Activation & Proliferation of Hematopoietic Stem Cells

描述（由申请人提供）：造血干细胞（HSC）与例如胚胎干细胞不同，具有有限的分裂潜力。造血干细胞复制能力的限制在再生障碍性贫血和慢性粒细胞白血病等血液病中似乎很重要。这些限制可能进一步阻碍新的治疗策略的发展，包括离体干细胞扩增和基因治疗。基于这些考虑，可能有助于定义和扩展HSC复制潜力的研究是重要的，也是普遍感兴趣的。以前的研究与纯化的人“候选人”HSC由该基金资助的已经表明，HSC的功能特性在个体发育过程中发生了显着变化，并在HSC增殖潜力与年龄的损失与可测量的端粒缩短。在这里，我们建议进一步研究端粒酶和端粒在造血中的作用。具体来说，我们想测试的假设，即HSC的复制历史可以追溯到他们的端粒长度的研究。为了验证这一假设，我们将使用我们实验室开发的改进的流式细胞术技术，检查纯化的HSC及其培养后代相对于更成熟细胞的定义群体的亚群中的端粒长度。这些技术还将用于进一步研究正常个体、患有各种血液疾病的患者和基因分型正常狒狒谱系的有核血细胞中端粒长度的年龄相关下降。我们将进一步尝试使用基因转移和蛋白转导策略来操纵HSC中的端粒长度，并在体外和体内研究具有延长的端粒的细胞的功能特性。具体目的是：1）研究正常人和各种血液病患者治疗前后的纯化HSC和有核血细胞的端粒长度; 2）研究狒狒有核血细胞端粒长度与年龄和基因型的关系; 3）研究人工端粒延长和端粒酶抑制对纯化“候选”HSC体外和体内增殖、分化和复制潜力的影响，这些研究将为端粒在HSC生物学中的作用提供关键的基线信息。这些信息是有关造血的基本理解，以及干细胞在血液学内外的应用。

DESCRIPTION (provided by applicant): Hematopoietic stern cells (HSC), unlike e.g. embryonic stem cells, have a finite potential to divide. Limitations in the replication potential of HSC appear to be important in hematological disorders such as aplastic anemia and chronic myeloid leukemia. Such limitations could furthermore hamper the development of novel therapeutic strategies, including ex vivo stem cell expansion and gene therapy. Based on these considerations, studies that may help define and extend the replicative potential of HSC are important and a general interest. Previous studies with purified human "candidate" HSC funded by this grant have shown that the functional properties of HSC change dramatically during ontogeny and that the loss in HSC proliferative potential with age correlates with measurable shortening of telomeres. Here we propose to further examine the role of telomerase and telomeres in hematopoiesis. Specifically, we want to test the hypothesis that the replication history of HSC can be traced by studies of their telomere length. In order to test this hypothesis, we will examine the telomere length in subsets of purified HSC and their cultured progeny relative to defined populations of more mature cells using refined flow cytometry techniques developed in our laboratory. These techniques will also be used to further study the age related decline in telomere length in nucleated blood cells from normal individuals, patients with various hematological disorders and pedigrees of genotyped normal baboons. We will furthermore attempt to manipulate the telomere length in HSC using gene transfer and protein transduction strategies and study the functional properties of cells with extended telomeres in vitro and in vivo. The specific aims are:1) To study the telomere length in purified HSC and nucleated blood cells from normal individuals and patients with various hematological disorders before and after therapy.2) To study the telomere length in nucleated blood cells from baboons in relation to their age and genotype.3) To study the effect of artificial telomere elongation and telomerase inhibition on the proliferation, differentiation and replicative potential of purified "candidate" HSC in vitro and in vivo.Taken together, these studies will provide crucial baseline information on the role of telomeres in the biology of HSC. Such information is relevant for a basic understanding of hematopoiesis as well as applications of stem cells in and outside hematology.