Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24 h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.     

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.     

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

Background

Circular 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.

Results

In 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.

Conclusion

We 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.

     

Functional neuronal cells generated by human parthenogenetic stem cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.     

ABC transporters, neural stem cells and neurogenesis--a different perspective

Stem cells intrigue. They have the ability to divide exponentially, recreate the stem cell compartment, as well as create differentiated cells to generate tissues. Therefore, they should be natural candidates to provide a renewable source of cells for transplantation applied in regenerative medicine. Stem cells have the capacity to generate specific tissues or even whole organs like the blood, heart, or bones. A subgroup of stem cells, the neural stem cells （NSCs）, is characterized as a self-renewing population that generates neurons and glia of the developing brain. They can be isolated, genetically manipulated and differentiated in vitro and reintroduced into a developing, adult or a pathologically altered central nervous system. NSCs have been considered for use in cell replacement therapies in various neurodegenerative diseases such as Parkinson＇s disease and Alzheimer＇s disease. Characterization of genes with tightly controlled expression patterns during differentiation represents an approach to understanding the regulation of stem cell commitment. The regulation of stem cell biology by the ATP-binding cassette （ABC） transporters has emerged as an important new field of investigation. As a major focus of stem cell research is in the manipulation of cells to enable differentiation into a targeted cell population; in this review, we discuss recent literatures on ABC transporters and stem cells, and propose an integrated view on the role of the ABC transporters, especially ABCA2, ABCA3, ABCB 1 and ABCG2, in NSCs＇ proliferation, differentiation and regulation, along with comparisons to that in hematopoietic and other stem cells.     

Quantitative analysis of gene expression in living adult neural stem cells by gene trapping

The potential of neural stem cells (NSCs) for the treatment of neurodegenerative diseases makes the identification and characterization of genes involved in neural stem cell responses therapeutically important. Although technologies exist for measuring gene expression in cells, they often provide only a representative expression profile specific to a stimulus and time. We developed a complementary technology based on a retroviral-vector gene-trap approach that uses beta-lactamase-induced disruption of fluorescence resonance energy transfer in the fluorophore CCF-2/AM. A library of 'tagged' adult rat NSCs was generated by transduction with gene-trap virus produced from a single-integrant packaging cell line that allowed us to quantitatively analyze dynamic gene expression changes in real time in living NSCs. Using this library we identified previously unknown genes regulated by oxidative stress, indomethacin and factors that induce differentiation, and show that one of the trapped genes, Sox6, is sufficient to induce astrocytic differentiation when overexpressed.     

TAM Receptors Support Neural Stem Cell Survival,Proliferation and Neuronal Differentiation

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III⁺) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.     

Properties of a fetal multipotent neural stem cell (NEP cell)

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFR alpha), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.     

Blocking BE301622 gene expression by RNAi initiates differentiation of neural stem cells in rat

Neural stem cells (NSCs) are capable of differentiating into neurons, astrocytes and oligodendrocytes. However, the molecular mechanisms regulating NSCs differentiation are not well understood. Our previous research by microarray analysis certified that a lot of genes are differentially expressed in the course of NSC differentiation. In this study we report the function of one of these genes, BE301622, by RNAi techniques. To silence the BE301622 gene, a long, double-stranded RNA (dsRNA) was synthesized by using a kit (Ambion T7 MegaScript) and transformed into NSCs. Expression of mRNA was tested through RT-PCR. The result showed the expression of BE301622 gene was specificially suppressed. This finding effectively validated that BE301622 is involved in the differentiation of NSCs.     

A proteomic approach to studying the differentiation of neural stem cells

The mechanisms that regulate the maintenance of stem cell self-renewal versus differentiation are complex and remain mostly unknown. Understanding neurogenesis and neural cell differentiation presents a unique challenge for the treatment of nervous system disorders. To gain more insight into molecular mechanisms of the differentiation of neural cells, we combined the advantage of porcine fetal neural stem cells (NSCs) in vitro differentiation model and proteomic analysis. Using 2-DE followed by MS, we profiled constituent proteins of NSCs and their differentiated progenies at first and then indicated protein species that were significantly up- or down-regulated during the differentiation. The largest identified group of constituent proteins was related to RNA and protein metabolism and processing, including chaperones, and the second largest consisted of proteins involved in cell organization (cytoskeleton and annexins). Differentiation of neural cells was found to be accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage, and redox regulation. Additional immunoblot analysis verified the induction of alpha-B crystallin and heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1. Furthermore, immunocytochemistry demonstrated specific localization of alpha-B crystallin in the cytoplasm or nucleus of glial cells and confirmed cellular expression patterns of hnRNPs A1 and A2/B1. These findings represent a significant step towards understanding neural cell differentiation and identification of the regulatory proteins associated with this process.     

Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro

Neural stem cells (NSCs) are currently considered powerful candidates for cell therapy in neurodegenerative disorders such as Parkinson's disease. However, it is not known when and how NSCs begin to differentiate functionally. Recent reports suggest that classical neurotransmitters such as acetylcholine (Ach) are involved in the proliferation and differentiation of neural progenitor cells, suggesting that neurotransmitters play an important regulatory role in development of the central nervous system (CNS). We have shown by calcium imaging and immunochemistry that proliferation and differentiation are enhanced by M2 muscarinic Ach receptors (mAchR) expressed on the NSC surface and on their neural progeny. Moreover, atropine, an mAchR antagonist, blocks the enhancement and inhibits the subsequent differentiation of NSCs. Further understanding of this neural-nutrition role of Ach might elucidate fetal brain development, the brain's response to injury, and learning and memory.     

Quantitative proteomic analysis of membrane proteins involved in astroglial differentiation of neural stem cells by SILAC labeling coupled with LC-MS/MS

Membrane proteins play a critical role in the process of neural stem cell self-renewal and differentiation. Here, we apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the self-renewing and the astroglial differentiating cells. High-resolution analysis on a linear ion trap-Orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 700 distinct membrane proteins during the astroglial differentiation. Of the 735 quantified proteins, seven cell surface proteins display significantly higher expression levels in the undifferentiated state membrane compared to astroglial differentiating membrane. One cell surface protein transferrin receptor protein 1 may serve as a new candidate for NSCs surface markers. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that most of overexpressed membrane proteins in the astroglial differentiation neural stem cells are involved in cellular growth, nervous system development, and energy metabolic pathway. Taken together, this study increases our understanding of the underlying mechanisms that modulate complex biological processes of neural stem cell proliferation and differentiation.