Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.     

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.     

脊髓源神经干细胞诱导分化为胆碱能神经元过程中Aldoc和Stmn1表达的研究

目的探讨脊髓源神经干细胞在诱导分化为胆碱能神经元过程中Aldoc和Stmn1的表达变化情况。方法从孕17天Wistar胚胎大鼠取出脊髓组织,制成细胞悬液,采用含EGF和bFGF的无血清限定性培养基培养,然后进行诱导分化,观察脊髓源神经干细胞向胆碱能神经元分化的情况,应用荧光定量PCR方法分析Aldoc和Stmn1基因在脊髓源神经干细胞诱导分化为胆碱能神经元的过程中的表达变化情况。结果神经干细胞在诱导分化为胆碱能神经元后,经免疫荧光检测有ChAT阳性细胞表达;Aldoc基因的表达量在胆碱能神经元较神经干细胞低(P<0.05);Stmn1基因的表达量则在诱导分化后较神经干细胞升高(P<0.05)。结论 Aldoc对神经干细胞的干性维持有重要作用,Stmn1在胆碱能神经元的成熟过程中起作用。     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Making connections in the inner ear: Recent insights into the development of spiral ganglion neurons and their connectivity with sensory hair cells

In mammals, auditory information is processed by the hair cells (HCs) located in the cochlea and then rapidly transmitted to the CNS via a specialized cluster of bipolar afferent connections known as the spiral ganglion neurons (SGNs). Although many anatomical aspects of SGNs are well described, the molecular and cellular mechanisms underlying their genesis, how they are precisely arranged along the cochlear duct, and the guidance mechanisms that promote the innervation of their hair cell targets are only now being understood. Building upon foundational studies of neurogenesis and neurotrophins, we review here new concepts and technologies that are helping to enrich our understanding of the development of the nervous system within the inner ear.     

Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina

One of the major roles of brain-derived neurotrophic factor (BDNF) is to promote the differentiation and support the survival of neurons in the central nervous system. The objective of the present study was to evaluate the effect of BDNF on the fate of adult rat hippocampus-derived neural stem cells (AHPCs) transplanted into the developing rat retina. Immunohistochemical analysis showed a significant increase in the ratio of grafted AHPCs stained for MAP2ab (P<0.05) and a marked decrease in the ratio of nestin-positive grafted cells in the slow-releasing BDNF group compared with the control group. The respective changes in the ratios of MAP5 and GFAP-positive grafted cells were comparable for the two groups. The results reported here suggest a potentially beneficial role for extended delivery of BDNF in the differentiation of grafted neural stem cells, which may lead to a novel modification of stem cell transplantation.     

神经干细胞体外增殖分化的钙成像研究

神经干细胞具有广阔的应用前景,但对于其增殖和分化的内源机制、外部环境信号还并不十分了解。研究表明,钙信号很可能在其中起到了调控作用。利用钙离子成像技术,观察神经干细胞的单细胞体外增殖和分化过程,记录了在细胞分裂过程中钙信号变化的曲线。发现细胞增殖和分化过程中都会产生钙浓度的变化,但在细胞分裂后期两者钙信号的模式却存在差别。实验结果提示,胞内钙水平的波动只是细胞增殖的伴随产物,但却是细胞分化的必要条件。由此提出钙信号对神经干细胞分化调控机制的假设,并指出其对今后研究的意义。     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.     

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.     

Plasticity of epidermal adult stem cells derived from adult goat ear skin

Here we report the isolation and characterization of pluripotent stem cells from adult goat skin. We found that these primary cells have the properties of embryonic stem cells (ESC), including the expression of appropriate immunological markers and the capability of forming embryoid bodies. The subcultured cells also show the characteristics of stem cells, such as the expression of CK19, beta(1-)integrin, P63, and formation of holo-clones in culture. Therefore, we termed these cells epidermal adult stem cells (EpiASC), although their origin was not identified. We have shown that clones of individual EpiASC proliferate and differentiate in culture to produce neurons, cardiomyocytes, osteoblasts, and occytes. Further, we cultivated EpiASC on bioengineered dermis and denuded human amniotic membrane (HAM), to reconstruct artificial skin and corneal epithelium. We successfully transplanted those artificial tissues in goats with acute full-thickness skin defect (AFTSD) and limbal stem cell deficiency (LSCD), respectively. Our results showed that indeed EpiASC reconstructed the skin (hair was observed in restored areas), and repaired the damaged cornea of goats with total LSCD. These data confirm that EpiASC can differentiate into different functional cell types in vivo or in vitro. Due to their high degree of inherent plasticity, and to their easy accessibility for collection from the skin, EpiASC are excellent candidate sources for diverse cell therapies.     

单细胞测序分析人类胚胎干细胞神经分化的分子机制

目的 探讨人类胚胎干细胞(ESCs)分化为神经细胞的关键性靶基因及分子机制,为临床靶向治疗神经康复患者提供分子理论依据.方法 基于GEO数据平台芯片,采用单细胞测序方法(scRNA-seq),利用R语言从多分子维度(单细胞差异基因、蛋白互作网络和基因通路等)分析人类ESCs分化过程中的关键Marker基因并利用质控和数...     

Embryonic stem cells in non-human primates: An overview of neural differentiation potential

Non-human primate (NHP) embryonic stem (ES) cells show unlimited proliferative capacities and a great potential to generate multiple cell lineages. These properties make them an ideal resource both for investigating early developmental processes and for assessing their therapeutic potential in numerous models of degenerative diseases. They share the same markers and the same properties with human ES cells, and thus provide an invaluable transitional model that can be used to address the safety issues related to the clinical use of human ES cells. Here, we review the available information on the derivation and the specific features of monkey ES cells. We comment on the capacity of primate ES cells to differentiate into neural lineages and the current protocols to generate self-renewing neural stem cells. We also highlight the signalling pathways involved in the maintenance of these neural cell types. Finally, we discuss the potential of monkey ES cells for neuronal differentiation.     

Discovery of small molecules that enhance astrocyte differentiation in rat fetal neural stem cells

1,3,4-Oxadiazole derivatives were found to enhance astrocyte differentiation in rat fetal neural stem cells (NSCs). Differentiation activity was assessed by immunocytochemistry and analysis of mRNA expression of astrocyte markers, GFAP and S100. Compounds 7 and 8 showed approximately a two-fold increase in astrocyte differentiation without engagement of neuronal differentiation and detectable cytotoxicity.     

Early identification of inner pillar cells during rat cochlear development

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Here, we examine its spatio-temporal development in rats from embryonic day 16 (E16) to E19 by using cytochemical and immunocytochemical methods at the light- and electron-microscope levels. We demonstrate that the organ of Corti develops from a non-proliferating cell zone that is located in the junctional region between two edges of the dorsal epithelium of the cochlear duct. We also reveal that the first cells to develop in this zone are the inner pillar cells, a particular type of non-sensory supporting cell, which arise in the base of the cochlear duct at the boundary between the two ridges at E16. Cell differentiation in this prosensory region continues according to a base-to-apex gradient; the inner hair cells appear in the greater epithelial ridge at E17 and the outer hair cells in the lesser epithelial ridge at E18. At E19, the various cell types of the organ of Corti are in place. Finally, we show that unlike the development of all the supporting cell types of the organ of Corti, the development of inner pillar cells within the prosensory domain seems not to involve Notch1 activation. These results highlight the central role that the inner pillar cells probably play in the development of the organ of Corti. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work received financial support from the Fonds de la Recherche Scientifique Médicale (grant no. 3.4540.06). N.T. is a PhD grant holder of the F.N.R.S.; B.M .is a Senior Research Associate of the F.N.R.S.     

干细胞向胰岛样细胞诱导分化的研究进展

目前,糖尿病患病率呈现上升趋势,它已成为严重危害人类健康的疾病之一。胰岛细胞移植可以用来治疗1型和部分2型糖尿病,但由于供体细胞来源匮乏,寻找新的细胞来源成了当务之急。近年来,随着细胞移植和组织工程的日益发展,干细胞研究为新型胰岛的来源开辟了新的途径。本文综述了近年来干细胞分化为胰岛样细胞的研究进展、存在问题和可能的解决途径。