Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells

Abstract

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 D7‐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.