Murine Models for Regenerative Medicine

描述（由申请人提供）：该提案将探索指定胚胎中胚层向模式生物（如鱼和小鼠）中造血命运的承诺的遗传途径，以鉴定可被工程化以驱动小鼠和人胚胎干细胞向造血干细胞分化的机制。迄今为止，我们已经成功地进行了初步努力来产生造血干细胞，当这些干细胞被同源异型选择基因hoxb 4修饰时，可以在受辐射的原代和次级小鼠中移植多谱系造血。最近从贫血斑马鱼突变体的数据已经确定cdx 4基因作为一个中央球员在指定造血中胚层通过激活hox基因。当cdx 4在胚胎干细胞中表达时，cdx 4刺激造血，并与hoxb 4联合促进增强的淋巴-骨髓移植。我们将检验cdx 4是小鼠胚胎和胚胎干细胞中指定造血的必需基因的假设。我们将研究cdx 4，hoxb 4和血液特异性因子scl是否通过直接蛋白转导来诱导造血分化，有效地验证这些因子作为发育开关来指定血液命运的假设。我们将确定胚胎形态发生蛋白Indian Hedgehog，骨形态发生蛋白4和Wnt 3a是否会指导血液发育并激活胚胎干细胞中的cdx-hox信号传导。最终，我们将测试来自胚胎干细胞的造血组织，包括通过治疗性克隆，孤雌生殖和体细胞重编程创建的细胞系，是否可以用于纠正小鼠模型中的遗传骨髓疾病。这些研究将为胚胎干细胞的治疗应用的可行性提供重要的测试。

DESCRIPTION (provided by applicant): This proposal will explore genetic pathways that specify the commitment of embryonic mesoderm toward hematopoietic fate in model organisms like the fish and mouse, in order to identify mechanisms that can be engineered to drive hematopoietic stem cell differentiation from mouse and human embryonic stem cells. To date, we have succeeded in preliminary efforts to generate hematopoietic stem cells that, when modified by the homeotic selector gene hoxb4, engraft multi-lineage hematopoiesis in irradiated primary and secondary mice. Recent data from anemic zebrafish mutants has identified the cdx4 gene as a central player in specifying hematopoietic mesoderm through activation of hox genes. When expressed in embryonic stem cells, cdx4 stimulates hematopoiesis and in combination with hoxb4 promotes enhanced lymphoid-myeloid engraftment. We will test the hypothesis that cdx4 is an essential gene for specifying hematopoiesis in mouse embryos and embryonic stem cells. We will investigate whether cdx4, hoxb4, and the blood specification factor scl will induce hematopoietic differentiation if delivered by direct protein transduction, effectively testing the hypothesis that these factors act as developmental switches to specify blood fate. We will determine whether the embryonic morphogens Indian Hedgehog, Bone Morphogenetic Protein 4, and Wnt3a will instruct blood development and activate cdx-hox signaling in embryonic stem cells. Ultimately, we will test whether hematopoietic tissues derived from embryonic stem cells, including lines created by therapeutic cloning, parthenogenesis, and somatic cell reprogramming, can be harnessed to correct genetic bone marrow disorders in murine models. These studies will provide an important test of the feasibility of therapeutic applications of embryonic stem cells.