EXPANSION OF HUMAN BONE MARROW CELLS CAPABLE OF REPOPULATING IN MICE BY COCULTURE WITH BONE MARROW STROMAL CELLS

我们测试了纯化骨髓细胞与基质细胞共培养对NOD/SCID小鼠再生能力的影响。基于CD34、Kit受体和CD38的表达，利用FACS Vantage对原始人骨髓细胞进行纯化。用培养皿贴壁法培养骨髓基质细胞。用基质细胞和CTLA4-Ig对小鼠进行预处理，观察人纯化骨髓细胞的植入情况。在基质细胞上培养的原始骨髓细胞可提高集落形成细胞的数量和存活率。在共培养体系中加入干细胞因子、flk2/flt3配体和血小板生成素显著增加了集落形成细胞的数量。然后，我们测试了NOD/SCID小鼠造血因子和基质细胞扩增细胞的再生能力。然而，没有小鼠移植扩增细胞成功植入。这些观察结果表明，需要适当的培养条件来扩增能够在NOD/SCID小鼠中再生的原始骨髓细胞。此外，我们研究了粒细胞集落刺激因子受体（G-CSFR）在原始髓系祖细胞中的表达及其对造血因子的反应，以确定严重先天性中性粒细胞减少症（SCN）患者粒细胞生成缺陷的基础。结果表明，表达G-CSFR的原始胶质样祖细胞的定性和定量异常可能在SCN患者粒细胞生成障碍中起重要作用。

We have tested the effects of coculture of purified bone marrow cells with stromal cells on the capability of repopulating in NOD/SCID mice. Primitive human bone marrow cells were purified based on the expression of CD34, Kit receptor and CD38 using the FACS Vantage. Bone marrow stromal cells were established by the adherence on dishes. The engraftment of human purified bone marrow cells were observed when mice were pretreated with the injection of stromal cells and CTLA4-Ig inducing anergy against human blood cells. The primitive bone marrow cells cultured on the stromal cells enhanced the number of colony-forming cells and the sruvival. The addition of stem cell factor, the ligand for flk2/flt3 and thrombopoietin to the coculture system significantly incrreased the number of colony-forming cells. Then, we tested NOD/SCID mice-repopulating ability of cells expanded by the hematopoietic factors and stromal cells. However, none of mice transplanted with expanded cells showed successful engraftment. These observations suggest the requirement of appropriate culture conditions for expanding the primitive bone marrow cells capable repopulating in NOD/SCID mice.Furthermore, we studied the expression of granulocyte colony-stimulating factor receptor (G-CSFR) in primitive myeloid progenitor cells and their responsiveness to hematopoietic factors to define the basis for the faulty granulopoiesis in patients with severe congenital neutropenia (SCN), . The results demonstrate that the presence of qualitative and quantitative abnormalities of primitive mveloid progenitor cells expressing G-CSFR may play an important role in the impairment of granulopoiesis in patients with SCN.