Bodger M P
Exp Hematol. 1987 Sep;15(8):869-76.
A two-stage procedure to enrich hemopoietic progenitor cells from human umbilical cord blood is described. Mononuclear cells isolated from 60% Percoll gradients were first treated with a panel of monoclonal antibodies and complement to remove lymphoid and myeloid cells and granulocyte-macrophage colony-forming cells (CFU-GM) and their immature progeny. Cells stained with monoclonal antibody RFB-1 were separated on the fluorescence-activated cell sorter and cultured in the mixed-colony assay. Progenitor cells were isolated in a high RFB-1 antigen density cell fraction containing greater than 95% undifferentiated blast cells and 32% +/- 12% colony-forming cells. Pluripotential progenitor cells (CFU mix) were enriched 229-fold and accounted for 3.2% +/- 1.2% of the isolated cells. Erythroid progenitor cells (BFU-E) were enriched 204-fold and accounted for 20.8% +/- 8.4% of the isolated cells. The procedure recovered 114% +/- 32% of CFU mix but the number of CFU mix in unseparated cord blood appears to be underestimated due to the presence of granulocytic cells identified by monoclonal antibody CMRF-7 (CD15) that decrease the cloning efficiency of CFU mix. Removal of CD15+ cells yielded a four- to fivefold improvement in the plating efficiency of CFU mix. Mixed-colony formation was completed in 9-11 days in cultures inoculated with enriched progenitor cell fractions, compared with 14-16 days in cultures of unseparated cord blood. The enriched progenitor cells could be useful for studying the regulation of hemopoiesis by recombinant growth factors.
本文描述了一种从人脐带血中富集造血祖细胞的两阶段程序。从60% Percoll梯度中分离出的单核细胞首先用一组单克隆抗体和补体处理，以去除淋巴细胞、髓细胞以及粒细胞-巨噬细胞集落形成细胞（CFU-GM）及其未成熟后代。用单克隆抗体RFB-1染色的细胞在荧光激活细胞分选仪上进行分离，并在混合集落测定中培养。祖细胞在高RFB-1抗原密度细胞组分中分离得到，该组分含有大于95%的未分化母细胞和32%±12%的集落形成细胞。多能祖细胞（CFU mix）富集了229倍，占分离细胞的3.2%±1.2%。红系祖细胞（BFU-E）富集了204倍，占分离细胞的20.8%±8.4%。该程序回收了114%±32%的CFU mix，但由于单克隆抗体CMRF-7（CD15）识别的粒细胞的存在降低了CFU mix的克隆效率，未分离脐带血中CFU mix的数量似乎被低估了。去除CD15+细胞后，CFU mix的接种效率提高了4至5倍。接种富集祖细胞组分的培养物中，混合集落在9至11天内形成，而未分离脐带血培养物中则为14至16天。富集的祖细胞可用于研究重组生长因子对造血的调节作用。
