HEMATOPOIETIC BLAST CELL COLONIES FUNCTIONAL CAPACITY

骨髓衰竭状态的分析因有限的模型而变得复杂 造血干细胞生理学的研究。承诺物的体外培养 祖细胞(CFU-GM、CFU-E)可能会也可能不会反映早期事件 造血术。小鼠骨髓衰竭状态(W/WV，CBA-N等)和 骨髓移植研究可以为了解 人类造血的生理学，尤其是与人类平行的模型 干细胞可以被开发出来。最近，原始造血干细胞 (CFU-BLAST)已经从小鼠和人类样本中培养出来；这些 细胞可能代表一个接近于全潜能干细胞的细胞 增殖和分化能力。人类和小鼠(摘自 用5-氟尿嘧啶[5-FU])预处理的动物将被 在半固体培养基中收获和培养。十到十六天后 原始的、无颗粒的、中等大小的、折射的、未分化的群体 “原始”细胞将从甲基纤维素中提取出来，它们的功能 容量将会确定。利用缺陷逆转录病毒介导的基因 转移作为克隆性的标志，自我更新能力和 这一早期干细胞的增殖潜力将被确定。For In 活体研究收获的原始细胞将被评估其 能够重建受到致命性照射的同基因或同种异体小鼠， 和有缺陷的突变小鼠。差异化和承诺的阶段 CFU-BLAST将与CFU-脾进行比较。爆炸将会是 从体外培养中获得并注射到受致死照射的小鼠体内 任何可检测到的脾克隆的数量和血统承诺都将 要下定决心。同样，来自5-FU处理的供者的脾克隆 骨髓细胞将被采集并在半固体培养基中进行培养 确定是否可以获得CFU-BLAST来源的克隆。虽然 不能进行类似的人体体内研究，而是平行的体外研究 将执行OF功能。半固体培养中的短期培养 在淋巴和髓系情况下将评估总潜力 此单元格的属性。人类和小鼠的原始集落细胞也将 用于启动和播种现有的长期连续骨髓培养，以及 他们从这些文化中恢复过来将是决定性的。这些项目的目标是 研究是为了确定真正的增殖和自我更新的能力 CFU-BLAST，探讨其在造血中的作用，并发展成平行 未来正常和无序造血研究的模型。

The analysis of bone marrow failure states is complicated by limited models of hematopoietic stem cell physiology. The in vitro culture of committed progenitor cells (CFU-GM, CFU-E) may or may not reflect early events in hematopoiesis. Murine bone marrow failure states (W/Wv, CBA-N, etc) and bone marrow transplantation studies can provide some insight into the physiology of human hematopoiesis, especially if parallel models with human stem cells can be developed. Recently, primitive hematopoietic stem cells (CFU-Blast) have been cultured from both murine and human samples; these cells may represent a cell close to the totipotential stem cell in their capacity for proliferation and differentiation. Human and murine (from animals pre-treated with 5-fluorouracil [5-FU]) hematopoietic cells will be harvested and cultured in semi-solid media. Ten to sixteen days later the colonies of primitive, agranular, medium-sized, refractile, undifferentiated "blast" cells will be plucked from the methylcellulose, and their functional capacity will be determined. Using defective-retrovirus-mediated gene transfer as a marker of clonality, the self-renewal capacity and proliferative potential of this early stem cell will be ascertained. For in vivo studies the harvested primitive cells will be evaluated for their ability to reconstitute lethally-irradiated syngeneic or allogeneic mice, and defective mutant mice. The stage of differentiation and commitment of the CFU-Blast will be compared to that of the CFU-Spleen. Blasts will be harvested from in vitro cultures and injected into lethally irradiated mice and the number and lineage commitment of any detectable spleen colonies will be determined. Similarly, spleen colonies derived from 5-FU treated donor bone marrow cells will be harvested and plated in semi-solid media to determine whether CFU-Blast-derived colonies can be obtained. Although similar human in vivo studies cannot be pursued, parallel in vitro studies of function will be performed. Short-term cultures in semi-solid media under lymphoid and myeloid conditions will asses the totipotential properties of this cell. Human and murine Blast colony cells will also be used to initiate and seed existing long-term continuous marrow cultures, and their recovery from these cultures will be determined. The goals of these studies are to determine the true proliferative and self-renewal capacity of the CFU-Blast, to explore its role in hematopoiesis, and to develop parallel models for future studies of normal and disordered hematopoiesis.