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。心脏和血管的缺陷是胚胎在子宫内死亡的主要原因。在卵黄囊发育中的血管网络以及某些胚胎内区域内，内皮细胞和造血细胞在空间和时间上紧密结合，被认为源自一种共同的中胚层祖细胞，即“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。成血管细胞和胚胎造血干细胞形成的调控和细胞命运规范仍不清楚。本研究的重点是在发育过程中产生造血细胞、血管内皮细胞和平滑肌细胞的中胚层细胞的特定亚群的特征和功能分析。首先，我们将评估从胚胎干细胞（胚状体）和早期小鼠胚胎中鉴定的中胚层干细胞/祖细胞群体的体外和体内造血潜能和功能活性。根据原始细胞表面标记的表达，通过流式细胞术分离细胞，并根据mMix的表达，使用GFP报告基因分离细胞，mMix是一种同源结构域转录因子，在胚状体亚群中表达，被认为含有成血管细胞。分选后的细胞将在小鼠培养和移植模型中使用几种不同的干细胞/祖细胞测定法进行分析。其次，在类似的研究中，我们将评估内皮和平滑肌电位以及前瞻性鉴定的中胚层干细胞/祖细胞群在体外和体内的功能活性。第三，我们将评估Mix同源结构域蛋白在胚胎体衍生的中胚层干细胞/祖细胞群体的体外和体内发育潜力和功能活性中的作用。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类胚胎干细胞和成体干细胞潜在治疗应用的兴趣的增加，中胚层干细胞/祖细胞群体的表征具有很高的意义。

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.