FACTORS INFLUENCING STEM CELL AND PLATELET PRODUCTION

描述：(改编自调查人员摘要) 移植后出现血小板恢复延迟 CD34+细胞的数量不理想，特别是从大量预处理的细胞中 供者或脐带血(UCB)移植后。体外实验 CD34+细胞的扩增被用来克服这个问题，但 在干细胞的质量和数量方面出现了一些问题 (HSC)的产生及其在体内的长期植入潜力。这个 PI建议在细胞因子中评估HSC功能的参数- Flk2/Flt3对脐血CD34+细胞的刺激培养及动员 配体(FL)和TPO促进HSC自我更新的比较 在骨髓基质或内皮细胞上培养。然后，HSC将被 在长期培养-启动和鹅卵石区域-形成中进行评估 检测和NOD-SCID再繁殖。如果发现缺陷，他将 确定这些是不是由于骨髓归巢缺陷所致 可能与趋化因子受体CXCR-4表达缺陷有关 造血干细胞与骨髓趋化因子SDF-1反应受损他会的 使用跨膜趋化试验和内皮细胞黏附 移行试验以确定CD34、c-kit是否存在缺陷 和黏附分子的表达。如果发现缺陷，他将使用 腺病毒基因传递(A)增强稳定的表达 C-kit配体和FL在基质和内皮细胞上的跨膜异构体 细胞；(B)增加HSC上Flk-2、c-kit和CXCR-4的表达 改善骨髓归巢和趋化作用，对抗转化生长因子β的抑制作用。 CD41a+巨核细胞的选择性扩增 评估他们对SDF-1的趋化反应和他们的能力 在体外和移植到NOD-SCID小鼠后产生血小板。 与进行性端粒缩短相关的增殖性衰老 在体外和体内可见于造血细胞和血管内皮细胞。这个 Pi建议转导催化组分的基因 端粒酶(HTERT)进入原始造血细胞、骨髓和 脐静脉内皮细胞和CD34+Flk-L+循环内皮细胞 使用逆转录病毒和腺病毒基因传递的祖细胞 可选标记。端粒酶活性将与端粒相关 我们的体外细胞扩增系统中的长度和测量 实现人口翻一番的数量。所有的化验系统 上面概述的干细胞和内皮功能的评估将是 适用于经过长时间的潜在“永生”的人群 通过判断是否有正常功能保留，是否 有功能的复兴--即成年人口获得 新生儿细胞的特征，或者是否有 “永生化”的细胞要经历肿瘤转化。

DESCRIPTION: (Adapted from investigator's abstract) Delayed platelet recovery is seen following transplantation with suboptimal numbers of CD34+ cells, particularly from heavily pretreated donors, or following umbilical cord blood (UCB) transplantation. Ex vivo expansion of CD34+ cells is being used to overcome this problem, but issues have arisen concerning the quality and quantity of stem cells (HSC) generated and their long-term engraftment potential in vivo. The PI proposes to evaluate parameters of HSC function in cytokine- stimulated cultures of UCB and mobilized PB CD34+ cells using Flk2/flt3 ligand (FL) and Tpo to enhance HSC self-renewal in comparison with co- cultures on marrow stroma or endothelial cells. HSC will then be evaluated in long term culture-initiating and cobblestone area-forming assays and NOD-SCID repopulation. If defects are seen, he shall determine whether these are due to defective marrow homing which in turn may relate to defective expression of the chemokine receptor CXCR-4 on HSCs and impaired response to marrow chemotactic factor SDF-1. He will use transmembrane chemotaxis assays and endothelial adhesion and transmigration assays to determine if there are defects in CD34, c-kit and adhesion molecule expression. If defects are found he will use adenoviral gene delivery to (a) enhance expression of the stable transmembrane isoforms of c-kit ligand and FL on stromal and endothelial cells; (b) increase flk-2, c-kit, and CXCR-4 expression on HSC to improve marrow homing and chemotaxis and counteract TGF beta inhibition. Selective expansion of CD41a+ megakaryocytes will be undertaken to evaluate their chemotactic response to SDF-1 and their ability to generate platelets in vitro and following transfer to NOD-SCID mice. Proliferative senescence related to progressive telomere shortening is seen in vitro and in vivo in hematopoietic cells and endothelium. The PI proposes to transduce the gene for the catalytic component of telomerase (hTERT) into primitive hematopoietic cells, marrow and umbilical endothelium, and CD34+Flk-l+ circulating endothelial progenitors using retroviral and adenoviral gene delivery with selectable markers. Telomerase activity will be correlated with telomere length in our ex vivo cell expansion systems with measurement of the numbers of population doublings be achieved. All of the assay systems outlined above for evaluation of stem and endothelial function will be applied to potentially "immortalized" populations after prolonged passage to determine if there is retention of normal function, whether there is functional rejuvenation - i.e., adult populations acquiring features of neonatal cells, or whether there is a tendency for "immortalized" cells to undergo neoplastic transformation.