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.

我们对 21 个布什时代的人类胚胎干细胞进行了广泛的表征，并将数据存入 NCBI GEO 供公众访问。我们创建了一个用户友好的基因表达搜索引擎，允许临时用户查询他们感兴趣的特定基因的数据。为了促进多能干细胞研究，我们对对照诱导多能干细胞 (iPSC) 系 BC1 进行了深入表征。这包括 FACS 分析和免疫细胞化学以及基因表达微阵列分析。我们与 NIH-CRM 合作，生成了源自 H1 ES 细胞系分化的神经元前体的 iPSC，并将其基因表达和甲基化谱与亲本系进行了比较。这提供了 ESC 与 iPSC 的直接比较，因为所有细胞系都具有相同的基因组。分析显示，虽然可能存在更微妙的影响有待确定，但没有全局差异。 与 NIH-CRM 合作，使用锌指核酸酶辅助基因靶向方法将转基因整合到人类基因组中已知的安全港位点之一的 AAVS1 中，生成了 5 个转基因 hESC 系，这些系表达来自细胞类型特异性启动子的可追踪标记。每个转基因包含一个 ZS green 基因和一个药物选择标记。每个细胞系均已确认正确的转基因整合以及正常的核型，并且已在 2 个细胞系中确认来自适当细胞类型的 ZS green 表达。所有线路将很快存入 WiCell，以便于分发到社区。 我们扩展了对使用不同小分子和替代底物进行多能细胞培养的新型非集落型（NCM）单层方法的研究。我们已经证明使用这种方法可以提高质粒 DNA、慢病毒颗粒和基于短寡核苷酸的 microRNA 的转染或转导效率。我们将继续改进该方法，以应用于药物筛选和治疗用途的高通量和可扩展性。此外，为了有效且可重复地产生特定细胞类型的同质群体，已尝试从 NCM 培养开始进行定向分化。这些分化策略不包括拟胚体的形成，而拟胚体是许多分化方案中异质性的主要来源。上述几种可追溯的转基因hESC系已成功分化为神经前体细胞（内胚层）、搏动心肌细胞（中胚层）和SOX17阳性细胞（内胚层），且具有相对较高的效率和同质性。将进一步优化和细化分化方案。 在将多能干细胞引入临床方面，我们一直在评估新型无异种基质、培养基和小分子抑制剂以及非整合重编程方法。这些方法包括仙台病毒和 microRNA 增强的基于 mRNA 的重编程。未来我们还将评估基于游离质粒的重编程策略，并测试重新编程血细胞的新策略。 最后，我们参与指导和教授标准和无饲养层多能干细胞培养，协助和建议从患者样本中生成 iPSC，并根据要求协助和建议分化策略。我们会更新 SCU 网站的协议和信息，以帮助其他研究人员进行研究。