Search

The distribution and functional characteristics of in vitro bone marrow (BM) endothelial colonies (CFU-En) were studied in 70 non-Hodgkin's lymphoma (NHL) patients in different phases of the disease to explore the association between CFU-En growth and angiogenesis, and between the number of CFU-En and the presence of hematopoietic and mesenchymal progenitor cells. The mean number of CFU-En/10(6) BM mononuclear cells seen in remission patients was significantly higher than that seen in newly diagnosed patients (P=0.04), and in normal subjects (P=0.008). Patients with low-grade NHL in remission displayed a higher CFU-En value compared with high-grade NHL (P=0.04). In the autograft group (40 patients), a significant reduction of CFU-En number was detected in the first 4-6 months after transplantation. In remission patients, the CFU-En number positively correlated with the incidence of BM colony-forming unit granulocyte-macrophage (CFU-GM) (P=0.013) and CFU-multilineage (CFU-GEMM) hematopoietic colonies (P=0.044). These in vitro data show that CFU-En numbers increase following standard-dose chemotherapy, thus providing a rationale for further investigating the effects of different cytostatic drugs on BM endothelial cells growth and function.

Background 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. Methods 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). Results 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. Discussion We report that immunomagnetic selection represents a valid tool for the selection of BM mesencbymal cells in samples obtained from both healthy subjects and patients with AML. This technique -was able to rescue two functional and immunophenotypic compartments related to two different selected populations. In particular, the CD105 + cells isolated in AML displayed, after stimulation with VEGF and ECGS, the ability to change towards an endothelial-like cell phenotype, thus revealing an unexpected plasticity. Both CD105 + and fibroblast + cells once successfully isolated might represent sources of mesencbymal cells populations useful for in vitro investigations and, above all, as therapeutic devices.