Origin of HSC from Embryonic Stem Cells

描述(由申请人提供)： 这项有指导的临床科学家发展奖(K08)的性质与对人类多能性和造血干细胞的理解有关。这项拟议的为期5年的研究培训计划将利用生物医学研究中最重要的新工具之一：人类胚胎干细胞(HES)来推进人类发育造血的研究。我的长期兴趣是促进临床干细胞移植，我打算将我职业生涯的很大一部分时间投入到干细胞生物学的基础研究中。这项建议将集中在从人类胚胎干细胞(HES)中衍生、培养和扩增原始人类造血干细胞(HSC)。鉴定早期人类HSC的策略将依赖于一个普遍的假设，即“血管母细胞”(HSC和内皮的双潜能前体)和/或“血源性内皮”前体可以被物理地分离出来，并通过与其他物种中描述的实验方法类似的实验方法来区分HES。这些来源于HES的祖细胞将用于人类淋巴造血发育的后续研究。该研究计划最初将重点放在扩增、纯化和鉴定HES来源的血管母细胞。随后的研究将探索SCL/TAL1在人类血管母细胞形成中的作用机制。SCL/TAL1是启动造血的主要调节因子。通过改进的同源重组方法，将报告基因定位于内源性SCL基因位点调控区，并纯化和扩增表达SCL的原始血内皮祖细胞，以进行进一步的表型和遗传分析。原始的HSC最终将在基质细胞和/或胎儿胸腺器官培养上培养，以衍生出人类T淋巴细胞发育的模型。Notch信号通路在HES驱动的淋巴-造血组织中的作用将作为一个长期目标进行研究。这些目标的成功将为确定人类干细胞的特性奠定基础，这是任何其他方法都无法获得的。

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.