Methods for Modulating Hemato-Vascular Development

描述(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。心脏和血管系统的缺陷在很大程度上导致了胚胎在子宫内的死亡。在卵黄囊发育中的血管网络中以及某些胚胎内区域，内皮细胞和造血细胞在空间和时间上密切相关，被认为来自共同的中胚层前体细胞--“血管母细胞”。最近的研究表明，血管母细胞也可能具有平滑肌细胞的潜能。血管母细胞和胚胎造血干细胞形成的调控以及细胞命运的指定仍然不是很清楚。这项建议侧重于特定的中胚层细胞亚群的特征和功能分析，这些细胞亚群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，我们将评估从分化中的胚胎干细胞(类胚体)和小鼠早期胚胎中筛选出的中胚层干细胞/祖细胞群体在体外和体内的造血潜力和功能活性。流式细胞术将根据原始细胞表面标记的表达和使用GFP报告转基因，基于它们表达的MMIX(一种在被认为含有血管母细胞的类胚体亚群中表达的同源结构域转录因子)来分离细胞。分离的细胞将在小鼠的培养和移植模型中使用几种不同的干细胞/祖细胞分析方法进行分析。其次，在类似的研究中，我们将评估在体外和体内预期确定的中胚层干细胞/祖细胞群体的内皮和平滑肌潜力和功能活性。第三，我们将评估混合同源结构域蛋白在体外和体内对类胚体来源的中胚层干/祖细胞群体的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。随着人们对再生医学的日益关注和对人类胚胎和成体干细胞潜在治疗应用的兴趣，中胚层干细胞/祖细胞群体的特征变得非常重要。

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.