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HEMATOPOIETIC BLAST CELL COLONIES FUNCTIONAL CAPACITY

造血母细胞集落功能能力

基本信息

批准号：
3192545
负责人：
SAUL Joseph SHARKIS
金额：
$ 19.71万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-04-05 至 1991-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3192545
关键词：
hematopoietic stem cells leukocyte activation /transformation

项目摘要

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.

有限的模型使骨髓衰竭状态的分析变得复杂造血干细胞生理学离体培养祖细胞（CFU-GM，CFU-E）可能反映或可能不反映早期事件，造血小鼠骨髓衰竭状态（W/Wv、CBA-N等）和骨髓移植研究可以提供一些见解，人体造血生理学，特别是如果与人体干细胞是可以发展的。最近，原始造血干细胞已经从鼠和人样品培养了CFU-Blast;这些细胞可以代表接近全能干细胞的细胞，增殖和分化的能力。人和鼠（来自用5-氟尿嘧啶[5-FU]）造血细胞预处理的动物，收获并在半固体培养基中培养。 10到16天后，原始、无颗粒、中等大小、无活性、未分化的菌落 “胚”细胞将从甲基纤维素中取出，能力将被确定。利用缺陷型逆转录病毒介导的基因转移作为克隆性的标志，自我更新能力和将确定该早期干细胞的增殖潜力。因为在体内研究将评价收获的原始细胞的重建致死性辐照的同基因或同种异体小鼠的能力，和有缺陷的突变小鼠。分化和承诺的阶段将CFU-Blast与CFU-Spleen进行比较。爆炸将是从体外培养物中收获并注射到致死辐射的小鼠中并且任何可检测到的脾集落的数量和谱系定型将被确定。类似地，来自5-FU处理的供体的脾集落收获骨髓细胞并接种在半固体培养基中，确定是否可以获得CFU-Blast衍生的集落。虽然不能进行类似的人体体内研究，平行的体外研究的功能将被执行。半固体培养基中的短期培养物在淋巴和骨髓条件下将评估全能性这个细胞的特性。人和鼠胚集落细胞也将被用于启动和接种现有的长期连续骨髓培养物，以及将测定它们从这些培养物中的回收率。这些目标研究是为了确定真正的增殖和自我更新能力， CFU-Blast，探讨其在造血中的作用，并平行开发为将来研究正常和异常造血提供模型。