Role of Endothelial Progenitors in Hematopoietic Stem Cell Expansion

人脐带血已被证明是一种有效的造血干细胞（HSC）来源，用于临床移植。这种材料在成人患者中更广泛使用的主要限制是脐带血样品中的HSC数量可能不足以移植到成人患者中，特别是不相关和不匹配的受体中。确定在脐带血中扩增HSC的方法将是该领域的一个重大进展。我们最近已经确定了一个层次的内皮祖细胞（EPC），在血液中循环，但也包括在人类和恒河猴血管内皮细胞内膜的一部分。与造血系统类似，EPC以层级组织，其中最增殖的祖细胞产生具有较少增殖潜力但具有更大成熟证据的许多子代，其中最成熟的内皮细胞显示无增殖潜力。在初步研究中，与成人血液相比，人脐带血富含EPC。我们推测，在个体发育过程中，EPC的频率和分布发生变化，从而使循环细胞和血管中EPC的数量随年龄的增长而下降，并且与端粒酶活性相关的增殖潜力丧失。在其他初步数据中，我们已经确定与人骨髓干细胞共培养的脐带血EPC与新鲜分离的骨髓HSC相比扩增HSC >260倍。我们现在假设具有最高增殖潜能的EPC比具有较低增殖潜能的EPC更能促进HSC的扩增。为了验证这些假设，我们提出了以下目标：具体目标1。确定人脐血EPC在体外扩增人脐血HSC中的作用。人脐带血与内皮祖细胞共培养？在子宫内注射到恒河猴胎儿宿主中后比较生长因子和再增殖能力。具体目标2。测定恒河猴个体发育过程中来源于血液和血管内皮的EPC的频率和增殖潜力。将检查从胎儿到老年人的恒河猴受试者的主动脉内皮细胞和外周血的EPC含量、增殖潜力和端粒酶活性。我们假设，EPC增殖潜力随着年龄的增长而下降，恒河猴模型使我们有机会在整个物种个体发育过程中对血管和血液进行采样。具体目标3。确定胚胎血管和成人血管来源的EPC在胚胎恒河猴脐带血和成人骨髓HSC体外扩增中的作用。我们假设胎儿EPC将比成人HSC更优化地扩增胎儿，但成人HSC将比成人EPC更好地扩增胎儿。

Human umbilical cord blood has been demonstrated to be an effective source of hematopoietic stem cells (HSC) for clinical transplantation. A major limitation to the more widespread use of this material for adult patients is that the number of HSC in a cord blood sample may be insufficient to engraft in adult patients, particularly unrelated and mismatched recipients. Determination of a method to expand HSC in cord blood would be a major advance in the field. We have recently identified a hierarchy of endothelial progenitor cells (EPC) that circulate in the blood, but also comprise a portion of the endothelial cell intima in human and rhesus macaque blood vessels. Similar to the hematopoietic system, EPC are organized in a hierarchy in which the most proliferative progenitors give rise to numerous progeny with less proliferative potential but greater evidence of maturation with the most mature endothelial cells displaying no proliferative potential. In preliminary studies, human umbilical cord blood was enriched in EPC compared to adult blood. We hypothesize that the frequency and distribution of EPC changes during ontogeny such that the number of circulating cells and EPC residing in vessels decline with age and there is loss of proliferative potential that correlates with telomerase activity. In other preliminary data, we have determined that cord blood EPC co- cultured with human marrow stem cells expands HSC >260 fold compared to freshly isolated marrow HSC. We now hypothesize that EPCwith the highest proliferative potential will better promote HSC expansion than EPC with lower proliferative potential. To test these hypotheses, we proposed the following aims: Specific Aim 1. Determine the role of human cord blood EPC in expanding human cord blood HSC ex vivo. Human cord blood will be co-cultured with EPC ? growth factors and the repopulating ability compared following injection into rhesus fetal hosts in utero. Specific Aim 2. Determine the frequency and proliferative potential of EPC derived from blood and vascular endothelium throughout rhesus ontogeny. Aortic endothelial cells and peripheral blood of rhesus subjects from fetal to aged adults will be examined for EPC content, proliferative potential, and telomerase activity. We hypothesize that EPC proliferative potential declines with age and the rhesus model permits us an opportunity to sample vessels and blood throughout the entire species ontogeny. Specific Aim 3. Determine the role of EPC derived from fetal versus adult blood vessels in the in vitro expansion of fetal rhesus cord blood and adult marrow HSC. We hypothesize that the fetal EPC will more optimally expand fetal than adult HSC but adult HSC will be better expanded on fetal than adult EPC.