Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain

Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. We now report that NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increased cell proliferation and decreased differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibited NPC proliferation and increased neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduced NPC proliferation and increased the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC was blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibited proliferation and induced neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis.     

Defective cholesterol traffic and neuronal differentiation in neural stem cells of Niemann-Pick type C disease improved by valproic acid, a histone deacetylase inhibitor

Niemann-Pick type C disease (NPC) is a neurodegenerative and lipid storage disorder for which no effective treatment is known. We previously reported that neural stem cells derived from NPC1 mice showed impaired self-renewal and differentiation. We examined whether valproic acid (VPA), a histone deacetylase inhibitor, could enhance neuronal differentiation and recover defective cholesterol metabolism in neural stem cells (NSCs) from NPC1-deficient mice (NPC1(-/-)). VPA could induce neuronal differentiation and restore impaired astrocytes in NSCs from NPC1(-/-) mice. Importantly, an increasing level of cholesterol within NSCs from NPC1(-/-) mice could be reduced by VPA. Moreover, essential neurotrophic genes (TrkB, BDNF, MnSoD, and NeuroD) were up-regulated through the repression of the REST/NRSF and HDAC complex by the VPA treatment. Up-regulated neurotrophic genes were able to enhance neural differentiation and cholesterol homeostasis in neural stem cells from NPC1(-/-) mice. In this study, we suggested that, along with cholesterol homeostasis, impaired neuronal differentiation and abnormal morphology of astrocytes could be rescued by the inhibition of HDAC and REST/NRSF activity induced by VPA treatment.     

A change in nuclear pore complex composition regulates cell differentiation

Nuclear pore complexes (NPCs) are built from ～30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination.     

Neural induction of porcine‐induced pluripotent stem cells and further differentiation using glioblastoma‐cultured medium

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine‐induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma‐ and Mad‐related protein (SMAD) inhibitors, a TGF‐β inhibitor and BMP4 inhibitor. The dSMADi‐derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid‐treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma‐cultured medium including injury‐related cytokines treated porcine iPSC‐NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.     

Comparison and Optimization of hiPSC Forebrain Cortical Differentiation Protocols

Several protocols have been developed for human induced pluripotent stem cell neuronal differentiation. We compare several methods for forebrain cortical neuronal differentiation by assessing cell morphology, immunostaining and gene expression. We evaluate embryoid aggregate vs. monolayer with dual SMAD inhibition differentiation protocols, manual vs. AggreWell aggregate formation, plating substrates, neural progenitor cell (NPC) isolation methods, NPC maintenance and expansion, and astrocyte co-culture. The embryoid aggregate protocol, using a Matrigel substrate, consistently generates a high yield and purity of neurons. NPC isolation by manual selection, enzymatic rosette selection, or FACS all are efficient, but exhibit some differences in resulting cell populations. Expansion of NPCs as neural aggregates yields higher cell purity than expansion in a monolayer. Finally, co-culture of iPSC-derived neurons with astrocytes increases neuronal maturity by day 40. This study directly compares commonly employed methods for neuronal differentiation of iPSCs, and can be used as a resource for choosing between various differentiation protocols.     

Abeta40 promotes neuronal cell fate in neural progenitor cells

Sequential cleavage of the amyloid precursor protein (APP) by beta- and then gamma- secretase gives rise to Abeta(1-40) (Abeta40), a major species of Abeta (beta-amyloid) produced by neurons under physiological conditions. Abeta(1-42) (Abeta42), a minor species of Abeta, is also produced by a similar but less understood mechanism of the gamma-secretase. The physiological functions of these Abeta species remain to be defined. In this report, we demonstrate that freshly prepared soluble Abeta40 significantly promotes neurogenesis in primary neural progenitor cells (NPCs). First, Abeta40 increases neuronal markers as determined by NeuN expression and Tuj1 promoter activity, differing from Abeta42, which induces astrocyte markers in NPCs. Second, Abeta40 induces neuronal differentiation at the end of S-phase in the cell cycle. Third, Abeta40 promotes NPC entry into S-phase, playing a role in NPC self-renewal. Interestingly, Abeta40 does not significantly increase apoptotic indexes such as DNA condensation and DNA fragmentation. In addition, Abeta40 does not augment caspase-3 activation in NeuN(+) or nestin(+) cells. Collectively, this report provides strong evidence that Abeta40 is a neurogenic factor and suggests that the debilitated function of Abeta40 in neurogenesis may account for the shortage of neurons in Alzheimer's disease.     

Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells

REST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes.     

Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.