Patricia Flores-Guzmán

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.

In Vitro Proliferation, Expansion, and Differentiation of a CD34+ Cell-Enriched Hematopoietic Cell Population from Human Umbilical Cord Blood in Response to Recombinant Cytokines

BACKGROUND The conditions and mechanisms that control the in vitro growth of hematopoietic stem/progenitor cells (contained within the population of CD34+ cells) are still not completely understood. METHODS By using an immunomagnetic system, we have enriched for umbilical cord blood (UCB)-derived CD34+ cells (55% of total cells recovered vs. 0.8% of total cells prior to the enrichment procedure) and analyzed their in vitro growth (proliferation, expansion, and differentiation) in a liquid culture system in the absence or presence of different recombinant cytokine combinations. RESULTS When the selected cells were cultured in the absence of recombinant cytokines, no proliferation or expansion was observed. In the presence of steel factor (SF) and interleukin-6 (IL-6), total cell number was increased nearly fourfold; however, no progenitor cell expansion took place. When cultures were supplemented with SF and IL-6 together with IL-3 and erythropoietin (EPO), a rapid proliferation of the CD34+ -enriched cell population was observed with a selective stimulation of erythropoiesis. However, this stimulation was only transient, suggesting that there was a rapid exhaustion of erythroid progenitor cells within the first 10 days. Significantly higher levels of proliferation and expansion of progenitor cells were observed in the presence of SF, IL-6, GM-CSF, and G-CSF with preferential stimulation of myelopoiesis. Interestingly, such stimulation of myelopoiesis was sustained for the entire culture period (>30 days). The highest levels of proliferation and expansion were observed in the presence of all six cytokines. Under these conditions, erythropoiesis was also sustained only transiently (10 days), whereas myelopoiesis was sustained for >30 days. CONCLUSIONS This study indicates that significant proliferation and expansion of hematopoietic progenitors can be achieved in vitro when culturing a cell population in which CD34+ cells comprise only >50% of the total cells. Our results also suggest that myeloid progenitors (those responding to GM-CSF and G-CSF) possess higher expansion potentials in vitro than their erythroid counterparts. The methods described here for the enrichment and culture of CD34+ cells may be relevant in the development of protocols for the ex vivo proliferation and expansion of hematopoietic progenitors for transplantation.

Concise Review: Ex Vivo Expansion of Cord Blood-Derived Hematopoietic Stem and Progenitor Cells: Basic Principles, Experimental Approaches, and Impact in Regenerative Medicine

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB‐derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self‐renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo‐expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

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.

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.

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.

In vitro effects of stromal cells expressing different levels of Jagged-1 and Delta-1 on the growth of primitive and intermediate CD34+ cell subsets from human cord blood

In trying to contribute to our knowledge on the role of Notch and its ligands within the human hematopoietic system, we have assessed the effects of the OP9 stroma cell line - naturally expressing Jagged-1 - transduced with either the Delta-1 gene (OP9-DL1 cells) or with vector alone (OP9-V), on the in vitro growth of two different hematopoietic cell populations. Primitive (CD34(+) CD38(-) Lin(-)) and intermediate (CD34(+) CD38(+) Lin(-)) CD34(+) cell subsets from human cord blood were cultured in the presence of 7 stimulatory cytokines under four different conditions: cytokines alone (control); cytokines and mesenchymal stromal cells; cytokines and OP9-V cells; cytokines and OP9-DL1 cells. Proliferation and expansion were determined after 7days of culture. Culture of CD34(+) CD38(-) Lin(-) cells in the presence of OP9-V or OP9-DL1 cells resulted in a significant increase in the production of new CD34(+) CD38(-) Lin(-) cells (expansion), which expressed increased levels of Notch-1; in contrast, production of total nucleated cells (proliferation) was reduced, as compared to control conditions. In cultures of CD34(+) CD38(+) Lin(-) cells established in the presence of OP9-V or OP9-DL1 cells, expansion was similar to that observed in control conditions, whereas proliferation was also reduced. Interestingly, in these latter cultures we observed production of CD34(+) CD38(-) Lin(-) cells. Our results indicate that, as compared to MSC, OP9 cells were more efficient at inducing self-renewal and/or de novo generation of primitive (CD34(+) CD38(-) Lin(-)) cells, and suggest that such effects were due, at least in part, to the presence of Jagged-1 and DL1.

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.

Comparative in vitro analysis of different hematopoietic cell populations from human cord blood: in search of the best option for clinically oriented ex vivo cell expansion

BACKGROUND: Ex vivo expansion of hematopoietic stem and progenitor cells has become a priority in the experimental hematology arena. In this study we have obtained different hematopoietic cell populations from umbilical cord blood and simultaneously assessed their proliferation and expansion kinetics. Our main goal was to determine which one of these cell populations would be more suitable for clinical‐grade ex vivo expansion.