Sox2 roles in neural stem cells

Throughout vertebrate evolution, Sox2 marks the developing nervous system from its earliest developmental stages and, therein, the most undifferentiated precursor cells, including stem cells. Recent gene targeting studies investigated the function of Sox2 in two neuronal systems: the developing eye and brain. These studies uncovered a requirement for Sox2 in the maintenance of neural stem cells, as well as a downstream role in the differentiation of specific neuron sub-types. In both systems, Sox2 action is markedly dose-dependent, and downstream-target gene studies are beginning to reveal the mechanisms of Sox2 function.     

神经干细胞来源的神经元的诱导分裂

为探讨神经干细胞分化成熟的神经元是否能够分裂。实验取材于成年哺乳动物,将神经干细胞体外培养8d后,诱导分化为神经元,然后进一步诱导其分裂。采用连续摄影与NF-160免疫细胞化学方法检测神经元的分裂过程,同时运用PCNA NF-160(或Chat、GABA、GAD)的免疫双标记证明分裂神经元是否为成熟神经元。将神经干细胞体外诱导分化培养8d,直至分化神经元外形成熟,进而加入EGF与bFGF诱导分裂。诱导分裂2d后,观察到有神经元样细胞分裂;同一区域内神经元样细胞的数量不断增加,表现为NF-160阳性。连续拍摄了神经元样细胞的分裂过程,分裂完成后的细胞同样表现为NF-160抗体反应阳性。PCNA NF-160(或Chat、GABA、GAD)的免疫双标记结果显示,一些细胞的胞浆显示为棕色的同时细胞核显示为黑色。结果提示,在一定的条件下,先前所认为的终末分化神经元可以重新进入细胞周期,成熟神经元仍然可以进行分裂增殖和自我更新。     

Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process

We examined the role of Notch signaling on the generation of neurons and glia from neural stem cells by using neurospheres that are clonally derived from neural stem cells. Neurospheres prepared from Dll1(lacZ/lacZ) mutant embryos segregate more neurons at the expense of both oligodendrocytes and astrocytes. This mutant phenotype could be rescued when Dll1(lacZ/lacZ) spheres were grown and/or differentiated in the presence of conditioned medium from wild-type neurospheres. Temporal modulation of Notch by soluble forms of ligands indicates that Notch signaling acts in two steps. Initially, it inhibits the neuronal fate while promoting the glial cell fate. In a second step, Notch promotes the differentiation of astrocytes, while inhibiting the differentiation of both neurons and oligodendrocytes.     

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.     

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.     

Noisy neurons keep neural stem cells quiet

Recently in Nature, Song et?al. (2012) show that the neurotransmitter GABA acts directly on radial glia-like neural stem cells to maintain quiescence and provide a mechanism for how neuronal activity controls the production of new neurons in the hippocampus.     

Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.     

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells

Abstract Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naïve hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.     

Region-specific generation of functional neurons from naive embryonic stem cells in adult brain

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.     

Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear

The cochlear sensory epithelium and spiral ganglion neurons (SGNs) in the adult mammalian inner ear do not regenerate following severe injury. To replace the degenerated SGNs, neural stem cell (NSC) is an attractive alternative for substitution cell therapy. In this study, adult mouse NSCs were transplanted into normal and deafened inner ears of guinea pigs. To more efficiently drive the implanted cells into a neuronal fate, NSCs were also transduced with neurogenin 2 (ngn2) before transplantation. In deafened inner ears and in animals transplanted with ngn2-transduced NSCs, surviving cells expressed the neuronal marker neural class III beta-tubulin. Transplanted cells were found close to the sensory epithelium and adjacent to the SGNs and their peripheral processes. The results illustrate that adult NSCs can survive and differentiate in the injured inner ear. It also demonstrates the feasibility of gene transfer to generate specific progeny for cell replacement therapy in the inner ear.     

miR-124 promotes neural differentiation in mouse bulge stem cells by repressing Ptbp1 and Sox9

Hair follicle stem cells (HFSCs) are able to differentiate into neurons and glial cells. Distinct microRNAs (miRNAs) regulate the proliferation and differentiation of HFSCs. However, the exact role of miR-124 in the neural differentiation of HFSCs has not been elucidated. HFSCs were isolated from mouse whisker follicles. miR-9, let-7b, and miR-124, Ptbp1 , and Sox9 expression levels were detected by real-time polymerase chain reaction (RT-PCR). The influence of miR-124 transfection was evaluated using immunostaining. We demonstrated that miR-124 and let-7b expression levels were significantly increased after the neural differentiation. Sox9 and Ptbp1 were identified as the target of miR-124 in the HFSCs. During neural differentiation and miR-124 mimicking, Ptbp1 and Sox9 levels were decreased. Moreover, the miR-124 overexpression increased MAP2 (58.43 ± 11.26) and NeuN (48.34 ± 11.15) proteins expression. The results demonstrated that miR-124 may promote the differentiation of HFSCs into neuronal cells by targeting Sox9 and Ptbp1.     

Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

Extracellular vesicles (EVs) are thought to mediate the transport of proteins and RNAs involved in intercellular communication. Here, we show dynamic changes in the buoyant density and abundance of EVs that are secreted by PC12 cells stimulated with nerve growth factor (NGF), N2A cells treated with retinoic acid to induce neural differentiation, and mouse embryonic stem cells (mESCs) differentiated into neuronal cells. EVs secreted from in vitro differentiated cells promote neural induction of mESCs. Cyclin D1 enriched within the EVs derived from differentiated neuronal cells contributes to this induction. EVs purified from cells overexpressing cyclin D1 are more potent in neural induction of mESC cells. Depletion of cyclin D1 from the EVs reduced the neural induction effect. Our results suggest that EVs regulate neural development through sorting of cyclin D1.     

Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro

Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons.     

Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells

Background

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Methodology/Principal Findings

We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184⁺/CD271⁻/CD44⁻/CD24⁺ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184⁻/CD44⁻/CD15^LOW/CD24⁺ and a population of glia that was CD184⁺/CD44⁺ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.

Conclusions/Significance

These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.