FACTORS INFLUENCING STEM CELL AND PLATELET PRODUCTION

影响干细胞和血小板生成的因素

• 批准号: 6343629
• 负责人: MALCOLM A. MOORE
• 金额: $ 33.32万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343629
• 关键词: Adenoviridae; CD34 molecule; NOD mouse; SCID mouse; cell adhesion; cell migration; chemoattractants; chemotaxis; cytokine receptors; enzyme activity; hematopoietic stem cells; human tissue; intermolecular interaction; messenger RNA; mixed tissue /cell culture; platelets; receptor expression; telomerase; transfection /expression vector; transforming growth factors; umbilical cord; vascular endothelium

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.

描述：（改编自研究者摘要） 移植后血小板恢复延迟， CD 34+细胞数量不理想，特别是来自重度预处理的 供体，或脐带血（UCB）移植后。离体 CD 34+细胞的扩增被用来克服这个问题，但 关于干细胞的质量和数量的问题已经出现， (HSC)产生和它们在体内的长期植入潜力。的 PI建议在细胞因子中评估HSC功能的参数- 使用Flk 2/flt 3刺激UCB和动员的PB CD 34+细胞的培养物 配体（FL）和Tpo增强HSC自我更新，与共 在骨髓基质或内皮细胞上培养。HSC将在 在长期的文化启动和鹅卵石区域形成中进行评估 测定和NOD-SCID再增殖。如果发现缺陷， 确定这些是否是由于有缺陷的骨髓归巢， 可能与趋化因子受体CXCR-4的表达缺陷有关， HSC和对骨髓趋化因子SDF-1的反应受损。他将 使用跨膜趋化性测定和内皮粘附， 确定CD 34、c-kit是否存在缺陷的迁移试验 和粘附分子表达。如果发现缺陷，他将使用 腺病毒基因递送以（a）增强稳定的 c-kit配体和FL在基质和内皮细胞上的跨膜异构体 （B）增加HSC上flk-2、c-kit和CXCR-4的表达， 改善骨髓归巢和趋化性并对抗TGF β抑制。 选择性扩增CD 41 a+巨核细胞， 评估它们对SDF-1的趋化反应以及它们 在体外产生血小板并在转移至NOD-SCID小鼠后产生血小板。 与进行性端粒缩短相关的退行性衰老是 在体外和体内的造血细胞和内皮中观察到。的 PI建议将该基因用于催化组分， 端粒酶（hTERT）进入原始造血细胞，骨髓和 脐带内皮和CD 34 + Flk-1+循环内皮 使用逆转录病毒和腺病毒基因递送的祖细胞 选择标记端粒酶活性与端粒长度相关 在我们的离体细胞扩增系统中， 人口的数量是不可能实现的。所有检测系统 上述用于评价股骨干和内皮功能的方法将 应用于潜在的“永生”人口后， 通过确定是否有正常功能的保留，是否 存在功能性复原-即，成年群体获得 新生儿细胞的特征，或者是否有一种倾向， 使“永生化”细胞经历肿瘤转化。