Phenotypic and Molecular Characterization of Bone Marrow L-NGFR + Mesenchymal Stem Cells.

In a previous report we demonstrated that the immunomagnetic sorting of bone marrow (BM) cells labeled with low-affinity nerve growth factor receptor (L-NGFR) antibodies allows the selection of phenotypically and functionally homogeneous cells that are capable of expansion, self-renewal and differentiation into multiple mesenchymal cells lineages. Furthermore, we reported the presence of a subpopulation of L-NGFR+ cells coexpressing CD133 and CD34, markers associated with a primitive hematopoietic stem cell phenotype. In the present study we expanded on the phenotypic characterization of these cells and investigated their potential for multilineage differentiation.

BM L-NGFR+ cells were analyzed by flow cytometry immediately after immunoseparation and the expression of a variety of stem cell markers was studied. In 12 subsequent experiments L-NGFR+ cells expressed CD45low (97.5% ±3), CD34 (19.9%±13), CD133 (10.4%±6), CD105 (46.8%±36%), P1H12 (50.5%±18), KDR (34%±18) and SSEA-3 (0.47%±0.41). In addition L-NGFR+ expressed high levels of the SCF ligand CD117 (40%±16%). As we previously demonstrated, L-NGFR antibodies identify a subpopulation of cells with a high proliferative capacity and potential for multilineage differentiation along the mesenchymal lineage. We now show, in accordance to these phenotypic data, that the L-NGFR+ cells in the presence of SCF (100 ng/ml) doubled the number of CFU-F and expanded both adipocytic and osteoblastic differentiation in comparison to mesenchymal cultures without growth factors or supplemented with Flt-3L+IL-6 (both 100 ng/ml). SCF seems therefore to act at least as a survival/proliferation factor for mesenchymal stem cells.

Transdifferentiation potentialities towards endothelium were determined incubating L-NGFR+ cells in M199 supplemented with 10% FBS, 50 ng/ml VEGF, 1 ng/ml bFGF and 2 ng/ml IGF-1. At confluence, the cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads and expanded with VEGF. Immunophenotypic analysis of 8 samples showed a variable expression of endothelial markers: P1H12 ranging from 17 to 58%, CD105 from 98–100% and CD202b from 18 to 100%.

L-NGFR+ cells, immediately after immunoseparation, were expressing Desmin but not MyoD, Miogenin, Mrf4, Myf5 by means of RT-PCR, while these cells were expressing NSE, TRKA and GalC, but not Nestin and GFAP. Experiments are ongoing to demonstrate muscle and neuron-glial differentiation in vitro using specific media (DMEM 10% FBS + 3 mM %-azacytidine, astrocyte conditioned media, neural stem cell conditioned media),

In conclusion, the expression on NGFR+ cells of a variety of markers, not exclusively related to the mesenchymal lineage, and the reproducible ability to differentiate endothelial cells suggest that these cells may represent a subset of adult MSC with some multipotentiality.