Human embryonic stem cell

人类胚胎干细胞

• 批准号: 8342311
• 负责人: Pamela Robey
• 金额: $ 137.34万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8342311
• 关键词: Address; Adherent Culture; Alleles; Antibodies; Australia; Bioinformatics; Bone Marrow; CD15 Antigens; Canada; Cell Line; Cells; Communities; Country; Data; Databases; Eligibility Determination; Embryo; FMR1; Fibroblasts; Fragile X Syndrome; Gene Silencing; Generations; Genes; Germ Layers; Growth; Guidelines; Human; Immunohistochemistry; In Vitro; Individual; International; Intramural Research Program; Israel; Laboratories; Lentivirus Vector; Maintenance; Messenger RNA; Molecular Genetics; Monitor; Mus; Mutation; N.I.H. Research Support; National Institute of Neurological Disorders and Stroke; Osteogenesis; Outcome Study; Pluripotent Stem Cells; Policies; Population; Process; Protocols documentation; Provider; Quality Control; Reagent; Registries; Research; Research Personnel; Resources; Retroviral Vector; Role; Sampling; Scientist; Series; Side; Site; Skin; Source; Staging; Stem Cell Development; Stem Cell Research; Stem cells; Stress; Stromal Cells; Suspension substance; Suspensions; Sweden; Teratoma; Training; UC06; Undifferentiated; United Kingdom; United States; United States National Institutes of Health; Variant; WA01 cell line; Work; adult stem cell; base; c-myc Genes; cell type; design; histone modification; human embryonic stem cell; human embryonic stem cell line; human stem cells; improved; in vivo; induced pluripotent stem cell; matrigel; novel; research study; stage-specific embryonic antigen 4; tool; web page

The NIH SCU has routinely grown hECs on the NIH registry since 2003, and the Unit has provided evidence that hESCs can be grown for long periods without genetic change. From this work, the SCU generated an mRNA database of information that includes both undifferentiated and differentiated samples. With help from the NINDS bioinformatics team, they have designed a public webpage in which this database of information may be easily interrogated and charts plotted. This provides an excellent reference source to which new hESC and iPSC lines can be compared. During the past year, the NIH SCU has also moved into the generation and characterization of induced pluripotent stem cells (iPSCs). Initially, they were involved with characterization of iPSCs derived by reprogramming fibroblasts using four reprogramming factors (Sox2, Oct4, c-Myc and Klf4) in individual retroviral vectors (NIH-i1-i10, the McKay lab), and compared them to hES cells. Their analyses indicate that there is strong similarity between iPSCs and hESs, and that any apparent variation is due to normal variation in the human population. In addition, the NIH SCU developed a new series of iPSCs (SCU-i1-10) from human bone marrow stromal cells and skin fibroblasts using a polycistronic lentiviral vector with the same four factors. All of these lines were initially propagated as colonies of mouse embryonic fibroblasts (MEFs), some were then transitioned as colonies onto MEF-free plates coated with Matrigel, and then finally into Matrigel coated plates as a novel monolayer culture generated by single cell suspensions. Of note, work has also focused on the role of stress reduction improving the growth of hESCs and iPSCs. By FACS, all SCU lines were negative for SSEA-1 and positive for SSEA-4, Tra-1-60 and Tra-1-81. By immunohistochemistry and qRT-PCR, POU5F1 and NANOG were at levels comparable to hESCs (WA01 and UC06). Current studies are in progress to determine if SCU-derived iPSCs are pluripotent based on differentiation into cell types representative of all three germ layers in vitro, and the ability to form teratomas in vivo in comparison to WA01. The SCU is also evaluating commercially available reagents needed for reprogramming, and subsequent growth in order to provide the best advice possible to the NIH community. The Unit has trained a number of investigators in the growth and maintenance of pluripotent stem cells, and have been cited for their contributions (Kumari D and Usdin K. (2010) The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome. Human Molecular Genetics. 19(23):463442; Kuznetsov SA, Cherman N, Robey PG. (2011) In vivo bone formation by progeny of human embryonic stem cells.Stem Cells and Development. 20(2):269-87).

自2003年以来，NIH SCU一直在NIH登记处常规培养hEC，并且该单位提供了hESC可以长时间生长而不发生遗传变化的证据。通过这项工作，SCU生成了一个mRNA信息数据库，其中包括未分化和分化的样本。 在NINDS生物信息学小组的帮助下，他们设计了一个公共网页，在这个网页上可以很容易地查询信息数据库并绘制图表。 这提供了一个很好的参考来源，新的hESC和iPSC系可以与之比较。 在过去的一年中，NIH SCU也进入了诱导多能干细胞（iPSC）的生成和表征。 最初，他们参与了在单个逆转录病毒载体（NIH-i1-i10，McKay实验室）中使用四种重编程因子（Sox 2，Oct 4，c-Myc和Klf 4）重编程成纤维细胞获得的iPSC的表征，并将其与hES细胞进行比较。 他们的分析表明，iPSC和hES之间存在很强的相似性，任何明显的变化都是由于人类群体中的正常变化。 此外，NIH SCU使用具有相同四种因子的多顺反子慢病毒载体，从人骨髓基质细胞和皮肤成纤维细胞中开发了一系列新的iPSC（SCU-i1-10）。 所有这些细胞系最初作为小鼠胚胎成纤维细胞（MEF）的集落繁殖，然后将一些细胞系作为集落转移到用Matrigel包被的无MEF的平板上，然后最终转移到Matrigel包被的平板上作为由单细胞悬浮液产生的新型单层培养物。 值得注意的是，工作还集中在减轻压力改善hESC和iPSC生长的作用上。 通过FACS，所有SCU系对SSEA-1呈阴性，对SSEA-4、Tra-1-60和Tra-1-81呈阳性。 通过免疫组织化学和qRT-PCR，POU 5 F1和NANOG处于与hESC（WA 01和UC 06）相当的水平。 目前的研究正在进行中，以确定SCU衍生的iPSC是否是多能的，其基于在体外分化成代表所有三个胚层的细胞类型，以及与WA 01相比在体内形成畸胎瘤的能力。 SCU还在评估重编程和后续生长所需的市售试剂，以便为NIH社区提供最佳建议。 该单位已经培训了许多研究人员在多能干细胞的生长和维持，并已被引用的贡献（Kumari D和Usdin K。（2010）抑制性组蛋白修饰在沉默的FMR 1等位基因上的分布为脆性X综合征的基因沉默机制提供了线索。人类分子遗传学。19（23）：463442; Kuznetsov SA，Cherman N，Robey PG.（2011）In vivo bone formation by professional of human embryonic stem cells.Stem Cells and Development. 20（2）：269-87）。