Juan José Montesinos

In vitro characterization of hematopoietic microenvironment cells from patients with myelodysplastic syndrome.

In vitro studies on the functional integrity of the hematopoietic microenvironment in myelodysplasia have been controversial. Although some of them suggest that such a microenvironment is functionally normal, there is increasing evidence indicating that there are alterations in the function of microenvironment (adherent) cell layers from myelodysplastic syndromes (MDS) marrow. Adherent cell layers developed in vitro, however, consist of a mixture of different cell types-mostly fibroblasts and macrophages-thus, it is not clear which cell type(s) is(are) functionally abnormal in this disorder. In order to address this issue, in the present study, we first assessed some functional properties of MDS-derived adherent cell layers, as a whole, and then we analyzed those same functional properties after separating these cells into two different populations: a fibroblast-enriched cell layer and a macrophage-enriched cell layer. When whole adherent layers from MDS patients were analyzed, no significant differences were observed, as compared to their normal counterparts, in terms of morphology and total cell number. A major difference, however, was observed when analyzing the production of the cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF-alpha). Indeed, adherent layers from MDS patients produced higher levels of these cytokines (2- and 22-fold, respectively), as compared to normal layers. When fibroblast- and macrophage-enriched cell layers were analyzed, a higher apoptotic index was observed in those derived from MDS marrow (4% of TUNEL-positive cells in normal fibroblast layers versus 27% in MDS-derived fibroblast layers; 7% of TUNEL-positive cells in normal macrophage layers versus 24% in MDS macrophage layers). Macrophages from MDS marrow produced significantly higher levels of TNF-alpha (nine-fold) than their normal counterparts. MDS-derived fibroblasts, on the other hand, produced higher levels of IL-6 (nine-fold), as compared to normal fibroblasts. Surprisingly, whereas normal fibroblasts showed a discrete production of TNF-alpha, we found a very high production of this cytokine in cultures of fibroblasts from MDS patients. In summary, in the present study we have demonstrated that, at least in vitro, both fibroblasts and macrophages from MDS bone marrow (BM) are functionally abnormal. Such abnormalities include an increased apoptotic index, as well as a high production of both IL-6 and TNF-alpha.

Immunoregulation by Mesenchymal Stem Cells: Biological Aspects and Clinical Applications

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into mesenchymal lineages and that can be isolated from various tissues and easily cultivated in vitro. Currently, MSCs are of considerable interest because of the biological characteristics that confer high potential applicability in the clinical treatment of many diseases. Specifically, because of their high immunoregulatory capacity, MSCs are used as tools in cellular therapies for clinical protocols involving immune system alterations. In this review, we discuss the current knowledge about the capacity of MSCs for the immunoregulation of immunocompetent cells and emphasize the effects of MSCs on T cells, principal effectors of the immune response, and the immunosuppressive effects mediated by the secretion of soluble factors and membrane molecules. We also describe the mechanisms of MSC immunoregulatory modulation and the participation of MSCs as immune response regulators in several autoimmune diseases, and we emphasize the clinical application in graft versus host disease (GVHD).

Functional analysis of myelodysplastic syndromes-derived mesenchymal stem cells.

Two different reports, including one from our own group, have recently demonstrated the presence of severe chromosomal abnormalities in mesenchymal stem cells (MSC) from patients with myelodysplastic syndromes (MDS). In the present study, we have assessed whether such cytogenetic abnormalities result in functional deficiencies in vitro. We found that both normal and MDS MSC showed similar expression patterns of cell adhesion molecules and extracellular matrix proteins. MDS MSC layers showed the capability to differentiate towards adipocytes, chondrocytes and osteoblasts, and supported the growth of early umbilical cord blood progenitors in a co-culture system. Unstimulated MDS MSC secreted more IL-1beta and after treatment with TNFalpha, they secreted more SCF, as compared to their normal counterparts. The present study demonstrates that, in spite of harboring severe chromosomal alterations, most of the functional properties of MDS-derived MSC remain normal, including their ability to support normal hematopoiesis in vitro.

Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood.

BACKGROUND AIMS We have previously characterized the in vitro growth of two cord blood-derived hematopoietic cell populations in liquid cultures supplemented with recombinant cytokines. In the present study, we assessed the effects of bone marrow-derived mesenchymal stromal cells (MSC) on the growth of such cells. METHODS CD34(+) CD38(+) Lin(-) and CD34(+) CD38(-) Lin(-) cells were obtained by negative selection, and cultured in the presence of marrow-derived MSC and/or early- and late-acting cytokines. Hematopoietic cell growth was assessed throughout a 30-day culture period. RESULTS In the presence of MSC alone, both populations showed significant proliferation. Direct contact between MSC and CD34(+) cells was fundamental for optimal growth, especially for CD34(+) CD38(-) Lin(-) cells. In the presence of early-acting cytokines alone, cell growth was significantly higher than in cultures established with MSC but no cytokines. In cultures containing both MSC and early-acting cytokines, a further stimulation was observed only for CD34(+) CD38(-) Lin(-) cells. The cytokine cocktail containing both early- and late-acting cytokines was significantly more potent at inducing hematopoietic cell growth than the early-acting cytokine cocktail. When cultures were supplemented with early- and late-acting cytokines, MSC had no further effect on the growth of hematopoietic cells. CONCLUSIONS MSC seem to play a key role, particularly on more primitive (CD34(+) CD38(-) Lin(-)) cells, only in the absence of cytokines or the presence of early-acting cytokines. When both early- and late-acting cytokines are present in culture, MSC seem to be unnecessary for optimal development of CFC and CD34(+) cells.

Comparative analysis of the in vitro proliferation and expansion of hematopoietic progenitors from patients with aplastic anemia and myelodysplasia

Aplastic anemia (AA) and myelodysplasia (MDS) show great similarities in their biology. To date, however, it is still unclear to what extent hematopoietic progenitor cells (HPCs) from AA and MDS share biological properties and what the functional differences are between them. In trying to address this issue, in the present study we have analyzed, in a comparative manner, the proliferation and expansion capacities of bone marrow (BM) progenitor cells from AA and MDS in response to recombinant cytokines. BM samples from normal subjects (NBM) and patients with AA and MDS were enriched for HPC by immunomagnetic-based negative selection. Selected cells were cultured in the absence (control) or in the presence of early-acting cytokines (Mix I), or early-, intermediate- and late-acting cytokines (Mix II). Proliferation and expansion were assessed periodically. In NBM and MDS cultures apoptosis was also determined. In NBM cultures, Mix I induced a nine-fold increase in total cell numbers and a 3.6-fold increase in colony-forming cell (CFC) numbers. In Mix II-supplemented cultures, total cells were increased 643-fold, and CFC 12.4-fold. In AA cultures, no proliferation or expansion were observed in Mix I-supplemented cultures, whereas only a four-fold increase in total cell numbers was observed in the presence of Mix II. In MDS cultures, a 12-fold increase in total cells and a 2.9-fold increase in CFC were observed in the presence of Mix I; on the other hand, Mix II induced a 224-fold increase in total cells and a 5.9-fold increase in CFC. Apoptosis was reduced in cytokine-supplemented cultures from NBM. In contrast, Mix II induced a significant increase in the rate of apoptosis in MDS cultures. Our results demonstrate that, as compared to their normal counterparts, AA and MDS progenitors are deficient in their proliferation and expansion potentials. Such a deficiency is clearly more pronounced in AA cells, which seem to be unable to respond to several cytokines. MDS progenitors, on the other hand, are capable to proliferate and expand in response to cytokines; however, their rate of apoptosis is increased by intermediate- and late-acting cytokines, so that the overall proliferation and expansion are significantly lower than those of normal progenitor cells.

In vitro evidence of the presence of mesenchymal stromal cells in cervical cancer and their role in protecting cancer cells from cytotoxic T cell activity.

Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSC populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papilloma virus, indicating that these cells are not infected by such a viral agent. Also, interestingly, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with the production of IL-10 in cell cocultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing downregulation of HLA class I molecules. This mechanism may have important implications in tumor growth.

Growth kinetics of progenitor cell-enriched hematopoietic cell populations in long-term liquid cultures under continuous removal of mature cells

BACKGROUND During long-term culture of primitive hematopoietic cells large numbers of mature cells are generated that, on the one hand, consume nutrients and cytokines present in the medium and, on the other hand, may produce or elicit the production of soluble factors that limit the growth of primitive cells. Thus it is possible that under standard culture conditions hematopoietic stem and progenitor cells are unable to display their true proliferation and expansion potentials. METHODS Hematopoietic cell populations, enriched for CD34+ cells, were obtained from both umbilical cord blood (UCB) and mobilized peripheral blood (MPB), and cultured in cytokine-supplemented liquid culture, under continuous removal of mature cells by means of weekly re-selection of primitive, lineage-negative (Lin-) cells. Proliferation and expansion capacities of such cells were determined weekly for a 42-day culture period. RESULTS As expected, based on our previous studies in standard liquid cultures, throughout the culture period there was a continuous decrease in the proportion of progenitor cells; however, after every re-selection on days 7, 14 and 21, there was a significant enrichment for both CD34+ cells and colony-forming cells (CFC). As a result of such an enrichment, the cumulative increase in the numbers of total cells and CFC in cultures with two, three or four selections was significantly higher than the increments observed in standard cultures, in which only a single selection was performed on day 0. Cultures of UCB cells showed consistently higher levels of both total cells and CFC than cultures of MPB cells. DISCUSSION Taken together, these results indicate that continuous removal of mature cells from liquid cultures of primitive progenitors results in higher increments in the levels of both total cells and CFC.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

BackgroundIn recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other “classic” sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway.MethodsWe determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado).ResultsWe detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides.ConclusionsThis study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

Deficient proliferation and expansion in vitro of two bone marrow cell populations from patients with acute myeloid leukemia in response to hematopoietic cytokines.

One has previously characterized two different hematopoietic cell populations (obtained by negative-selection) from normal bone marrow. Population I was enriched for CD34+ Lin− cells, whereas Population II was enriched for CD34+ CD38− Lin− cells. Both populations showed elevated proliferation and expansion potentials in serum-free liquid cultures, supplemented with a combination of eight different cytokines, with the latter displaying more immature features than the former. One has also characterized the chronic myeloid leukemia (CML) counterparts of these two populations and demonstrated functional deficiencies in terms of their growth in culture. In keeping with this line of research, the goal of the present study was to obtain the same two populations (Populations I and II) from acute myeloid leukemia (AML) bone marrow and to characterize their biological behavior under the same culture conditions. The results demonstrated that AML-derived Populations I and II were unable to proliferate in culture conditions that allowed significant proliferation of Populations I and II from normal marrow. Population I from AML also showed a deficient expansion capacity; in contrast, Population II cells were able to expand to a similar extent to the one observed for Population II from normal marrow. Both normal and AML populations were highly sensitive to the inhibitory effects of TNF-α; interestingly, whereas in normal fractions TNF-α showed a more pronounced inhibitory effect on more mature cells (Population I), this cytokine inhibited proliferation and expansion of AML Populations I and II in a similar degree. It is noteworthy that the functional deficiencies observed in AML cells were even more pronounced than those previously reported for cultures of CML cells. The results reported here may be of relevance considering the interest by several groups in developing methods for the in vitro purging of leukemic cells, as part of protocols for autologous transplantation of hematopoietic cells in leukemic patients.

Effect of rhGM-CSF on the kinetics of hematopoiesis in long-term marrow cultures from patients with acute myelogenous leukemia.

In the present study, we have assessed the effects of recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) in Dexter-type long-term marrow cultures (LTMC) from patients with acute myelogenous leukemia (AML). Addition of rhGM-CSF to AML LTMC resulted in a significant increase in the number of total nucleated cells (1.3-4.3-fold, as compared to untreated cultures). However, a simultaneous decrease in the numbers of myeloid progenitor cells (CFU) was observed. Interestingly, there was a selective stimulation of the growth of leukemic progenitors (AML-CFU). Indeed, whereas on day 0 these cells were detected in only 2 patients, between weeks 1 and 5 they were detected in 10 of the 14 patients included in the study. It is noteworthy that around 50% of the cells detected in the non-adherent fraction of rhGM-CSF-treated AML LTMC were blasts, whereas in untreated cultures, blasts corresponded to only 23% of the non-adherent cells, and the majority corresponded to cells of the monocyte-macrophage lineage. These results indicate that rhGM-CSF is a cytokine with a significant stimulatory activity for the in vitro growth of AML progenitor and blast cells, and, together with previous reports in the literature, suggest that the use of rhGM-CSF in clinical settings must be taken with caution since this cytokine, although beneficial in reducing the risk of infections after chemotherapy, may induce the reappearance of the disease after treatment. Further studies should be encouraged to understand in greater detail the effects of rhGM-CSF, and other cytokines, on the hematopoietic system of AML patients.