DIRECTED STEM CELL DIFFERENTIATION - HUMAN APPLICATION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。简介：只有更好地了解造血干细胞和造血调控，才能提高骨髓移植技术。涉及分析造血干细胞通过细胞周期转运分化的功能能力的研究已经阐明了发生向特定谱系的显著分化的点。分子研究证实，干细胞在周期中具有变化的受体表达;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，（谱系阴性的罗丹明低Hoechst低（LRH））细胞可以在细胞周期的特定位置定向谱系分化为巨核细胞或粒细胞。一个关键的方面是，这些变化不是固定的分化步骤，而是随着细胞周期的持续转运而可逆的。这些数据表明，细胞不是一个层次上定义的祖细胞类别，而是处于表型流动的恒定状态。为了将这些发现转化为人类应用，同时研究人类干细胞至关重要。该试点项目旨在将正在进行的小鼠研究与人类研究相平行。研究方法：我们将首先评估不同的细胞群，并观察它们的细胞周期阶段，细胞倍增时间，培养中的活力并优化培养条件。我们将测试不同群体的纯化骨髓抽吸物和G-CSF致敏的CD 34 + PBSC的临床样本。细胞群将基于DNA含量基于细胞周期阶段立即分选并置于分化培养物中，或者它们将与细胞因子一起培养，并且在原代培养后的连续时间，细胞亚群将置于分化培养物中，例如具有G-CSF、GM-CSF和SCF的巨噬细胞-粒细胞培养物。结果：在体内亚致死辐射恢复模型已经开发，并将允许我们测试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.