Methods for Modulating Hemato-Vascular Development

描述（由申请人提供）：在发育中的小鼠胚胎中形成的第一个器官系统是心血管系统。心脏和脉管系统的缺陷是子宫内胚胎死亡的主要原因。在卵黄囊的发育血管网络内以及在某些胚胎内区域，内皮细胞和造血细胞以紧密的空间和时间关联出现，并且被认为源自共同的中胚层祖细胞“成血管细胞”。“最近的研究表明，成血管细胞也可能具有平滑肌细胞的潜力。成血管细胞和胚胎造血干细胞形成的调控和细胞命运的规范仍然没有很好地理解。这项建议的重点是中胚层细胞的特定亚群的特征和功能分析，这些细胞在发育过程中产生造血细胞、血管内皮细胞和平滑肌细胞。首先，我们将评估造血潜能和功能活性在体外和体内的中胚层干/祖细胞群体的前瞻性识别分化胚胎干细胞（胚状体）和早期小鼠胚胎。基于原始细胞表面标志物的表达，并使用GFP报告基因转基因，基于mMix的表达，通过流式细胞术分离细胞，mMix是一种在被认为含有成血管细胞的胚状体亚群中表达的同源结构域转录因子。在小鼠的培养和移植模型中，将使用几种不同的干/祖细胞测定法分析分选的细胞。其次，在类似的研究中，我们将评估内皮和平滑肌的潜力和功能活性在体外和体内的中胚层干/祖细胞群体的前瞻性识别。第三，我们将评估Mix同源结构域蛋白对胚状体衍生的中胚层干/祖细胞群体的体外和体内发育潜力和功能活性的作用。胚胎造血/血管干细胞的起源及其与成人干细胞的关系尚不清楚。随着对再生医学的关注和对人胚胎干细胞和成体干细胞潜在治疗应用的兴趣的增加，中胚层干/祖细胞群体的表征具有重要意义。

DESCRIPTION (provided by applicant): The first organ system to form in the developing mouse embryo is the cardiovascular system. Defects within the heart and vasculature are largely responsible for embryonic lethality in utero. Within the developing vascular network of the yolk sac as well as in certain intra embryonic regions, endothelial and hematopoietic cells arise in close spatial and temporal association and are thought to derive from a common mesodermal progenitor, the "hemangioblast." Recent work has indicated that hemangioblasts may also have smooth muscle cell potential. Regulation of hemangioblast and embryonic hematopoietic stem cell formation and cell fate specification are still not well understood. This proposal focuses on the characterization and functional analysis of specific subsets of mesodermal cells that give rise to hematopoietic, vascular endothelial, and smooth muscle cells during development. First, we will evaluate the hematopoietic potential and functional activity in vitro and in vivo of prospectively identified mesodermal stem/progenitorcell populations from differentiating embryonic stem cells (embryoid bodies) and from early mouse embryos. Cells will be isolated by flow cytometry on the basis of their expression of primitive cell surface markers and, using a GFP reporter transgene, on the basis of their expression of mMix, a homeodomain transcriptionfactor expressed in embryoid body subsets thought to contain hemangioblasts. The sorted cells will be analyzed using several different assays for stem/progenitor cells in culture and transplantation models in the mouse. Second, in analogous studies, we will evaluate the endothelial and smooth muscle potential and functional activity in vitro and in vivo of prospectively identified mesodermal stem/progenitor cell populations. Third, we will evaluate the role of the Mix homeodomain protein on the developmental potential and functional activity in vitro and in vivo of embryoid body derived mesodermal stem/progenitor cell populations. The origin of embryonic hematopoietic / vascular stem cells and their relationship to stem cells of the adult are unknown. With the increasing focus on regenerative medicine and interest in potential therapeutic applications of human embryonic and adult stem cells, the characterization of mesodermal stem/progenitor cell populations takes on high significance.