Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.

Abstract

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into multiple mesodermal tissues. We previously reported that monoclonal antibodies to the low-affinity nerve growth factor receptor (alpha-LNGFR) stain bone marrow (BM) mesenchymal cells. We now show that LNGFR antibodies label primitive MSCs with high specificity and purity in adult BM, and compare these cells to those isolated by plastic adherence (PA) and CD45(-)anti-glycophorin A(-) selection. Low-density mononuclear cells (LD-MNCs) from normal BM were separated by PA or immunomagnetic selection for NGFR(+) or CD45(-)alpha-glycophorin A(-) cells. The three fractions were grown in Iscove's modified Dulbecco medium + 20% fetal bovine serum +/- basic fibroblast growth factor (bFGF) in order to assess their proliferative capacity and evaluate their phenotype during culture. The clonogenic potential of the MSCs was assessed using a colony-forming unit fibroblast (CFU-F) assay, whereas multipotential differentiation was determined after culture in adipocytic and osteoblastic conditioned media. The NGFR(+) mesenchymal cells grown without growth factors showed persistent NGFR expression (rapidly down-regulated after the addition of bFGF) and persistent CFU-F activity. The NGFR(+) fractions were rich in clonogenic precursors: CFU-F median frequency was 1584/1 x 10(6) cells (range 325-13,793) in the NGFR(+) cells and 35/1 x 10(6) cells (range 27-112) in the LD-MNCs. The NGFR(-) fraction never showed any residual CFU-F activity. Compared with the other two fractions, the NGFR(+) cells (+/- bFGF) showed a 1 to 3 log greater expansion in the number of fibroblastic cells and a greater capacity to give rise to adipocyte colonies and induce osteoblastic differentiation, and they had similar effects in supporting the growth of hematopoietic precursors. The data suggest that positive selection using low-affinity NGFR antibodies makes it possible to obtain homogeneous multipotent MSCs.