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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.

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.

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.

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35) - except for samples having a leukocyte count >25 x 10 9 /L or a CD34 count

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/10(6) BM mononucleated cells in untreated MM samples (2.07 s.d. +/- 1.3) was significantly higher than in normal BM (0.28 +/- 0.48), while no difference was seen between normal BM and MGUS (0.28 +/- 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 +/- 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 +/- 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.

Objective The aim of this study was to evaluate which CD34 + cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT). Materials and Methods Based on data from 34 patients with hematologic malignancies, doses of CD34 + cells and CD34 + cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (α subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34 + cell growth was comparatively evaluated in the LTC assay. Results Our data demonstrated that some of the most primitive progenitor subsets (CD34 + CD117 − HLA-DR − , and CD34 + CD38 + HLA-DR − ) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34 + CD117 − HLA-DR − phenotype; long-term erythrocyte engraftment was correlated with CD34 + CD38 + HLA-DR − cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34 + CD38 + HLA-DR − cell subtype and the total LTC-CFC quantity infused. Conclusions These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34 + cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34 + cell quantity.