Human embryonic stem cell

人类胚胎干细胞

• 批准号: 8746882
• 负责人: Pamela Robey
• 金额: $ 77.49万
• 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/8746882
• 关键词: Blood Cells; Cardiac Myocytes; Cell Culture Techniques; Cell Death; Cell Line; Cell Survival; Cells; Chromosomal Instability; Clinic; Collaborations; Communities; Controlled Study; Data; Deposition; Differentiation Antigens; ES Cell Line; Educational process of instructing; Embryo; Endoderm; Endoderm Cell; Evaluation; Future; Gene Expression; Gene Expression Microarray Analysis; Gene Targeting; Generations; Genes; Genome; Genomics; Goals; Heterogeneity; Human Genome; Karyotype; MDM2 gene; Mentors; Mesoderm; Messenger RNA; Methods; Methylation; MicroRNAs; Mission; Monitor; Neurons; Oligonucleotides; Pathway interactions; Patients; Pharmaceutical Preparations; Plasmids; Pluripotent Stem Cells; Population; Preclinical Drug Evaluation; Process; Protocols documentation; Quality Control; Reporter; Research Personnel; Role; Sampling; Sendai virus; Side; Site; Somatic Cell; Sorting - Cell Movement; Source; Staging; Stem Cell Research; Stem cells; Surface; System; Technology; Testing; Therapeutic Uses; Transfection; Transgenes; Transgenic Organisms; United States National Institutes of Health; Update; Xeno; Zinc Fingers; base; biological adaptation to stress; cell type; human embryonic stem cell; immunocytochemistry; improved; induced pluripotent stem cell; inhibitor/antagonist; interest; monolayer; neural precursor cell; new technology; novel; novel strategies; nuclease; particle; plasmid DNA; progenitor; promoter; scale up; small molecule; stem cell technology; user-friendly; web site

We have performed extensive characterization of the 21 Bush-era human embryonic stem cells and deposited the data with NCBI GEO for public access. We have created a user-friendly gene expression search engine which allows a casual user to interrogate the data for their particular gene of interest. In our mission to facilitate pluripotent stem cell research, we have performed in-depth characterization of a control induced pluripotent stem cell (iPSC) line, BC1. This includes FACS analysis and immunocytochemistry as well as gene expression microarray analysis. In collaboration with the NIH-CRM, we generated iPSCs derived from neuronal precursors differentiated from H1 ES cell lines and compared their gene expression and methylation profiles to the parental line. This provides a direct comparison of ESCs to iPSCs as all lines have the same genome. Analysis shows no global difference although there may be more subtle effects to be determined. In collaboration with NIH-CRM, 5 transgenic hESC lines, which express traceable markers from cell type-specific promoters, have been generated using a Zinc Finger nuclease-assisted gene-targeting method to integrate the transgenes into AAVS1, one of the known safe harbor sites in the human genome. Each transgene contains a gene for ZS green and a drug-selection marker. Correct transgene integration as well as normal karyotype has been confirmed for each line and expression of ZS green from appropriate cell types has been confirmed in 2 lines. All lines will be deposited with WiCell shortly to facilitate distribution to the community. We have extended our studies on the novel non-colony type (NCM) monolayer method for pluripotent cell culture using different small molecules and alternative substrates. We have demonstrated improved efficiency of transfection or transduction of plasmid DNAs, lentiviral particles, and short oligonucleotide-based microRNAs using this method. We will continue to improve the method for application to high thoughput and scalability for drug screening and therapeutic use. In addition, to generate homogeneous populations of specific cell types efficiently and reproducibly, directed differentiation has been attempted starting from NCM culture. These differentiation strategies do not include the formation of embryoid bodies, which are a major source of heterogeneity in many differentiation protocols. Several of the traceable transgenic hESC lines described above have been successfully differentiated into neural precursor cells (endoderm), beating cardiomyocytes (mesoderm), and SOX17-positive cells (endoderm) with relatively high efficiency and homogeneity. Further optimization and refinement of the differentiation protocols will be pursued. In terms of bringing pluripotent stem cells to the clinic, we have been evaluating novel xeno-free substrates, media and small molecule inhibitors as well as non-integrating methods of reprogramming. These methods include Sendai virus and microRNA boosted mRNA- based reprogramming. We will also be evaluating episomal plasmid-based reprogramming strategies in the future and testing novel strategies to reprogram blood cells. Finally, we have been involved in mentoring and teaching standard and feeder-free, pluripotent stem cell culture, assisting and advising on the generation of iPSCs from patient samples as well as assisting and advising on differentiation strategies as requested. We update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.

我们已经对21个灌木时代的人类胚胎干细胞进行了广泛的表征，并将数据沉积在NCBI GEO中以进行公共通道。我们创建了一个用户友好的基因表达搜索引擎，该引擎允许休闲用户为其特定的感兴趣基因询问数据。为了促进多能干细胞研究，我们对对照诱导的多能干细胞（IPSC）系（BC1）进行了深入的表征。这包括FACS分析和免疫细胞化学以及基因表达微阵列分析。与NIH-CRM合作，我们生成了与H1 ES细胞系不同的神经元前体衍生的IPSC，并将其基因表达和甲基化谱与父母系进行了比较。这提供了ESC与IPSC的直接比较，因为所有线都有相同的基因组。分析表明，尽管可能有更多细微的效果要确定，但没有全球差异。 与NIH-CRM合作，使用锌指特异性启动子表达可追溯标记的5种转基因hESC线，已使用锌指核酸酶辅助基因靶向方法来将转染物集成到AAVS1中，这是人类基因组中已知的安全港口的一个。每个转基因都包含一个用于ZS绿色的基因和一个药物选择标记。已确认了正确的转基因整合以及正常的核型，并在两条线中证实了适当细胞类型的ZS绿色的表达。所有线路都将在不久后与Wicell一起存放，以促进向社区分发。 我们已经扩展了有关使用不同的小分子和替代底物的多能细胞培养的新型非颜色类型（NCM）单层方法的研究。我们已经证明，使用这种方法，我们已经证明了质粒DNA，慢病毒颗粒和短寡核苷酸的质粒DNA，慢性病毒颗粒的转染或转导效率提高。我们将继续改善用于高含量和可扩展性的药物筛查和治疗用途的方法。此外，为了有效且可重复地产生特定细胞类型的均匀种群，从NCM培养开始尝试了定向分化。这些分化策略不包括形成胚胎体，这是许多分化方案中异质性的主要来源。上述的几种可追溯的转基因hESC系已成功区分为神经前体细胞（内胚层），击败心肌细胞（中胚层）和具有相对较高的效率和同质性的Sox17阳性细胞（内胚层）。将追求分化协议的进一步优化和完善。 在将多能干细胞带到诊所方面，我们一直在评估新颖的无XENO底物，培养基和小分子抑制剂以及不整合的重新编码方法。这些方法包括仙台病毒和microRNA增强基于mRNA的重编程。我们还将在将来评估基于二种质粒的重编程策略，并测试重新编程血细胞的新型策略。 最后，我们参与了指导和教学标准和不含喂食器，多能干细胞培养，为患者样本中的IPSC产生和建议，并根据要求协助和提供有关分化策略的建议。我们可以使用协议和信息更新SCU网站，因为它可以帮助其他研究人员进行研究。