Mesenchimal stem cells (MSC) are a population of multipotent cells which can be isolated from the adult bone marrow (BM) and retain the capacity to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, tendon, muscle and fat. We previously reported that a monoclonal antibody (MoAb) to the low-affinity nerve growth factor receptor (anti-NGFR) stains BM mesenchimal cells (Cattoretti et al, Blood 81(7):1726-1738,1993). In this study we now describe the mesenchimal stem cells isolated with this antibody and compare them to MSC isolated by plastic adherence and immunomagnetic sorting of CD45 Va-glycoforin cells. Comparison of the MSC obtained with different methods with the same BM cells was performed by the analysis of multiple parameters: immunophenotypic characteristics, clonogenic potential, proliferation and differentiation capability. Low-density mononuclear cells (LD-MNCs) from normal BM were immunomagnetically separated (MACS, Miltenyi Biotech, Germany) with a-NGFR MoAbs, with OC-CD45 plus a-glycoforin MoAbs or by plastic adherence and trypsinization steps and grown in IMDM + 30% FBS. Immunophenotypic analysis by the APAAP technique of cells grown in presence of bFGF showed a population of NGFR derived cells uniformly TE-7V5B5VNGFR/CD14-/CD45', while other methods gave rise to fibroblast cultures sometimes containing residual monocytic cells even after multiple passages. To evaluate the clonogenic potential of MSC, CFU-F were assessed in limiting dilution assays. The frequency of stromal precursors was very high in the NGFR fraction (up to 1 colony/72 cells seeded), but much lower in the CD457oc-glycoforin" population and in MSC obtained from plastic adherence (average frequency of 1 colony/10,000 cells seeded). The NGFR fraction never gave rise to CFU-F colonies. Purified NGFR cells showed fold expansion values up to 1.5xl08 after 2 months of culture, while during the same period the CD45Voc-glicoforin' cells expanded only up to 2.3x106 fold and plastic adherence cells 3xl05 fold. To assess the multipotential capacity of these cells, adipogenic and osteogenic differentiation assays were performed. Anti-NGFR+ cells gave rise to a much higher number of adipocyte colonies (50 colonies/SxlO4 cells seeded) and formed a higher number of aggregates with an increased expression of alkaline phosphatase activity and calcium accumulation when compared to CD45"/cc-glycoforin- and plastic adherence MSC. All these data suggest that the low affinity NGFR is a marker of primitive, multipotent MSC, which can be isolated with high purity and show great expansion potential.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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