Functional and immunophenotypic characteristics of isolated CD105+ and fibroblast+ stromal cells from AML: implications for their plasticity along endothelial lineage

In vitro cultures of BM cells firm newly diagnosed patients with AML displayed a defective BM stromal compartment, with a reduced number offibroblast-colony-forming unit (CFU-F: 1±1.25 SD) and a decreased proliferatrve ability. The purposes of our study were: 1) to select BM mesencbymal stem cells (MSC) and BM-derived stromal cells (BMDSCs) from AML patients at diagnosis and from healthy subjects, using an immunomagnetic system and either anti-CD105 or anti-fibroblast MAbs; 2) to study the immunophenotypic and functional properties of freshly isolated and cultured mesencbymal cells; 3) to test the in vitro plasticity of the selected cells to differentiate towards an endothelial phenotype. Fresh mononuclear cells obtained from BM of 20 patients newly diagnosed with AML and from eight healthy subjects were selected by using anti-fibroblast and anti-CD105 MAbs. Freshly isolated cells were analyzed, characterized by flow cytometry using a wide panel of MAbs and seeded in long-term culture medium to assess CFU-F formation. The level of confluence after 30 days and functional capacity in a long-term colony-forming cell culture (LTC-CFC) were tested. Furthermore, the cultured selected cell populations were assayed for their ability to differentiate into an endothelial-like cell pbenotype with the addition of vascular endothelial growth factor (VEGF) and endothelial cell growth supplement (ECGS). In normal subjects the selection produced an increase of the CFU-F number of 2.6-fold with anti-fibroblast MAb and 2.1-fold with the anti-CDIOS MAb. Anti-fibroblast and anti-CD105 MAb selection from AML BM cells resulted in a statistically significant greater count ofCFU-F that was respectively 10.6-fold (P =0.04) and 14.4-fold (P =0.00001) higher in comparison with the unsdected AML samples. Interestingly, in 80% of AML samples immunoselection was also able to restore the capacity of the CFU-F to proliferate and form confluent stromal layers. The isolation of those layers sustained the proliferation and differentiation of bematopoietic stem cells in the LTC-CFC. The phenotypic profile of cultured BMDSCs was different from that of the freshly isolated cells, and changed in relation to the culture conditions: CD105+ selected cells cultured with VEGF and ECGS expressed endothelial markers, a finding that suggests that this cell subpopulation may have the potential to differentiate toward an endothelial-like phenotype. We report that immunomagnetic selection represents a val