Guadalupe Martínez-Jaramillo

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 characterization of the hematopoietic system in pediatric patients with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) has been recognized as a hematologic neoplasia that originates at the level of a primitive lymphoid stem/progenitor cell. To date, however, the biology of the hematopoietic system in this disorder is still not fully understood. In the present study, we have determined the progenitor cell content (including myeloid, erythroid and multipotent progenitors) in 14 children with ALL and followed the proliferation kinetics of these cells in Dexter-type long-term marrow cultures. We have also characterized some aspects related to the composition and function of the hematopoietic microenvironment developed in vitro. All patients included in this study showed extremely reduced levels of progenitor cells (median of 6.2% of the levels found in normal marrow). Proliferation of these cells in long-term cultures was markedly deficient, since they showed very low numbers - compared to normal cultures - and reached undetectable levels after only a few weeks. Regarding the microenvironment developed in vitro, whereas normal marrow samples contained a median of 8 fibroblastic progenitors/10(5) marrow cells and the stromal cell layers developed in culture contained a median of 341000 adherent cells per well, ALL marrow samples showed no fibroblastic progenitors and the numbers of adherent cells were 21% of those in normal cultures. Interestingly, the levels of TNFalpha and IL-6 in ALL culture supernatants were significantly increased, compared to normal cultures. Bone marrow samples from all 14 children were also analyzed once they reached a complete clinical and hematological remission. Myeloid, erythroid and multipotent progenitor cell levels were significantly increased, compared to patients at diagnosis, and proliferation of myeloid progenitors in long-term cultures was also improved. In contrast, proliferation of erythroid progenitors showed no difference to that in cultures from patients at diagnosis. The numbers of fibroblastic progenitors and adherent cells were significantly increased, compared to patients at diagnosis, and TNFalpha and IL-6 levels returned to normal. In summary, in the present study, we have demonstrated significant in vitro alterations of the hematopoietic system, both in terms of its composition and function, in pediatric patients with ALL. Importantly, most of these alterations are corrected, at least partially, after chemotherapy.

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 recombinant human granulocyte macrophage-colony stimulating factor in long-term marrow cultures from patients with aplastic anemia

The hematopoietic system in patients with aplastic anemia (AA) shows both quantitative and qualitative deficiencies, i.e., reduced numbers of hematopoietic progenitor cells (HPC) and impaired HPC proliferation in long‐term marrow cultures (LTMC). Since recombinant human granulocyte macrophage‐colony stimulating factor (rhGM‐CSF) has been shown to be a potent stimulator of normal hematopoiesis, both in vivo and in vitro, in the present study we wanted to assess the possibility of stimulating hematopoiesis in LTMC from 17 patients with AA, by weekly addition of rhGM‐CSF (10 ng/ml). In LTMC from 11 patients (group of responders), rhGM‐CSF induced a significant increase (4.8‐fold, compared with untreated cultures) in the levels of myeloid progenitor cells; in contrast, in six patients (group of nonresponders), myeloid progenitors were refractory to this cytokine. In the group of responders, rhGM‐CSF also induced a pronounced increment in the levels of nonadherent and adherent cells (5.99‐ and 5.18‐fold, respectively, compared with untreated cultures). Among the different myelopoietic lineages, rhGM‐CSF preferentially stimulated the macrophagic lineage; this was evident both at the progenitor and mature cell levels. Interestingly, the effect of rhGM‐CSF in LTMC from AA patients was only transient. Indeed, the effects mentioned above were observed only during the first three weeks of culture; afterwards, myeloid progenitor and nonadherent cell levels in treated cultures declined, practically reaching the levels observed in untreated cultures. At the moment, we do not know whether this transient stimulatory effect is due to the production of inhibitory cytokines, by macrophages generated in response to rhGM‐CSF, or to the exhaustion of the HPC pool in AA cultures. In all 17 patients, rhGM‐CSF had no effect on the kinetics of erythroid or multipotent progenitor cells. These results are in keeping with clinical studies in which it has been observed that most AA patients treated with rhGM‐CSF show increments in circulating monocytes and granulocytes, as well as in bone marrow cellularity. However, little or no effect is observed on erythropoiesis. The actual mechanisms involved in the in vitro effects of rhGM‐CSF on myeloid progenitor cells from AA bone marrow are still not completely understood. Future studies on this issue should be encouraged, since they may help to understand the in vivo (clinical) effects of this cytokine. Am. J. Hematol. 61:107–114, 1999.

Deficient proliferation of myeloid, erythroid, and multipotent progenitor cells in long-term marrow cultures from patients with aplastic anemia treated with immunosuppressive therapy

By using Dexter‐type long‐term marrow cultures (D‐LTMC), it has been shown previously that hematopoietic progenitor cells (HPC) from patients with aplastic anemia (AA) have a deficient proliferation in vitro. The studies reported to date, however, have focused exclusively on granulomonocytic progenitors and no information exists on erythroid or multipotent progenitor cells. On the other hand, in such studies, the input progenitor cell numbers were significantly below normal levels, thus suggesting that the rapid disappearance of myeloid progenitor cells from AA D‐LTMC could also be due, at least in part, to their reduced number at culture onset. In the present study, we have followed the kinetics of myeloid, erythroid, and multipotent progenitors, from 24 AA patients subjected to immunosuppressive therapy (including patients that achieved complete, partial, or no remission at all), throughout a seven‐week culture period. For analysis, we grouped all the patients based on their initial content of all three types of progenitors. Thus, we were able to evaluate separately the kinetics of these cells in D‐LTMC from patients with normal and subnormal levels of progenitor cells. At the time of marrow sampling, most patients showed decreased levels of HPC; in fact, only 21%, 8%, and 16% of them showed normal levels of myeloid, erythroid, and multipotent progenitors, respectively. When cultured in D‐LTMC, HPC from all AA patients analyzed showed a relatively fast disappearance from the cultures. Indeed, myeloid progenitors could be detected for only six weeks, whereas erythroid and multipotent progenitors disappeared from the cultures after two and one weeks of culture, respectively. In contrast, in normal marrow D‐LTMC, myeloid, erythroid, and multipotent progenitors were detected for at least seven, five, and three weeks, respectively. Such a deficient proliferation was observed even in cultures of AA patients that contained normal levels of HPC at culture onset. Interestingly, no correlation was found between HPC proliferation in D‐LTMC and response to treatment. Thus, the results of this study indicate the presence of a functional in vitro deficiency in the hematopoietic system of patients with AA, including those that achieved partial or complete remission after immunosuppressive treatment. Furthermore, this work suggests that such a proliferation deficiency is more pronounced in erythroid and multipotent progenitors than in their myeloid counterparts. Am. J. Hematol. 59:149–155, 1998.

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells. When cell fractions enriched for CD34(+) Lin(-) cells were obtained, the levels of hematopoietic progenitors from MM marrow were within the normal range, and so was their growth kinetics in liquid suspension cultures. The levels of fibroblast progenitors in MM were not statistically different from those in normal marrow; however, their proliferation potential was significantly reduced. Conditioned media from MM-derived MNC and stroma cells contained factors that inhibited normal progenitor cell growth. Our observations suggest that hematopoietic progenitors in MM marrow are intrinsically normal; however, their growth in LTMC may be hampered by the presence of abnormal accessory and stroma cells. These results suggest that besides its role in the generation of osteolytic lesions and the expansion of the myeloma clone, the marrow microenvironment in MM may have a negative effect on hematopoiesis.

Severe hematopoietic alterations in vitro, in bone marrow transplant recipients who develop graft-versus-host disease.

Graft-versus-host disease (GVHD) is currently one of the major obstacles for successful allogeneic bone marrow transplantation (BMT). GVHD results from a complex set of interactions between donor T cells and a variety of target tissues from the host. To gain a better understanding of the biology of the human hematopoietic system in GVHD patients, in the present study we have determined the progenitor cell content in bone marrow (BM) samples from BMT recipients, with and without GVHD, and followed their growth kinetics in Dexter-type long-term marrow cultures (LTMC). We have also assessed some aspects regarding the composition of the hematopoietic microenvironment developed in vitro. As compared to normal subjects, BMT recipients showed decreased numbers of myeloid, erythroid, and multipotent progenitor cells. Interestingly, progenitor levels were significantly lower in GVHD patients (7% of the levels in normal marrow) than in those without GVHD (44% of the levels in normal marrow). When marrow cells from BMT recipients were cultured in LTMC, hematopoiesis was sustained at lower levels and for shorter periods of time, as compared to cultures from normal subjects. The hematopoietic deficiencies observed in this in vitro system were also more pronounced in GVHD patients. In terms of the microenvironment elements, reduced numbers of fibroblastic progenitors and adherent stromal cells were observed in BMT recipients, as compared to normal subjects, who showed 7 colony-forming unit fibroblast (CFU-F)/10(5) marrow cells and 320,000 adherent cells in LTMC. Again, GVHD patients showed more severe deficiencies (0.16 CFU-F/10(5) marrow cells and 34,000 adherent cells in LTMC) than patients without GVHD (2 CFU-F/10(5) marrow cells and 122,000 adherent cells in LTMC). Our results demonstrate that the hematopoietic system of BMT recipients is impaired, both in terms of its in vitro composition and function, and that these deficiencies are clearly more pronounced in patients with GVHD than in those without GVHD. Finally, although the evidence is still preliminary, our results also indicate that the severity of the hematopoietic alterations may be greater in acute GVHD than in chronic GVHD.