Human pluripotent stem cells

人类多能干细胞

• 批准号: 9157598
• 负责人: Pamela Robey
• 金额: $ 124.74万
• 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/9157598
• 关键词: Agreement; Area; Biological Assay; Biological Models; Brain; Cell Culture Techniques; Cell Line; Cell Therapy; Cell physiology; Cells; Chromosomal Instability; Clinic; Clinical; Collaborations; Communities; Controlled Study; Deposition; Derivation procedure; Development; Differentiation Antigens; Disease model; Educational process of instructing; Embryo; Endoderm; Extracellular Matrix; Generations; Genes; Genomics; Goals; Hepatic; Human; Individual; International; Intestines; Knowledge; Laminin; Manuscripts; Mentors; Methods; Mission; Modeling; Monitor; Organoids; Parkinson Disease; Pathogenesis; Patients; Pharmaceutical Preparations; Pluripotent Stem Cells; Population; Process; Production; Progeria; Protocols documentation; Publications; Publishing; Quality Control; Reporter; Research Personnel; Role; Sampling; Severity of illness; Side; Site; Societies; Stem Cell Research; Stem cells; Structure of retinal pigment epithelium; Surface; Symptoms; Syndrome; System; Technology; Testing; Tetracyclines; Transgenes; Transgenic Organisms; United States National Institutes of Health; Update; WA09 Cell Line; Work; Zinc Fingers; base; biological adaptation to stress; cell type; glucose metabolism; human disease; human embryonic stem cell; improved; induced pluripotent stem cell; inhibitor/antagonist; integration site; interest; meetings; mutant; nerve stem cell; nervous system disorder; novel; overexpression; pathogen; pluripotency; promoter; scale up; small molecule; stem cell technology; transgene expression; web site

During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below. In collaboration with NIH-CRM, 5 transgenic hESC lines, which express traceable markers from cell type-specific promoters, were generated and deposited with WiCell for distribution. This manuscript was published in 2015. To study the role of individual genes in human disease pathogenesis, two inducible model cell lines expressing ALPHA-SYNUCLEIN or mutant LAMIN-A (PROGERIN) were generated in H9 (WA09) hESCs using a tetracycline-inducible system. Overexpression of each transgene models Parkinson's disease or Hutchinson-Gilford Progeria syndrome respectively with the level of transgene expression, regulated by the inducible system, anticipated to mimic the severity of the disease symptoms. However, analysis of transgene expression showed that the level of expression induced by the drug was not consistent within the cell population, possibly due to random integration of the transgene. This work was presented at the International Society for Stem Cell Research (ISSCR) annual meeting in 2015. To overcome this problem, another set of tetracycline-inducible lines have been generated in WA09 and an induced pluripotent stem cell (iPSC) line by targeting safe-harbor sites for integration. Analysis is underway. We have continued to develop protocols to optimize human PSC culture on different extracellular matrices, particularly regarding the use of laminin-based methods to facilitate definitive endoderm differentiation and hepatic lineage maturation. This work was also presented at the ISSCR annual meeting in 2015. Other assays in development include the use of multiple panel surface markers in monitoring cellular differentiation and functional maturation and glucose metabolism assays to monitor cellular energetic states in hPSC culture and differentiation. 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. We are advising on the establishment of GMP protocols related to Dr. Kapil Bhartis (NEI) clinical initiative regarding iPSC derivation and differentiation into retinal pigmented epithelial cells. Finally, we continued to mentor and teach standard and feeder-free, pluripotent stem cell culture, provided assistance and advise on the generation of iPSCs from collaborators samples, as well as assistance and advise on differentiation strategies as requested. Such differentiation strategies have included the derivation of human intestinal organoids for pilot testing culture of gut pathogens as well as cortical spheroids to model brain development. We have also assisted collaborators by providing material and neural stem cell lines derived from PSCs, resulting in the publication of 2 other manuscripts in 2015. As always, we update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.

在上一财年，NIH SCCF 在许多领域取得了进展，如下所示。 与 NIH-CRM 合作，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，并存放在 WiCell 中进行分发。这篇手稿发表于 2015 年。 为了研究单个基因在人类疾病发病机制中的作用，使用四环素诱导系统在 H9 (WA09) hESC 中生成了表达 ALPHA-SYNUCLEIN 或突变体 LAMIN-A (PROGERIN) 的两种诱导模型细胞系。每个转基因的过度表达分别模拟帕金森病或哈钦森-吉尔福德早衰综合症，转基因表达水平受诱导系统调节，预计可模拟疾病症状的严重程度。然而，转基因表达分析表明，药物诱导的表达水平在细胞群内并不一致，这可能是由于转基因的随机整合所致。这项工作在 2015 年国际干细胞研究学会 (ISSCR) 年会上发表。为了克服这个问题，在 WA09 中生成了另一组四环素诱导细胞系，并通过靶向安全港位点进行整合生成了诱导多能干细胞 (iPSC) 细胞系。分析正在进行中。 我们继续开发方案来优化不同细胞外基质上的人类 PSC 培养，特别是使用基于层粘连蛋白的方法来促进定形内胚层分化和肝谱系成熟。这项工作也在 2015 年 ISSCR 年会上进行了展示。其他正在开发的检测方法包括使用多组表面标记来监测细胞分化和功能成熟，以及使用葡萄糖代谢检测来监测 hPSC 培养和分化中的细胞能量状态。 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。我们建议建立与 Kapil Bhartis 博士 (NEI) 临床倡议相关的 GMP 协议，该协议涉及 iPSC 衍生和分化为视网膜色素上皮细胞。 最后，我们继续指导和教授标准和无饲养层多能干细胞培养，提供有关从合作者样本中生成 iPSC 的帮助和建议，以及根据要求提供有关分化策略的帮助和建议。这种分化策略包括衍生人类肠道类器官以用于肠道病原体培养的初步测试，以及皮质球体以模拟大脑发育。我们还通过提供源自 PSC 的材料和神经干细胞系来协助合作者，导致 2015 年发表了另外 2 篇手稿。 一如既往，我们会在 SCU 网站上更新协议和信息，以帮助其他研究人员进行研究。