OPTIMIZE EMBRYONIC STEM CELL CULTURE MEDIA

优化胚胎干细胞培养介质

• 批准号: 7349414
• 负责人: James Alexander Thomson
• 金额: $ 2.72万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7349414
• 关键词: OPTIMIZE; EMBRYONIC; STEM; CELL; CULTURE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To develop a media formulation and protocol for the human ES cell culture that will improve human ES cell viability and promote uniformity, consistency and ease of use across a variety of users in different locations. Some of the specific objectives necessary to accomplish this goal are to: 1) optimize the physiochemical environment, 2) optimize the basal media formulation and 3) eliminate undefined media components. Human embryonic stem (ES) cells already provide a powerful research tool for understanding the human body, the current sub-optimal conditions for human ES cell culture place significant limitations on their use in basic research and on their large-scale expansion and distribution. The labor required to continuously prepare MEF feeder layers is a major limitation in large-scale production of human ES cells. Combined with concerns surrounding cross- species contamination that may arise from growth of human ES cells on murine feeder layers, the elimination of fibroblast feeder cells would greatly improve the efficiency and consistency of ES cell culture. We have previously reported that high bFGF concentrations support feeder-independent growth of human ES cells, but those conditions included poorly defined serum and matrix components. In this budget year we reported a feeder- independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material (Nature Biotechnology, February 2006). Key signaling molecules included are bFGF, GABA, LiCl, pipecolic acid, and TGF_, and removal of any one of these components decreased culture performance. For each cell line tested (H1, H7, H9, and H14) proliferation rates and the percentage of cells maintaining expression of human ES cell markers were higher in this medium compared to fibroblast-conditioned medium. As a proof of principle, we used these conditions to derive two new cell lines (these cell lines were obtained using private funding at a private research laboratory).

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。开发用于人类胚胎干细胞培养的培养基配方和方案，以提高人类胚胎干细胞的活力，促进不同地区各种用户使用的统一性、一致性和易用性。实现这一目标所必需的一些具体目标是：1)优化理化环境，2)优化基本培养基配方，3)消除未定义的培养基成分。人类胚胎干细胞（ES）已经为了解人体提供了一个强大的研究工具，目前人类胚胎干细胞培养的次优条件对其在基础研究中的应用以及其大规模扩展和分布造成了重大限制。连续制备MEF喂料层所需的劳动力是大规模生产人类胚胎干细胞的主要限制。考虑到人类胚胎干细胞在小鼠饲养层上生长可能引起的跨物种污染，消除成纤维细胞饲养细胞将大大提高胚胎干细胞培养的效率和一致性。我们之前报道过高bFGF浓度支持人类胚胎干细胞不依赖饲料生长，但这些条件包括不明确的血清和基质成分。在本预算年度，我们报告了一种不依赖饲养器的人类胚胎干细胞培养，其中包括完全来自重组来源或从人类材料中纯化的蛋白质成分（自然生物技术，2006年2月）。关键信号分子包括bFGF、GABA、LiCl、果酸和TGF_，去除其中任何一种成分都会降低培养性能。对于所测试的每个细胞系（H1, H7， H9和H14），与成纤维细胞条件培养基相比，该培养基中的增殖率和维持人类胚胎干细胞标记物表达的细胞百分比更高。作为原理证明，我们利用这些条件获得了两种新的细胞系（这些细胞系是在一家私人研究实验室使用私人资助获得的）。