Molecular Mechanisms Regulating Stem Cell Plasticity

调节干细胞可塑性的分子机制

• 批准号: 6527874
• 负责人: Michael A Rudnicki
• 金额: $ 25万
• 依托单位: OTTAWA HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527874
• 关键词: cell differentiation; cell population study; developmental genetics; flow cytometry; laboratory mouse; microarray technology; molecular cloning; muscle satellite cell; myogenesis; representational difference analysis; transcription factor; transfection

DESCRIPTION (provided by applicant): Recent work has identified the presence of adult stem cells within most if not all adult tissues that contribute to many tissues following reintroduction in vivo. Our laboratory has identified the paired box transcription factor Pax7 as being required for the specification of myogenic satellite cells from progenitor adult satellite cells. In the absence of Pax7, adult stem cells in muscle appear to undergo default differentiation towards the hematopoietic lineages. In this application, we outline a research program designed to further our understanding of the molecular mechanisms that regulate the lineage commitment of adult stem cells. To elucidate the mechanisms that direct the specification of adult stem cells, we propose to characterize the inductive signal, test candidate factors such as Shh, Wnts, and BMPs, and finally molecularly clone the inducing factors using expression-based screens. Candidate factors with inductive activity will be tested on adult stem cells from different tissues. To investigate whether expression of Pax7 in adult stem cells directly induces their myogenic specification, viral vectors will be used to ectopically express Pax7 in adult stem cells from diverse tissues. The ability of viral vectors expressing Pax7 to induce myogenic specification will be assessed both in vitro and in vivo. Lastly, this approach will be used to elucidate the function of the alternative splice forms of Pax7. To elucidate the broader mechanisms that regulate specification, a PCR-based differential cloning procedure called representational difference analysis (RDA) and microarray analysis will be used to identify potential Pax7 target genes. RDA and microarray analysis will also be used to perform comparative expression profiling between muscle, marrow and neural stem cells. Lastly, microarray profiling and cluster analysis will be performed on adult stem cells undergoing myogenic specification. To investigate the mechanism by which Pax7 enforces lineage commitment, FACS/Hoechst enriched adult stem cells from Pax7-/- mice will be transplanted either intravenously or intramuscularly.

描述（由申请人提供）： 最近的工作已经确定了大多数人在大多数人中的存在 重新引入后，所有有助于许多组织的成年组织 体内。 我们的实验室已经确定了配对盒转录因子PAX7 这是从 祖先成人卫星细胞。 在没有PAX7的情况下，成年干细胞中 肌肉似乎对造血的默认区分差异 血统。 在此应用程序中，我们概述了一个旨在的研究计划 进一步我们对调节分子机制的理解 成年干细胞的谱系承诺。 阐明的机制 指导成人干细胞的规范，我们建议表征 归纳信号，测试候选因素，例如SHH，WNT和BMP，以及 最后，使用基于表达的筛选将诱导因子克隆。 具有诱导活性的候选因素将在成年干细胞上进行测试 来自不同的组织。 调查PAX7在成人中的表达是否 干细胞直接诱导其肌源规范，病毒载体将 用于在不同组织的成年干细胞中异位表达PAX7。 表达PAX7诱导肌源规范的病毒向量的能力 将在体外和体内评估。 最后，这种方法将是 用于阐明PAX7的替代剪接形式的功能。 到 阐明调节规范的更广泛的机制，一种基于PCR的规范 差分克隆程序称为表示差分析 （RDA）和微阵列分析将用于识别潜在的PAX7目标 基因。 RDA和微阵列分析也将用于进行比较 肌肉，骨髓和神经干细胞之间的表达分析。 最后， 将对成人茎进行微阵列分析和聚类分析 经过肌源规范的细胞。 通过调查机制 哪个PAX7执行谱系承诺，FACS/HOECHST富含成年干细胞 从pax7 - / - 小鼠将被静脉注射或 肌肉内。