Endothelial Cell Role in Yolk Sac Stem Cell Development

描述(申请人提供)：我们工作的总体目标是阐明内皮细胞和造血细胞相互作用在建立小鼠造血系统中的作用和分子基础。研究卵黄囊血岛器官发生的机制对于了解小鼠第一个器官是如何形成的以及作为研究造血系和内皮系如何从共同的前体产生的模型是重要的。然而，目前尚缺乏鉴定卵黄囊造血早期事件的试剂。我们提供了新的初步数据，CD41的表达标志着原始和明确的小鼠胚胎造血的开始。我们利用共聚焦显微镜来确定原始和最终祖细胞的祖细胞出现的位置。我们还开发了一种策略，允许从胚胎或成年小鼠器官中分离出纯净的内皮细胞群体。我们现在建议解决我们领域中存在重大信息差距的以下领域：1.卵黄囊血岛发育的机制尚不清楚。我们假设血细胞的形成伴随着血管的形成，但是原始的红系祖细胞是在任何内皮衬里的管腔外形成的。我们进一步假设，最终的造血祖细胞是以血岛内皮细胞群的形式出现的。因此，原始的和确定的祖先是通过不同的机制产生的，这就提出了他们是否共享共同的前体的问题。2.缺乏肝脏造血祖细胞种植的直接证据。我们假设第10天胎肝接种循环卵黄囊CD41光明定向祖细胞，在体外卵黄囊CD41光明祖细胞将优先黏附于胎肝内皮细胞。3.移植血管的细胞可保护小鼠免受辐射死亡，但不具有造血干细胞活性。我们提出的初步证据表明，与成人肾或肝内皮细胞相比，卵黄囊内皮细胞在体外优先维持HSC的再繁殖能力。我们推测卵黄囊和胚胎适当的内皮细胞将促进体内移植的HSC的植入。

DESCRIPTION (provided by applicant): The overall goal of our work is to elucidate the roles and molecular basis of endothelial and hematopoietic cell interactions in the establishment of the hematopoietic system in the mouse. Examination of the mechanisms of yolk sac blood island organogenesis is important to understand how the first organ in the mouse is formed and as a model to investigate how the hematopoietic and endothelial lineages arise from a common precursor. However, reagents to identify the earliest events in yolk sac hematopoiesis have been lacking. We present novel preliminary data that CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo. We have utilized confocal microscopy to identify the site of emergence of the progenitor cells of both primitive and definitive progenitors. We have also developed a strategy that permits isolation of pure populations of endothelial cells from embryonic or adult mouse organs. We now propose to address the following areas where significant information gaps exist in our field: 1. The mechanisms of yolk sac blood island development remain undefined. We hypothesize that blood cell formation occurs concomitant with vasculogenesis but that primitive erythroblast progenitor cells are formed outside of any endothelial lined lumen. We further hypothesize that definitive hematopoietic progenitor cells emerge as clusters of cells from the endothelial lining of the blood islands. Thus, primitive and definitive progenitors arise via different mechanisms, raising the question of whether they share a common precursor. 2. Direct evidence of hematopoietic progenitor cell seeding of the liver is lacking. We hypothesize that the day 10 fetal liver is seeded with circulating yolk sac CD41bright definitive progenitor cells in vivo and that yolk sac CD41 bright progenitor cells will preferentially adhere to fetal liver-derived endothelial cells in vitro. 3. Cells from vascular grafts protect mice from radiation-induced death but do not possess hematopoietic stem cell activity (HSC). We present preliminary evidence that yolk sac endothelial cells preferentially maintain HSC repopulating ability ex vivo compared to adult kidney or liver endothelial cells. We hypothesize that yolk sac and embryo proper endothelial cells will facilitate HSC engraftment upon transplantation in vivo