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)

Abstract

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

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