentiation potential based on neural marker expression profiles. For both cell types, their capabilities of differentiating into neuron-, astrocyte- and oligodendrocytes-like cells (NLC, ALC and OLC, respectively) were analyzed. Our methodologies were capable of producing NLC, ALC and OLC from adult murine and human transdifferentiated NSC. NSC showed augmented gene expression of NES, TUJ1, GFAP and PDGFRA/Cnp. Following differentiation induction into NLC, OLC or ALC, specific neural phenotypes were obtained expressing MAP2, GalC/O4 or GFAP with compatible morphologies, respectively. Accordingly, immunostaining for nestin+ in NSC, GFAP+ in astrocytes and GalC/O4+ in oligodendrocytes was detected. Co-cultured NLC and OLC showed excitability in 81.3% of cells and 23.5% of neuron/oligodendrocyte marker expression overlap indicating occurrence of in vitro myelination. We show here that hADSC can be transdifferentiated into NSC and distinct neural phenotypes with the occurrence of neuron myelination in vitro, providing novel strategies for CNS regeneration therapy. Superior Part: Schematic organization of obtaining and generating hNSC from hADSC and differentiation processes and phenotypic expression of neuron, astrocyte and oligodendrocyte markers (MAP2, GFAP and O4, respectively) and stem cell marker (NES) of differentiating hNSC 14days after induction. The nuclear staining in blue corresponds to DAPI. bar = 100μm. Inferior part: Neural phenotype fates in diverse differentiation media. NES: nestin; GFAP: Glial fibrillary acidic protein. MAP2: Microtubule-associated protein 2. TUJ1: β-III tubulin. PDGFRA: PDGF receptor alpha. Two-way ANOVA with Bonferroni post-test with n = 3. * p < 0.05 and ** p < 0.01: (NSCiM1 NSC induction medium 1) vs differentiation media.

Single-cell RNA sequencing of mouse neural stem cell differentiation reveals adverse effects of cadmium on neurogenesis

Cadmium (Cd) is a toxic heavy metal and widely exists in the environment. Extensive studies have revealed that Cd exposure can elicit neurotoxicity and potentially interfere with neurogenesis. However, underlying mechanisms by which Cd exposure affects neurogenesis remain unclear. In this study, we performed single-cell RNA sequencing (scRNA-seq) of the differentiated mixture from neonatal mouse Neural Stem Cells (mNSCs) that were exposed to Cd for 24 h and differentiated for 7 days. Our results showed that Cd exposure led to an increase in the differentiation of NSCs into astrocytes while a decrease into neurons. Besides, Cd induced subtype-specific response and dysregulated cell-to-cell communication. Collectively, our scRNA-seq data suggested that Cd had toxic effects on NSCs differentiation at the single-cell level, which offered insight into the potential molecular mechanism of Cd on neurogenesis. Furthermore, our findings provided a new method for assessing the neurodevelopmental toxicity of environmental pollutants.

Circular RNA expression profiles during the differentiation of mouse neural stem cells

BackgroundCircular RNAs (circRNAs) have recently been found to be expressed in human brain tissue, and many lines ofevidence indicate that circRNAs play regulatory roles in neurodevelopment. Proliferation and differentiation of neural stem cells (NSCs) are critical parts during development of central nervous system (CNS).To date, there have been no reports ofcircRNA expression profiles during the differentiation of mouse NSCs. We hypothesizethat circRNAs mayregulate gene expression in the proliferation anddifferentiation of NSCs.ResultsIn this study, we obtained NSCs from the wild-type C57BL/6 J mouse fetal cerebral cortex. We extracted total RNA from NSCs in different differentiation stagesand then performed RNA-seq. By analyzing the RNA-Seq data, we found 37circRNAs and 4182 mRNAs differentially expressedduringthe NSC differentiation. Gene Ontology (GO) enrichment analysis of thecognate linear genes of these circRNAsrevealed that some enriched GO terms were related to neural activity. Furthermore, we performed a co-expression network analysis of these differentially expressed circRNAs and mRNAs. The result suggested a stronger GO enrichmentin neural features for both the cognate linear genes of circRNAs and differentially expressed mRNAs.ConclusionWe performed the first circRNA investigation during the differentiation of mouse NSCs. Wefound that12 circRNAs might have regulatory roles duringthe NSC differentiation, indicating that circRNAs might be modulated during NSC differentiation.Our network analysis suggested the possible complex circRNA-mRNA mechanisms during differentiation, and future experimental workis need to validate these possible mechanisms.

Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling

The low survival and differentiation rates of stem cells after either transplantation or neural injury have been a major concern of stem cell-based therapy. Thus, further understanding long-term survival and differentiation of stem cells may uncover new targets for discovery and development of novel therapeutic approaches. We have previously described the impact of mitochondrial apoptosis-related events in modulating neural stem cell (NSC) fate. In addition, the endogenous bile acid, tauroursodeoxycholic acid (TUDCA) was shown to be neuroprotective in several animal models of neurodegenerative disorders by acting as an anti-apoptotic and anti-oxidant molecule at the mitochondrial level. Here, we hypothesize that TUDCA might also play a role on NSC fate decision. We found that TUDCA prevents mitochondrial apoptotic events typical of early-stage mouse NSC differentiation, preserves mitochondrial integrity and function, while enhancing self-renewal potential and accelerating cell cycle exit of NSCs. Interestingly, TUDCA prevention of mitochondrial alterations interfered with NSC differentiation potential by favoring neuronal rather than astroglial conversion. Finally, inhibition of mitochondrial reactive oxygen species (mtROS) scavenger and adenosine triphosphate (ATP) synthase revealed that the effect of TUDCA is dependent on mtROS and ATP regulation levels. Collectively, these data underline the importance of mitochondrial stress control of NSC fate decision and support a new role for TUDCA in this process.

Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress

Apoptosis regulatory proteins, such as p53, play a pivotal role in neural differentiation, through mechanisms independent of cell death. In addition, p53 has been identified as an important regulator of mitochondrial survival response, maintaining mitochondrial DNA (mtDNA) integrity and oxidative protection. The aim of this study was to determine the role of mitochondrial p53 in organelle damage and neural differentiation. Our results show that mitochondrial apoptotic events such as reactive oxygen species production, mitochondrial membrane permeabilization, and cytochrome c release are typical of early-stage mouse neural stem cell differentiation, which occurs 3-18 h after induction of differentiation, with no evidence of cell death. In addition, decreased mtDNA content, lipidated LC3 (LC3-II), colocalization of mitochondria and LC3-II puncta, and mitochondria-associated Parkin are consistent with activation of mitophagy. Importantly, at early stages of neural differentiation, p53 was actively translocated to mitochondria and attenuated mitochondrial oxidative stress, cytochrome c release, and mitophagy. Forced mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth. In conclusion, our results reveal a novel role for mitochondrial p53, which modulates mitochondrial damage and apoptosis-related events in the context of neural differentiation, thus enhancing neuronal fate.

The characterization of gene expression during mouse neural stem cell differentiation in vitro

Neural stem cells (NSCs) are tissue-specific, multipotent stem cells that can differentiate into three cell lineages in the central nervous system: neurons, astrocytes and oligodendrocytes. The therapeutic potential of NSCs has fueled attempts to characterize the expression of genes that regulate their fate. In this study, NSCs from embryonic day 15 (E15) mouse embryos were differentiated for 1 (DF-1) or 2 (DF-2) days, and the gene expression patterns in the NSCs and in the DF-1 and DF-2 cells were measured by microarray and real-time RT-PCR. Among the analyzed genes, 1898 genes were up-regulated in the DF-1 and DF-2 cells relative to the NSCs, whereas 1642 genes were down-regulated. The up-regulated genes included Gfap, Smad6, Fst, Tgfb2 and Cdkn2. The down-regulated genes included Ccnb1, Ccnd1 and Ccnd2. We identified gene networks that were associated with BMP and TGF-beta2 signaling pathways using Ingenuity Pathway Analysis. Our results suggest that the differentiation of E15 NSCs into astrocytes is based on a combinatorial network of various signaling pathways, including cell cycle, BMP and TGF-beta2 signaling.

MiR-34a Regulates Mouse Neural Stem Cell Differentiation

BackgroundMicroRNAs (miRNAs or miRs) participate in the regulation of several biological processes, including cell differentiation. Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. In addition, members of the miR-34 family have been identified as direct p53 targets. However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. Here, we investigated the role of miR-34a in regulating mouse neural stem (NS) cell differentiation.Methodology/Principal FindingsmiR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism.ConclusionsOur results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity.

P53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis.

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

BackgroundTo investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs.Principal FindingsDicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia.ConclusionsThe block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation.

Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation

BackgroundMicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However, evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently, we have shown that apoptosis-associated factors, such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development, we performed global miRNA expression profiling experiments in NS cells. Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct models of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. In addition, the expression of antiapoptotic miR-19a and 20a was also evaluated.ResultsThe expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast, expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly, differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells.ConclusionsIn conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs.

Caspases and p53 modulate FOXO3A/Id1 signaling during mouse neural stem cell differentiation

Neural stem cells (NSCs) differentiate into neurons and glia, and a large percentage undergoes apoptosis. The engagement and activity of apoptotic pathways may favor either cell death or differentiation. In addition, Akt represses differentiation by up-regulating the inhibitor of differentiation 1 (Id1), through phosphorylation of its repressor FOXO3A. The aim of this study was to investigate the potential cross-talk between apoptosis and proliferation during mouse NSC differentiation. We determined the time of neurogenesis and gliogenesis using neuronal beta-III tubulin and astroglial GFAP to confirm that both processes occurred at approximately 3 and 8 days, respectively. p-Akt, p-FOXO3A, and Id1 were significantly reduced throughout differentiation. Caspase-3 processing, p53 phosphorylation, and p53 transcriptional activation increased at 3 days of differentiation, with no evidence of apoptosis. Importantly, in cells exposed to the pancaspase inhibitor z-VAD.fmk, p-FOXO3A and Id1 were no longer down-regulated, p53 phosphorylation and transcriptional activation were reduced, while neurogenesis and gliogenesis were significantly delayed. The effect of siRNA-mediated silencing of p53 on FOXO3A/Id1 was similar to that of z-VAD.fmk only at 3 days of differentiation. Interestingly, caspase inhibition further increased the effect of p53 knockdown during neurogenesis. In conclusion, apoptosis-associated factors such as caspases and p53 temporally modulate FOXO3A/Id1 signaling and differentiation of mouse NSCs.

The Role of MICRORNA 34-A During Apoptosis-Mediated Mouse Neural Stem Cell Differentiation

The Role of MICRORNA 34-A During Apoptosis-Mediated Mouse Neural Stem Cell Differentiation

Neural stem cell differentiation is mediated by integrin β4 in vitro

Neural stem cells are capable of differentiating into three major neural cell types, but the underlying molecular mechanisms remain unclear. Here, we investigated the mechanism by which integrin β4 modulates mouse neural stem cell differentiation in vitro. Inhibition of endogenous integrin β4 by RNA interference inhibited the cell differentiation and the expression of fibroblast growth factor receptor 2 but not fibroblast growth factor receptor 1 or fibroblast growth factor receptor 3. Overexpression of integrin β4 in neural stem cells promoted neural stem cell differentiation. Furthermore, integrin β4-induced differentiation of neural stem cells was attenuated by SU5402, the inhibitor of fibroblast growth factor receptors. Finally, we investigated the role of integrin β4 in neural stem cell survival: knockdown of integrin β4 did not affect survival or apoptosis of neural stem cells. These data provide evidence that integrin β4 promotes differentiation of mouse neural stem cells in vitro possibly through fibroblast growth factor receptor 2.