神经干细胞向神经元分化调控的研究进展

神经干细胞是一类具有分裂潜能和自更新能力的母细胞,它可以通过对称分裂和不对称分裂方式产生神经组织的各类细胞,包括神经元、星形胶质细胞和少突胶质细胞。中枢神经系统受到损伤后,神经元和胶质细胞的损伤导致了临床症状,内源性神经干细胞的修复作用不大,原因是干细胞的数量有限,微环境的不允许。移植的神经干细胞进入体内后,由于受到多种因素的影响,常保持未分化状态或大部分分化为胶质细胞。神经干细胞向神经元分化的调控机制及其影响因素直接决定神经干细胞源性神经元的比例和神经元之间功能性突触的数量。现就其研究进展做一综述。     

肠上皮干细胞研究进展

肠上皮干细胞是位于肠黏膜陷窝内的具有自我更新和增殖分化为成熟肠上皮细胞功能的细胞。肠黏膜上皮细胞不断更新及肠黏膜损伤的修复均通过肠上皮干细胞增殖、分化完成。根据抗损伤能力的不同肠上皮干细胞可分为三级,一定程度内可适应不同生理、病理变化。陷窝内干细胞之间存在竞争,占优势的干细胞迅速分裂、增殖,使陷窝表现为单克隆性。肠上皮干细胞所处微环境内的各种细胞因子及端粒酶共同影响肠上皮干细胞的分裂、增殖与分化。目前尚缺乏确切的肠上皮干细胞标记,Msi-1、Hes-1和D整合素可能是肠上皮干细胞的自然标记物,但有待进一步研究证实。     

Myoepithelial Cells of Submucosal Glands Can Function as Reserve Stem Cells to Regenerate Airways after Injury

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The Use of Genetically Modified Embryonic Stem Cells to Generate Transgenic Mice

Targeted genetic modification of embryonic stem cells (ESC) was used to obtain nondifferentiated cell clones containing the foreign genetic material in the genome. It was demonstrated that transgenic animals may be obtained by ESC injection in preimplantation embryos and subsequent transplantation of the embryos into a recipient female. Using this method, we constructed chimeric animals with a modified genome.     

大鼠内细胞团和胎儿神经干细胞构建嵌合体的潜力(英文）

嵌合体大鼠是研究人类疾病的重要动物模型。用囊胚注射法研究了大鼠内细胞团（ICM）和胎儿神经干细胞（FNS）构建嵌合体的潜力。结果发现来自黑色（DA）大鼠第5天（D5）和第6天（D6）囊胚的ICM细胞注入D5 Sprague-Dawley（SD）大鼠囊胚后得到3只嵌合体大鼠;D5 SD大鼠ICM细胞注射入D5 DA囊胚后得到4只嵌合体大鼠;而体外培养的DA或SD大鼠ICM细胞注射后均未能获得嵌合体大鼠。本研究用大鼠胎儿神经干细胞（rFNS）和LacZ转染的rFNS构建嵌合体,未能获得嵌合体大鼠;但在LacZ转染的SD rFNS注射到DA大鼠囊胚后发育来的41只胎儿中,有2只胎儿其组织切片中发现少量LacZ阳性细胞。结果表明DA和SD大鼠ICM具有参与嵌合体发育的潜力,但ICM细胞经体外培养后构建嵌合体的潜力显著下降（P<0.05）;大鼠胎儿神经干细胞构建嵌合体的潜力较低,可能仅具有参与早期胚胎发育的潜力。     

大鼠内细胞团和胎儿神经干细胞构建嵌合体的潜力

嵌合体大鼠是研究人类疾病的重要动物模犁.用囊胚注射法研究了大鼠内细胞团(ICM)和胎儿神经干细胞(FNS)构建嵌合体的潜力.结果发现来自黑色(DA)大鼠第5天(D5)和第6天(D6)囊胚的ICM细胞注入D5 Sprague-Dawley(SD)大鼠囊胚后得到3只嵌合体大鼠:D5 SD大鼠ICM细胞注射入D5 DA囊胚后得到4只嵌合体大鼠:而体外培养的DA或SD人鼠ICM细胞注射后均未能获得嵌合体大鼠.本研究用大鼠胎儿神经干细胞(rFNS)和LacZ转染的rFNS构建嵌介体,未能获得嵌合体人鼠:但在LacZ转染的SD rFNS注射到DA大鼠囊胚后发育来的41只胎儿中,有2只胎儿其组织切片中发现少量LacZ阳性细胞.结果表明DA和SD大鼠ICM具有参与嵌合体发育的潜力,但ICM细胞经体外培养后构建嵌合体的潜力显著F降(P＜0.05);大鼠胎儿神经干细胞构建嵌合体的潜力较低,可能仅具有参与早期胚胎发育的潜力.     

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche^1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment⁴ such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures². These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded⁵. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions² to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes¹. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.     

胶质细胞的神经干细胞/祖细胞特性

近年来,关于胶质细胞有许多令人惊奇的发现。其中最令人感兴趣的是部分胶质细胞在体内外都表现出神经千细胞／祖细胞的特性,在适当条件下能分化成神经元、星形胶质细胞和／或少突胶质细胞。不仅存在于非哺乳类脊椎动物整个生命周期的放射胶质显示出这一特性,存在于成年哺乳动物脑室下区和颗粒下层的星形胶质细胞也是如此。在体外培养中,部分胶质细胞具有形成多潜能神经球的能力。在体内,胶质细胞充当前驱细胞时的命运受到细胞间相互作用、细胞因子、血脉系统、胞外基质以及基膜等所构建的微环境的影响。胶质细胞的这些特性将对神经修复产生深远影响。     

Pancreatic Insulin-Producing Cells Differentiated from Human Embryonic Stem Cells Correct Hyperglycemia in SCID/NOD Mice,an Animal Model of Diabetes

Background

Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes.

Methods

We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5–7×10⁶ differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels.

Results

The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature β cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P<0.0001).

Conclusions

The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for ≥8 weeks.     

Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells

Diverse metabolic pathways, such as the tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation, regulate the differentiation of induced pluripotent stem cells (iPSCs) to cells of specific lineages and organs. Here, the protein dynamics during cardiac differentiation of human iPSCs into cardiomyocytes (CMs) are characterized. The differentiation is induced by N‐(6‐methyl‐2‐benzothiazolyl)‐2‐[(3,4,6,7‐tetrahydro‐4‐oxo‐3‐phenylthieno[3,2‐d]pyrimidin‐2‐yl)thio]‐acetamide, a Wnt signaling inhibitor, and confirmed by the mRNA and protein expression of cTnT and MLC2A in CMs. For comparative proteomics, cells from three stages, namely, hiPSCs, cardiac progenitor cells, and CMs, are prepared using the three‐plex tandem mass tag labeling approach. In total, 3970 proteins in triplicate analysis are identified. As the result, the upregulation of proteins associated with branched chain amino acid degradation and ketogenesis by the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis are observed. The levels of 3‐hydroxymethyl‐3‐methylglutaryl‐CoA lyase, 3‐hydroxymethyl‐3‐methylglutaryl‐CoA synthase 2, and 3‐hydroxybutyrate dehydrogenase 1, involved in ketone body metabolism, are determined using western blotting, and the level of acetoacetate, the final product of ketogenesis, is higher in CMs. Taken together, these observations indicate that proteins required for the production of diverse energy sources are naturally self‐expressed during cardiomyogenic differentiation. Furthermore, acetoacetate concentration might act as a regulator of this differentiation.     

Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

食蟹猴精原干细胞体外培养体系的初步建立

为了掌握食蟹猴(Macaca fascicularis)精原于细胞(spermatogonial stem cells,SSCs)体外培养生长特性,并建立其培养体系.手术法获得幼年期食蟹猴单侧睾丸,改良的两步酶消化法获得其细胞悬液,添加特定培养液进行体外培养,以碱性磷酸酶(AKP)染色鉴定培养细胞,并评价不同饲养层细胞...