Origin of HSC from Embryonic Stem Cells

描述（由申请人提供）： 该指导临床科学家发展奖（K 08）的性质涉及对人类多能和造血干细胞的理解。 这项为期5年的研究培训计划将推进人类发育造血的研究，其中一个最重要的新工具是生物医学研究：人类胚胎干细胞（hES）。 我的长期兴趣是临床干细胞移植的发展，我打算把我职业生涯的很大一部分投入到干细胞生物学的基础研究中。 本提案将集中于从人胚胎干细胞中衍生、培养和扩增原始人造血干细胞（HSC）。 鉴定早期人HSC的策略将依赖于一般假设，即“成血管细胞”（HSC和内皮的双能祖细胞）和/或“生血内皮”前体可以使用与在其他物种中描述的那些类似的实验方法从分化的hES中物理分离和表征。 这些hES衍生的祖细胞将用于人类淋巴造血发育的后续研究。 该研究计划最初将专注于扩大，纯化和表征hES衍生的成血管细胞。 随后的研究将探讨SCL/TAL 1（启动造血的主要调节因子）在人类成血管细胞形成中的机制作用。 用改良的同源重组方法将报告cDNA基因靶向内源SCL基因座调控区（“敲入”），纯化并扩增表达原始SCL的血液内皮祖细胞用于进一步的表型和遗传分析。 原始HSC最终将在基质细胞系和/或胎儿胸腺器官培养物上培养，用于衍生人T淋巴细胞发育的模型。 Notch通路信号传导在hES驱动的淋巴造血组织中的作用将作为长期目标进行研究。 这些目标的成功将为表征无法通过任何其他方式获得的人类干细胞奠定基础。

DESCRIPTION (provided by applicant): The nature of this Mentored Clinical Scientist Development Award (K08) pertains to the understanding of human pluripotent and hematopoietic stem cells. This proposed 5-year research training program will advance the study of human developmental hematopoiesis with one of the most important new tools in biomedical research: the human embryonic stem cell (hES). My long-term interest is in advancement of clinical stem cell transplantation, and I intend to devote a large part of my career to basic research in stem cell biology. This proposal will focus on the derivation, culture, and expansion of primitive human hematopoietic stem cells (HSC) from hES. The strategy for identifying early human HSC will rely on the general hypothesis that a "hemangioblast" (bipotential progenitor of HSC and endothelium) and/or a "hemogenic endothelial" precursor can be physically isolated and characterized from differentiating hES using experimental approaches similar to those described in other species. These hES-derived progenitors will be used for subsequent studies in human lympho-hematopoietic development. The research program will initially focus on expanding, purifying, and characterizing hES-derived hemangioblasts. Subsequent studies will probe the mechanistic role SCL/TAL1, a master regulator for initiating hematopoiesis, plays on the formation of the human hemangioblast. A reporter cDNA will be gene-targeted to the endogenous SCL locus regulatory region ("knock-in") with modified methods of homologous recombination, and primitive SCL-expressing hemato-endothelial progenitors will be purified and expanded for further phenotypic and genetic analysis. Primitive HSC will ultimately be cultured on stromal lines and/or fetal thymic organ cultures for the derivation of a model for human T-lymphocyte development. The role of Notch pathway signaling in hES-drived lymph-hematopoietic tissue will be studied as a long-term goal. The success of these goals will lay the groundwork for characterizing human stem cells not available by any other means.