DIRECTED STEM CELL DIFFERENTIATION - HUMAN APPLICATION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。前言：只有对造血干细胞和造血调控有更深入的了解，才能改进骨髓移植技术。涉及通过细胞周期传递分析造血干细胞分化功能的研究已经阐明了向特定谱系发生戏剧性分化的点(S)。分子研究证实，干细胞通过周期改变了受体的表达；反映了对特定微环境影响的敏感性。这可能会对造血干细胞的后代产生直接影响。小鼠研究证实(LRH)细胞可在细胞周期的特定位置定向向巨核细胞或粒细胞分化。一个关键的方面是，这些变化不是固定的分化步骤，而是随着持续的细胞周期传递而可逆的。这一数据表明，这些细胞处于表型流动的恒定状态，而不是按等级定义的祖细胞类别。要将这些发现转化为人类应用，同时研究人类干细胞是至关重要的。这一试点项目旨在将正在进行的小鼠工作与人体研究的启动同步进行。方法：首先评估不同群体的细胞，观察它们的细胞周期、细胞倍增时间、培养活力，并优化培养条件。我们将测试不同人群的纯化骨髓抽吸物和G-CSF刺激的CD34+PBSC的临床样本。细胞群将立即根据DNA含量的细胞周期时相进行分选并置于分化培养中，或者将其与细胞因子一起培养，并在原代培养后连续几次将亚群细胞置于分化培养中，如含G-CSF、GM-CSF和SCF的大噬细胞-粒细胞培养。结果：建立了体内亚致死剂量辐射恢复模型，可用于检测不同群体的培养细胞。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction: Improvement in bone marrow transplantation techniques can only be made with a better understanding of the hematopoietic stem cell and the regulation of hematopoiesis. Studies involving analyzing the functional ability of hematopoietic stem cell differentiation through cell cycle transit have elucidated points where dramatic differentiation toward specific lineage(s) occurs. Molecular studies confirm that stem cells have changing receptor expression through cycle; a reflection of sensitivity to specific microenvironmental influences. This may have a direct impact on the progeny of the hematopoietic stem cell. Murine studies confirmed that (lineage negative rhodamine low Hoechst low (LRH)) cells can be directed towards lineage differentiation into megakaryocytes or granulocytes at specific positions in celly cycle. A critical aspect is that these changes are not fixed differentiation steps but rather are reversible with continued cell cycle transit. This data suggests that instead of a hieracrhally defined progenitor class, the cells are in a constant state of phenotype flux. To translate these findings to human application; it is crucial to concurrently study human stem cells. This pilot project aims to parallel ongoing murine work with initiation into human studies. Methods: We will first evaluate different populations of cells and look at their cell cycle phase, cell doubling time, viability in culture and optimize the culturing conditions. We will test different populations of purified marrow aspirates and clinical samples of G-CSF primed CD34+ PBSC. Cell populations will either be sorted immediately based on cell cycle phase based on DNA content and placed in differentiation cultures, or they will be cultured with cytokines and at serial times after primary culture, sub-populations of cells will be placed in differentiation cultures such as a machrophage-granulocyte culture with G-CSF, GM-CSF and SCF. Results: An in vivo sub-lethal radiation recovery model has been developed and will allow us to test different populations of cultured cells.