Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells
Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells
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DOI：
10.1002/art.10696
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发表时间：
2002-12-01
影响因子：
--
通讯作者：
McGonagle, D
中科院分区：
文献类型：
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作者：
Jones, EA;Kinsey, SE;McGonagle, D
Objective.间充质祖细胞/干细胞（MPCs）及其在风湿性疾病中的作用越来越受到风湿病学的关注，但对这些细胞在体内的表型知之甚少。本研究的目的是分离和表征人骨髓（BM）MPC。荧光显微镜用于鉴定粘附BM细胞中推定的MPC。为了纯化它们，使用抗成纤维细胞微珠的阳性选择，结合微珠+、CD 45（低）细胞的荧光激活细胞分选（FACS）。采用四色流式细胞术测定这些细胞的更详细的表型，并采用标准的软骨形成、成骨和成脂试验研究其分化潜能。采用W-FIB偶联（抗成纤维细胞）微珠进行阳性选择，然后通过FACS对特异性结合的微珠+、CD 45（低）细胞进行纯化，从而对显微镜下鉴定为大的成纤维细胞样D 7-FIB+细胞的推定MPC进行纯化。这些细胞占BM中单核细胞的0.01%。CD 105、LNGFR、HLA-DR、CD 10、CD 13、CD 90、STRO-1和骨形态发生蛋白受体IA（BMPRIA）均呈阳性，CD 14、CD 34、CD 117和CD 133均呈阴性。只有具有这种表型的细胞才能增殖并产生能够分化成软骨、成骨和成脂的贴壁细胞单层。BM中的D 7-FIB-细胞缺乏任何MPC活性。未培养的皮肤成纤维细胞具有与BM MPCs相似的表型，但LNGFR、STRO-1、HLA-DR和BMPRIA均为阴性。本研究显示了不同的表型，形态学和方法的BM MPC的分离。这一发现可能对确定MPCs在关节炎、骨疾病和关节再生中的生理作用有意义。
Objective. There is an increased interest in rheumatology in mesenchymal progenitor/stem cells (MPCs) and their roles in rheumatic diseases, but little is known about the phenotype of these cells in vivo. The aim of this study was to isolate and characterize human bone marrow (BM) MPCs.Methods. Fluorescence microscopy was used to identify putative MPCs among adherent BM cells. To purify them, a positive selection with antifibroblast microbeads was used, combined with fluorescence-activated cell sorting (FACS) for microbead+,CD45(low) cells. A more detailed phenotype of these cells was determined using 4-color flow cytometry, and standard chondrogenic, osteogenic, and adipogenic assays were used to investigate their differentiation potentials.Results. Putative MPCs microscopically identified as large, fibroblast-like, D7-FIB+ cells were purified using positive selection with W-FIB-conjugated (antifibroblast) microbeads followed by FACS for specifically bound microbead+,CD45(low) cells. These cells represented 0.01% of mononuclear cells in the BM. They were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, STRO-1, and bone morphogenetic protein receptor type IA (BMPRIA) and were negative for CD14, CD34, CD117, and CD133. Only cells with this phenotype could proliferate and produce adherent cell monolayers capable of chondrogenic, osteogenic, and adipogenic differentiation. D7-FIB- cells in the BM lacked any MPC activity. Uncultured skin fibroblasts had a phenotype similar to that of BM MPCs, but were negative for LNGFR, STRO-1, HLA-DR, and BMPRIA.Conclusion. This study shows the distinct phenotype, morphology, and method of isolation of BM MPCs. The findings may have implications for defining the physiologic roles of MPCs in arthritis, bone diseases, and joint regeneration.