Murine Models for Regenerative Medicine

描述(申请人提供)：这项提案将探索在鱼类和小鼠等模式生物中指定胚胎中胚层对造血命运的承诺的遗传途径，以确定可以通过工程手段推动小鼠和人类胚胎干细胞分化为造血干细胞的机制。到目前为止，我们已经成功地进行了初步的努力来产生造血干细胞，当被同源异型选择器基因HOXB4修饰时，在受照射的初级和次级小鼠中植入多系造血。来自贫血斑马鱼突变体的最新数据表明，cdx4基因是通过激活Hox基因来指定造血中胚层的中心角色。当在胚胎干细胞中表达时，cdx4刺激造血，并与HOXB4结合促进增强的淋巴-髓系植入。我们将检验这一假设，即cdx4是小鼠胚胎和胚胎干细胞中指定造血的必要基因。我们将研究cdx4、HOXB4和血液规范因子scl如果通过直接的蛋白质转导传递是否会诱导造血细胞分化，有效地检验了这些因子作为发育开关来决定血液命运的假设。我们将确定胚胎形态因子印度刺猬、骨形态发生蛋白4和WNT3a是否会指导血液发育并激活胚胎干细胞中的CDX-HOX信号。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新编程创建的系，是否可以在小鼠模型中用于纠正遗传性骨髓疾病。这些研究将为胚胎干细胞治疗应用的可行性提供重要的检验。

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.