Culture of bone marrow CD105+ cells allows rapid selection of pure BM-stromal cells for chimerism studies in patients undergoing allogeneic bone marrow transplantation (original) (raw)

2005, Bone Marrow Transplantation

Stromal tissue derived from adult bone marrow (BM) contains clonogenic progenitor cells (CFU-F), some of which are considered to be multi-potent MSCs, capable of differentiating into a range of mesenchymal cell lineages. Current methods for the isolation of BM-MSCs rely upon the rapid adhesion of the stromal progenitor populations to tissue culture plastic and their subsequent rapid proliferation in vitro, 2 resulting, however, in a heterogeneous starting population of adherent BM cells. A significant proportion of the latter represent adherent monocytic cells, the major cause of false-positive results in studies investigating the origin of BM-stromal cells following SCT. In addition, BM-MNCs from patients post allogeneic transplantation show a significant impairment in the ability to generate confluent SC-layers in long-term Dexter-type cultures preventing molecular assessment of chimerism. Therefore, studies based on these methods are limited by monocyte-macrophage contamination and defective SC growth. Recent studies have demonstrated that positive selection using a commercialized anti-endoglin (CD105) antibody enables to obtain homogenous SC-cultures to be obtained without contamination with hematopoietic-derived cells. We hypothesized that cultures initiated with immunomagnetically isolated BM-CD105 þ cells, a cell fraction enriched in mesenchymal progenitor cells (MPC), from patients after hematopoietic SCT would efficiently generate SC-layers without cells of hematopoietic origin, suitable for accurate SC-chimerism analysis. We chose to use the anti-CD105 monoclonal antibody for MPC enrichment for two reasons: First, it was the only commercially available antibody for direct immunomagnetic positive selection of MPCs and secondly preliminary work in our lab has shown that a sample of 4-5 ml BM would be enough for obtaining SC-layers in cultures supplemented with bFGF. We obtained 4-6 ml BM samples from 12 patients 1-24 months post matched sibling allogeneic SC. Five of these patients had suffered from b-thalassemia and the rest from malignant hematological diseases. Initially, we performed immunomagnetic isolation of BM-CD105 þ cells with a cell recovery rate of 0.270.12%. Two immunophenotypic types of CD105 þ cells were detected: (1) CD105 þ GlycophorinA þ CD45 À (22.574.2%), 'erythroid cells' phenotype, (2) CD105 þ GlycophorinA À CD45 low/moderate (20.676.8%), 'mesenchymal cells' phenotype. Further flow cytometric analysis of MACS-isolated cells demonstrated no CD14, CD19, CD31, or CD10 expression. Light microscopic examination of cytospins prepared with freshly