Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stromal cells from patients with hematologic disorders: correlation with bone marrow microenvironment.

The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alterations in the expression of CD105, CD90, CD184, and HLA-DR molecules. The decrease in the percentage of CD105+ and CD90+ MSC correlated with an increased bone marrow angiogenesis. This paper provides evidence that multiparametric flow cytometry is essential for the establishment of a standardized protocol to identify various MSCs subsets and aberrant phenotypes.

"In vitro" evaluation of bone marrow angiogenesis in myelodysplastic syndromes: a morphological and functional approach.

In myelodysplastic syndromes (MDS), ineffective hematopoiesis and cytopenia may arise either from increased susceptibility to apoptosis of hematopoietic cells, or possibly from alterations of bone marrow stroma that contributes to defective development of marrow lineages. In order to test the significance of the endothelial growth in MDS patients, two in vitro assays are presented in this study: a long-term culture method for the detection of human bone marrow endothelial colonies (CFU-En) (77 patients) and a human primary model for the evaluation of the influence of a "bone marrow conditioned medium" on the formation of new vessels in a culture matrix ("angio-kit assay") (in 24 out 77 patients). The in vitro growth pattern of bone marrow CFU-En as well as the generation of microvessels in the angio-kit system was increased in RA, RARS and RAEB in comparison with controls. No CFU-En were detected in RAEB-T. The occurrence of large islands, formed by clusters of endothelial cells, unable to generate microcapillaries was also reported. Chromosomal abnormalities did not correlate with the angiogenetic pattern. These in vitro assays may constitute an useful approach to the analysis of angiogenesis in MDS.

CXCR-4 expression on bone marrow CD34+ cells prior to mobilization can predict mobilization adequacy in patients with hematologic malignancies.

To investigate the mechanisms of mobilization and of the factors implicated in the homing of progenitors and possibly understand the reasons for unpredicted mobilization failure, we analyzed CXCR-4 (CD184) expression on bone marrow (BM) CD34+ cells prior to peripheral blood stem cell (PBSC) mobilization in 24 patients affected by hematologic malignancies (non-Hodgkin lymphoma, multiple myeloma, and acute myeloid leukemia). We wanted to determine whether the level of CXCR-4 expressed by hematopoietic stem cells could influence mobilization process and therefore could be considered a predictive factor for mobilization adequacy. These data were also compared with stromal cell function as assessed by colony forming unit-fibroblast (CFU-F) and CFU endothelial cells (CFU-En) assays and stromal layer confluence capacity exhibited by patients' BM cells. In this study, we also compared CXCR-4 expression on CD34+ cells from different sources and at different migration stages specifically bone marrow (BM), steady state peripheral blood (SSPB), fetal cord blood (FCB), cord blood (CB), and mobilized PBSC. Seven (29%) of the 24 patients undergoing mobilization failed to achieve an adequate number of CD34+ stem cells (5 x 10(6)/kg CD34+ cells) and showed a very high expression frequency of CXCR-4 on BM CD34(+) stem cells (mean number of positive cells, 97%) investigated before the mobilization regimen. We also found that high expression intensity per cell for CXCR-4 was associated with lower amounts of mobilized CD34+ cells whereas those patients (17 out of 24 patients, 71%) with lower expression intensity per cell of CD184 on BM CD34+ cells prior to mobilization harvested at least 5 x 10(6)/kg CD34+ cells. Setting a cut off of 5 x 10(6)/kg CD34+ cells harvested, patients mobilizing less had a mean value of 97% CD34+ cells expressing CXCR-4 with a relative mean channel fluorescence of 458 whereas patients mobilizing more than 5 x 10(6)/kg CD34+ progenitors showed a mean value of 59.8% CD34+/CXCR4+ cells with a relative mean channel fluorescence value of 305. Interestingly, in the poor mobilizers group, the marrow stromal microenvironment was found to be more severely damaged in comparison with that of good mobilizers. The comparative analysis of CXCR-4 expression showed no difference in percentage values between steady-state PB (87.4%) and BM (85.1%) stem cells whereas mobilized CD34+ stem cells have a lower expression frequency of CXCR-4 (71.6%) compared to that of progenitors from other sources. Fetal blood CD34+ stem cells had the lowest mean expression frequency of CD184 antigen (36.3%), while CB cells had the highest (94.8%). In conclusion, this study provides evidence that monitoring CXCR-4 CD34 double positive cells before mobilization can be regarded as a predictive factor for mobilization outcome, giving us directional cues for the choice of the best stem cell mobilization regimens.