PROLIFERATION OF PURIFIED HEMATOPOIETIC STEM CELLS

血细胞来源于造血干细胞群体，其通常被定义为具有自我更新能力的多能细胞。 虽然造血系统的再生能力令人印象深刻，但干细胞的复制潜力似乎有限。 因为干细胞可以分裂的次数是开发新的治疗策略（包括干细胞移植、离体扩增和基因治疗）的重要考虑因素，所以可能有助于定义或扩展干细胞的复制潜力的研究受到普遍关注。 以前的研究由该基金资助的纯化的人类候选干细胞已经表明，它们的功能特性在个体发育过程中发生了显着变化，其增殖潜力的丧失与端粒重复序列数量的可测量变化相关，并且它们表达低水平的端粒酶。 在这里，我们建议进一步研究端粒在造血干细胞复制和分化中的作用。 为此，我们将使用我们实验室开发的新技术来测量移植和体外扩增前后单个细胞和造血细胞特定染色体上端粒重复序列的长度。 我们将使用逆转录病毒介导的人类端粒酶逆转录酶基因转移，以检查人工端粒延长对干细胞特性的影响，可以在体外测量。最后，我们将专门研究l7 p端粒在造血细胞的分化，复制和衰老中的作用。 该提案的具体目标是：（1）研究同种异体移植的短期和长期存活者的骨髓和淋巴供体细胞中的端粒长度，并与移植供体的细胞进行比较;（2）研究人工端粒延长对纯化的“候选”干细胞的增殖、分化和复制潜力的影响;（3）研究L7 p端粒在造血细胞增殖和复制性衰老中的作用。

Blood cells originate from a population of hematopoietic stem cells which are typically defined as pluripotent cells with self-renewal capacity. Although the regenerative capacity of the hematopoietic system is impressive, the replicative potential of stem cells nevertheless appears finite. Because the number of times stem cells can divide is an important consideration in the development of novel therapeutic strategies, including stem cell transplantation, ex vivo expansion and gene therapy, studies that may help define or extend the replicative potential of stem cells are of general interest. Previous studies funded by this grant with purified human candidate stem cells have shown that their functional properties change dramatically during ontogeny, that loss in their proliferative potential correlates with measurable changes in the number of telomere repeats and that they express low levels of telomerase. Here we propose to further examine the role of telomeres in the replication and differentiation of hematopoietic stem cells. For this purpose, we will use novel techniques developed in our laboratory to measure the length of telomere repeats in individual cells and on specific chromosomes in hematopoietic cells before and after transplantation and ex vivo expansion. We will use retroviral-mediated transfer of the human telomerase reverse transcriptase gene to examine the effect of artificial telomere elongation on stem cell properties that can be measured in vitro. Finally, we will specifically examine the role of l7p telomeres in the differentiation, replication and senescence of hematopoietic cells. The specific aims of the proposal are 1) to study the telomere length in myeloid and lymphoid donor cells of short- and long-term survivors of allotransplants in comparison with cells of the transplant donor; 2) to study the effect of artificial telomere elongation on the proliferation, differentiation and replicative potential of purified "candidate" stem cells; and 3) to study the role of l7p telomeres in the proliferation and replicative senescence of hematopoietic cells.