Decreased frequency, but normal functional integrity of mesenchymal stromal cells derived from untreated and Imatinib-treated chronic myeloid leukemia patients.

In vitro, Imatinib inhibits the proliferation and stimulates the osteogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). However, it is unknown whether Imatinib affects the biology of MSC in vivo. We asked whether MSC from long-term Imatinib-treated CML patients were affected by the in vivo treatment. MSC from untreated and Imatinib-treated patients displayed normal functional properties (i.e. proliferation, immunophenotype, differentiation and hematopoietic supportive capacity) - but a decreased frequency. In vitro, Imatinib lost its effect when discontinued; which suggest that it has a reversible effect on MSC. Therefore it might lose its effect on MSC after discontinuation in vivo.

Human mesenchymal stromal cells from adult and neonatal sources: comparative analysis of their morphology, immunophenotype, differentiation patterns and neural protein expression.

BACKGROUND: Bone marrow (BM) has been recognized as the main source of mesenchymal stromal cells (MSC); however, MSC have also been detected in umbilical cord blood (UCB) and placenta (PL). In the present study, we obtained MSC from these three sources and characterized them in a comparative manner. METHODS: MSC were obtained from BM, UCB and PL samples and analyzed to determine their morphology, cell-surface antigen (Ag) expression and differentiation potential. Particular emphasis was placed on the expression of neural markers. RESULTS: MSC were detected in 9/9, 11/104 and 5/5 samples from BM, UCB and PL, respectively. MSC populations comprised several morphologically distinct cell types, including neural-like cells. MSC were positive for 'mesenchymal' Ag (CD105, CD73 and CD90), although CD90 expression was very heterogeneous. Interestingly, CD13 expression was high in all three sources. In all cases, MSC showed osteogenic and chondrogenic differentiation; however, UCB MSC showed no adipogenic potential. Furthermore, MSC from UCB produced a different type of cartilage compared with MSC from BM and PL. It is noteworthy that in all three sources we detected the expression of neural proteins without any neural differentiation stimuli. A significant increase in the proportion of neural marker-positive MSC was observed in the presence of neural inducers. DISCUSSION: Our results indicate that PL may prove to be a more appropriate source for obtaining MSC than UCB, and suggest the possibility that a subpopulation of MSC may possess neural potential, which is favored by neural inducers.

In vitro characterization of two lineage-negative CD34+ cell-enriched hematopoietic cell populations from human UC blood.

BACKGROUND: During the last few years there has been increasing interest, from both biologic and clinical points of view, in the ex vivo expansion of umbilical cord blood (UCB)-derived hematopoietic cells. This has brought about the need to characterize different cell populations present in UCB, and to explore different ex vivo approaches for the culture, expansion and biologic manipulation of these cells. METHODS: By using a negative-selection method, two UCB cell populations were obtained that were enriched for primitive lineage-negative (Lin-) cells, including those expressing the CD34 Ag (35-93% of the total cells in each fraction). Population I was enriched for CD34+ Lin- cells, whereas population II was enriched for CD34+ CD38- Lin- cells. Both populations were cultured in serum-free liquid cultures supplemented with different combinations of early and late-acting recombinant cytokines (all of them added at 10 ng/mL). Every 5-7 days proliferation, expansion and differentiation capacities of each population were determined, for a total period of 25-42 days. RESULTS: Both cell populations showed extensive proliferation and expansion capacities; however, population II [2300- and 232-fold increase in nucleated and colony-forming cell (CFC) numbers, respectively] was clearly superior in both parameters compared with population I (1120- and 20-fold increase in nucleated and CFC numbers, respectively). Depending on the cytokine combination used, granulocytes, macrophages and erythroblasts were preferentially produced. We also observed that both populations were highly sensitive to the inhibitory effects of tumor necrosis factor-alpha, even in the presence of stimulatory cytokines. DISCUSSION: This study demonstrates that the two progenitor cell-enriched populations obtained by negative selection possess extensive proliferation and expansion potentials in vitro, generating significant numbers of both primitive and mature cells. These cells may be a good alternative to purified CD34+ cells, obtained by positive selection, for pre-clinical and clinical protocols aimed at the ex vivo expansion of UCB cells.

Tumor necrosis factor-alpha levels in long-term marrow cultures from patients with aplastic anemia: modulation by granulocyte-macrophage colony-stimulating factor.

We have previously shown that the levels of hematopoietic progenitors in long-term marrow cultures (LTMC) from patients with aplastic anemia (AA) are drastically reduced, as compared to normal LTMC. We have also reported that when LTMC from AA patients are supplemented with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) there is an increase in colony-forming cell (CFC) levels. However, such a stimulation is only transient and it is followed by an inhibition in CFC growth. Based on these observations, in the present study we have tested the hypothesis that the levels of tumor necrosis factor-alpha (TNF-alpha), an inhibitor of hematopoiesis, are increased in AA LTMC and that such levels are further increased after rhGM-CSF has been added to the cultures for several weeks. Accordingly, we have determined the levels of TNF-alpha in the supernatant of LTMC established from normal (n = 8) and AA (n = 6) bone marrow and in AA LTMC supplemented with rhGM-CSF (n = 6). At the time of culture initiation, TNF-alpha levels were below detection in all the samples analyzed. After 5 weeks of culture, TNF-alpha levels in normal LTMC were very low, with a median of 7.3 pg/mL. In contrast, AA LTMC contained higher levels of TNF-alpha (median of 49.6 pg/mL). In keeping with our hypothesis, addition of rhGM-CSF to AA LTMC resulted in a significant further increase of TNF-alpha levels (median of 135.4 pg/mL). Our results demonstrate an inverse correlation between reduced hematopoiesis in AA LTMC and increased levels of TNF-alpha in this culture system. Based on the results presented here, together with previous reports indicating that TNF-alpha is a potent inducer of apoptosis in hematopoietic progenitor cells, it seems reasonable to suggest that TNF-alpha is implicated in the pathophysiology of AA.

Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long-term proliferation.

It is known that the levels of hematopoietic progenitor cells (HPC) are greatly reduced in the majority of patients with myelodysplastic syndromes (MDS). To date, however, only limited information exists on the growth kinetics of these cells in long-term marrow cultures (LTMC), particularly in terms of erythroid and multipotent progenitors. In the present study, we have determined the HPC content in the bone marrow of 12 MDS patients and followed the proliferation kinetics of myeloid (including granulocyte, macrophage and granulocyte macrophage), erythroid (including early and late) and multipotent progenitor cells in LTMC throughout a 7-week culture period. Both the non-adherent and adherent fractions of the cultures were analyzed, so we were able to look at progenitor cells in suspension and those that physically associated to the stromal cell layer developed in culture. All 12 patients were grouped based on their FAB subtype and the in vitro growth of the HPC was analyzed accordingly. The results presented here indicate that in the majority of MDS patients, pronounced deficiencies exist both in the content and the long-term proliferation of marrow HPC. Such deficiencies were particularly evident for multipotent progenitors and those committed to the erythroid lineage, in which alterations in the maturation process also seem to be present. Our results suggest that, at least in some patients, HPC--besides showing an impaired proliferative capacity--lose their ability to adhere to the stromal cell layers developed in culture. RA patients showed the less affected in vitro HPC growth, whereas HPC from RAEB and RAEB-t showed a markedly deficient growth in culture. Interestingly, myelopoiesis was significantly increased in cultures of CMML patients. These results give some new insights into the biology of MDS-derived HPC.