Embryonic and adult neural stem cell research in China

Neural stem cells(NSCs) are one specific type of multipotential stem cells that have the ability to proliferate for a long time and to differentiate into neural cells,including neurons,astrocytes and oligodendrocytes.These NSCs exist in both the embryonic and adult central nervous system(CNS) of all mammalian species.Progress has been made in the understanding of the developmental regulation of NSCs and their function in neurogenesis.This review discusses recent progress in this area,with emphasis on work d...     

Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

室管膜下区神经干细胞与神经发生的研究进展

室管膜下区(subventricular zone,SVZ)存在着神经干细胞(nueral stem cells,NSCs),是成年哺乳动物脑内重要的神经发生区域。神经发生过程极为复杂,包括一系列的生物学事件。在病理状态下,SVZ区的细胞增殖,新生的神经细胞迁移到病灶处,取代或修复受损的细胞,起到保护脑组织的作用。该文就SVZ区的神经干细胞、神经发生过程及病理状态下神经发生的相关研究做一综述。     

Ogt controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling

1. Download : Download high-res image (190KB)
2. Download : Download full-size image

     

p57 controls adult neural stem cell quiescence and modulates the pace of lifelong neurogenesis

Throughout life, neural stem cells (NSCs) in the adult hippocampus persistently generate new neurons that modify the neural circuitry. Adult NSCs constitute a relatively quiescent cell population but can be activated by extrinsic neurogenic stimuli. However, the molecular mechanism that controls such reversible quiescence and its physiological significance have remained unknown. Here, we show that the cyclin‐dependent kinase inhibitor p57^kip2 (p57) is required for NSC quiescence. In addition, our results suggest that reduction of p57 protein in NSCs contributes to the abrogation of NSC quiescence triggered by extrinsic neurogenic stimuli such as running. Moreover, deletion of p57 in NSCs initially resulted in increased neurogenesis in young adult and aged mice. Long‐term p57 deletion, on the contrary, led to NSC exhaustion and impaired neurogenesis in aged mice. The regulation of NSC quiescence by p57 might thus have important implications for the short‐term (extrinsic stimuli‐dependent) and long‐term (age‐related) modulation of neurogenesis.     

胚胎干细胞

胚胎干细胞具有自我复制并分化为人体各种功能细胞的潜能。胚胎干细胞具有的独特生物学特性使其被广泛应用于生物学研究的各个领域,特别是发育学。同时,它潜在的医学应用也成为世界范围内的研究热点。但是,由于人胚胎干细胞的来源为植入前的早期胚胎,人胚胎干细胞自诞生之日起便倍受争议。本文将从胚胎干细胞的来源、特性、鉴定标准、增殖机理、应用前景以及研究本身涉及的伦理学争论给予概述。     

Embryonic stem cells: a promising tool for cell replacement therapy

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.     

Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult‐derived neural stem cell cultures

The adult rat hippocampus contains fibroblast growth factor 2–responsive stem cells that are self‐renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation. Hippocampus‐derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up‐regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up‐regulate trkA, trkB, trkC, and p75NGFR expression. Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain‐derived neurotrophic factor or NT‐3 led to a significant increase in neurons displaying mature γ‐a‐minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes. Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA‐dependent expression of the Trk receptors. In combination, these sequential effects were sufficient to stimulate stem cell–derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 65–81, 1999     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis

According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular–subventricular zone (V‐SVZ). Both processes will ultimately lead to the generation of adult‐born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult‐born OB interneurons largely derive from an additional NSC‐type resident in the basal V‐SVZ. Despite being both capable of self‐renewal and long‐term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch‐related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch‐effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation‐dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch‐mediated lateral inhibition across the V‐SVZ.     

Regulatory genes controlling cell fate choice in embryonic and adult neural stem cells

Neural stem cells are the most immature progenitor cells in the nervous system and are defined by their ability to self-renew by symmetric division as well as to give rise to more mature progenitors of all neural lineages by asymmetric division (multipotentiality). The interest in neural stem cells has been growing in the past few years following the demonstration of their presence also in the adult nervous system of several mammals, including humans. This observation implies that the brain, once thought to be entirely post-mitotic, must have at least a limited capacity for self-renewal. This raises the possibility that the adult nervous system may still have the necessary plasticity to undergo repair of inborn defects and acquired injuries, if ways can be found to exploit the potential of neural stem cells (either endogenous or derived from other sources) to replace damaged or defective cells. A full understanding of the molecular mechanisms regulating generation and maintenance of neural stem cells, their choice between different differentiation programmes and their migration properties is essential if these cells are to be used for therapeutic applications. Here, we summarize what is currently known of the genes and the signalling pathways involved in these mechanisms.     

Embryonic stem cell differentiation: The role of extracellular factors

Embryonic stem (ES) cells have the capacity to self renew and to differentiate into cellular derivatives of the endodermal, ectodermal, and mesodermal lineages. Therefore, ES cells have been used to analyse the effects of exogenous factors on the developmental pattern during in vitro differentiation. By using an in vitro loss-of-function approach based on beta1 integrin-deficient ES cells, it was found that integrin-dependent mechanisms are involved in the regulation of Wnt-1 and BMP-4 expression. Antagonistic effects of the signalling molecules Wnt-1 and BMP-4, morphogens involved in early differentiation events, have been observed in vivo and in vitro: BMP-4 acts as a potent mesoderm inducer, whereas Wnt-1 plays a critical role in the determination of neuroectoderm. Here, we summarise data of ES cell-derived cardiac, myogenic, and neuronal differentiation of wild type and beta1 integrin-deficient ES cells. We present evidence that the interaction of cells with the extracellular matrix via integrins determines the expression of the signalling molecules BMP-4 and Wnt-1, resulting in the activation of the mesodermal and neuroectodermal lineage, respectively. The results support the idea that the influence of the extracellular 'niche' on the developmental fate of pluripotent stem cells is determined not only by soluble factors, but also by the extracellular matrix.     

Phenotypical and functional heterogeneity of neural stem cells in the aged hippocampus

Adult neurogenesis persists in the hippocampus of most mammal species during postnatal and adult life, including humans, although it declines markedly with age. The mechanisms driving the age‐dependent decline of hippocampal neurogenesis are yet not fully understood. The progressive loss of neural stem cells (NSCs) is a main factor, but the true neurogenic output depends initially on the actual number of activated NSCs in each given time point. Because the fraction of activated NSCs remains constant relative to the total population, the real number of activated NSCs declines in parallel to the total NSC pool. We investigated aging‐associated changes in NSCs and found that there are at least two distinct populations of NSCs. An alpha type, which maintains the classic type‐1 radial morphology and accounts for most of the overall NSC mitotic activity; and an omega type characterized by increased reactive‐like morphological complexity and much lower probability of division even under a pro‐activation challenge. Finally, our results suggest that alpha‐type NSCs are able to transform into omega‐type cells overtime and that this phenotypic and functional change might be facilitated by the chronic inflammation associated with aging.     

小鼠胚胎干细胞结合丝素材料向神经细胞分化的实验研究

以小鼠胚胎干细胞（ES）为种子细胞,使用改良的4-/4+ RA方案,诱导小鼠ES细胞在丝素材料上向神经细胞分化,探讨丝素材料对其生长、黏附、分化等情况的影响。将小鼠ES细胞悬浮培养4 d得到的拟胚体（EBs）分别接种到经丝素膜和明胶包被的培养皿上进行诱导,比较不同材料上EBs的贴壁率及向神经元分化的比率。结果表明EBs在明胶和柞蚕丝素蛋白膜（TSF）上贴壁较快,平均贴壁率为90.3%和84.4%,在桑蚕丝素蛋白膜（SF）上贴壁较慢,贴壁率低,仅为38.5%,同时三者神经元的分化比率均能达到40%以上,无明显差异。通过以上实验,我们得出,TSF有望成为小鼠ES细胞向神经细胞分化的支架材料。     

MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism‐like behaviors

The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2‐deficient mice exhibit autism‐like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders.