Stem/Progenitor Cell Inversions With Cell Cycle Transit

描述(由申请人提供)： 血细胞形成(造血)的过程已被广泛研究，但许多基本机制仍不清楚。以前的研究表明，在细胞周期的不同时间点，骨髓细胞的植入是不同的，而且这些变化是可逆的。在最近的工作中，我们已经表明，小鼠的祖细胞数量也随着细胞因子诱导的细胞周期传递而变化。研究未分离的骨髓细胞，我们发现7因子反应性祖细胞(hpp-cfc和cfu-c)在第一个细胞周期中显着增加，然后回到基线。值得注意的是，在竞争性移植模型中，这些增加与干细胞植入能力的降低密切相关。这种现象被称为干细胞/祖细胞倒置。这些数据表明，早期骨髓干细胞的功能表型在穿越细胞周期时可逆地从可移植干细胞到祖细胞再到造血干细胞，而先前设想的干细胞到祖细胞的单向层次结构是可逆表型转变的连续体。这项资助的长期目标是进一步表征细胞周期相关的转换点，包括植入、祖细胞水平和骨髓细胞的分化能力，研究Flt-3配体、血小板生成素和钢铁因子中未分离和高度纯化的干细胞。我们计划评估细胞周期中不同时间点的(阴性)罗丹明(Low)Hoechst(Low)(LRH)骨髓细胞在竞争性移植模型中的短期和长期植入，评估7因子反应的祖细胞，以及在存在7因子细胞因子鸡尾酒和两种旨在促进粒-巨噬细胞或巨核细胞分化的鸡尾酒时，评估纯化干细胞的分化能力。在植入研究中，将重点放在细胞周期的G1期。分化研究将在特氟龙批量培养和单细胞克隆的基础上进行。为了进一步了解干细胞在细胞周期不同时间点的表型。为了指出替代诱导研究的方法，我们将对细胞表面的细胞因子受体、黏附蛋白、整合素和其他表位进行表征。这些研究使用了高速细胞分选、荧光细胞成像和各种体外和体内干细胞/祖细胞分析。他们承诺进一步确定造血骨髓干细胞的基本性质，并可能导致有趣的临床前模型，以选择性地支持各种清髓治疗方法的谱系。

DESCRIPTION (provided by applicant): The process of blood cell formation (hematopoiesis) has been extensively studied but many basic mechanisms continue to be unknown. Previous studies have shown that marrow cell engraftment varies at different points in cell cycle and that these changes are reversible. In recent work we have shown that murine progenitor numbers also vary with cytokine induced cell cycle transit. Studying unseparated marrow cells we demonstrated that 7-factor responsive progenitors (HPP-CFC and CFU-c) increased markedly during the first cell cycle transit and then returned to baseline. Remarkably, these increases were tightly linked to the decreased ability of stem cells to engraft in a competitive transplant model. This phenomenon is termed stem/progenitor cell inversions. These data suggest that the functional phenotype of early marrow stem cells shifts reversibly from engraftable stem cell to progenitor and back to hematopoietic stem cell as it traverses cell cycle, and that the previously conceived stem-cell-to-progenitor unidirectional hierarchy is instead a continuum of reversible phenotypic shifts. The long term objective of this grant are to further characterize the cell cycle related inversions points as to engraftment, progenitor levels and differentiation capacity of the marrow cells studying both unseparated and highly purified stem cells in FLT-3 ligand, thrombopoietin and steel factor. We plan to assess lineage(negative) rhodamine(low) Hoechst(low) (LRH) marrow cells at different points in cell cycle as to short and long-term engraftment in a competititive transplant model, to assess 7-factor-responsive progenitors and to evaluate the capacity of purified stem cells to differentiate in the presence a 7-factor cytokine cocktail and two cocktails designed to promote granulocyte-macrophage or megakaryocyte differentiation. In the engraftment studies there will be an emphasis on G1 phase of cell cycle. The differentiation studies will be carried out in bulk Teflon cultures and on a single cell clonal basis. In order to further understand the phenotype of the stem cell at different points in cell cycle. To point ways to alternative induction studies we will characterize cytokine receptors, adhesion proteins, integrins and other epitopes on the cell surface. These studies employ high-speed cell sorting, fluorescent cell imaging and a variety of in vitro and in vivo stem/progenitor assays. They promise to further define the basic nature of the hematopoietic marrow stem cells and could lead to interesting preclinical models for selective lineage support of various myeloablative therapy approaches .