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.

In vitro functional alterations in the hematopoietic system of adult patients with acute lymphoblastic leukemia.

In previous studies, we have demonstrated that progenitor cell-enriched marrow cell populations from patients with myeloid leukemia - including both acute (AML) and chronic (CML) - show severe functional alterations when cultured in stroma-free liquid cultures supplemented with stimulatory cytokines. In trying to expand our characterization of the biology of leukemic cells, in the present study we have used a similar approach and analyzed the in vitro growth of equivalent cell populations from patients with acute lymphoblastic leukemia (ALL). ALL marrow cell populations -enriched for hematopoietic progenitors by means of a negative selection procedure- were assessed for their proliferation and expansion potentials, in liquid cultures supplemented with a mixture of early- and late-acting recombinant stimulatory cytokines, throughout a 25-day culture period. ALL cells, although capable of responding to the stimulatory signals provided by hematopoietic stimulators, showed deficient proliferation potentials (reduced capacity to generate more nucleated cells), as compared with their normal counterparts. The capacity to generate myeloid and erythroid progenitors was also significantly reduced in ALL cultures. Interestingly, the functional alterations observed in ALL cultures (i.e., deficient proliferation and expansion potentials) were more pronounced in those from Ph+ patients than in those from Ph- patients. This study indicates that bone marrow cell populations - enriched for hematopoietic progenitor cells - from ALL patients possess deficient proliferation and expansion potentials in vitro, and that such functional alterations are more severe when cells are derived from Ph+ patients, as compared to their Ph- counterparts.

Expression of ice, bcl-2, c-myc and p53 in different bone marrow cell populations from patients with diffuse large B-cell lymphoma.

We have previously reported functional alterations in vitro in the hematopoietic compartment of patients with diffuse large B-cell lymphoma (DLBCL). In the present study, we assessed the presence of molecular alterations in hematopoietic cells derived from DLBCL marrow. Accordingly, the expression of four genes (i.e. ice, bcl-2, c-myc and p53) was assessed both, at the mRNA and protein levels, in three cell populations: (i) population I, consisting of morphologically recognizable precursor and mature cells; (ii) population II, enriched for CD34+ Lineage-negative (Lin-) cells; and (iii) population III, enriched for CD34+ CD38- Lin- cells. By using a multiplex reverse transcriptase-polymerase chain reaction system, we observed reduced expression of bcl-2 in population I, and c-myc in populations I and II from lymphoma marrow compared to their normal counterparts. On the other hand, expression of ice and p53 was not significantly different when comparing normal and DLBCL samples. At the protein level, all four molecules were expressed in a higher proportion of samples from DLBCL patients than in marrow samples from normal subjects. Interestingly, these proteins were expressed predominantly in primitive cells (population III), whereas the proportion of positive samples was reduced in population II, and even more in population I. Taken together, our results indicate that, in DLBCL, molecular alterations are present in hematopoietic cells from bone marrow, including morphologically recognizable precursor and mature cells, as well as primitive hematopoietic progenitors (CD34+ cells). To date, the physiological implications of these alterations are still unclear, and further studies should be undertaken to address this issue.

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-alpha; interestingly, whereas in normal fractions TNF-alpha 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.

In vitro proliferation and expansion of hematopoietic progenitors present in mobilized peripheral blood from normal subjects and cancer patients.

In the present study, we have assessed, in a comparative manner, the in vitro proliferation and expansion potentials of hematopoietic progenitor cells (HPC) present in mobilized peripheral blood from normal subjects (MPB-n; n = 18) and cancer patients (MPB-c; n = 18). The latter included patients with breast cancer (BrCa; n = 8), Hodgkin disease (HD; n = 4), non-Hodgkin lymphoma (NHL; n = 3), and acute myeloid leukemia (AML; n = 3). Progenitor cells from normal bone marrow (BM) and umbilical cord blood (UCB) were included as controls. HPC, enriched by a negative selection procedure, were cultured for 25 days, in serum-free liquid media in the presence of a cytokine combination including early- and late-acting cytokines. Our results demonstrate that the in vitro biological properties of progenitor cells present in MPB differ, depending on whether they are derived from healthy individuals, from patients with solid tumors, or from patients with hematological neoplasias. Among all cell sources analyzed, UCB-derived progenitors showed the greatest proliferation and expansion potentials (1000-fold increase in total cell numbers on day 15, and a 22-fold increase in myeloid progenitor cell numbers, at day 10). Progenitor cells present in MPB from hematologically normal individuals showed proliferation and expansion potentials comparable to those of HPC from normal BM (500-fold increase in total cell numbers on day 15, and a 14-fold increase in myeloid progenitor cell numbers, at day 10). The proliferation/expansion potentials of MPB progenitors from BrCa patients were also within the normal range, although in the lower levels (327-fold increase in total cell numbers, on day 15, and 11.8-fold increase in myeloid progenitors, at day 10). In contrast, progenitors present in MPB from patients with HD, NHL, and especially AML, showed reduced in vitro capacities (119-, 102-, and 51-fold increase in total cell numbers, respectively; and 8-, 4-, and 2.6-fold increase in myeloid progenitor cells, respectively). To our knowledge, this is the first report in which the in vitro proliferation and expansion potentials of HPC from MPB from normal subjects and cancer patients are assessed simultaneously in a comparative manner.

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 proliferation and expansion of hematopoietic progenitors from patients with diffuse large B-cell lymphoma before and after chemotherapy.

We have previously demonstrated that when cultured in Dexter-type Long-Term Marrow Cultures (LTMC), hematopoietic progenitor cells (HPC) from patients with Diffuse Large B-Cell Lymphoma (DLBCL) show a defective proliferation, as compared to HPC from normal marrow. In that study it was also demonstrated that functional alterations were present in the hematopoietic microenvironment developed in culture; thus, it was not clear whether such a defective proliferation in vitro was due to an intrinsic defect in the HPC compartment of DLBCL patients, or to an altered microenvironment, or both. In order to address this question, in the present study we have assessed the proliferation and expansion potentials of HPC present in bone marrow from patients with DLBCL, in cytokine-supplemented liquid cultures initiated with a cell population enriched for CD34+ Lin- cells, in the absence of stromal cells and in the presence of reduced numbers of accessory cells. Our results demonstrated that bone marrow-derived HPC from patients with DLBCL, both before and right after chemotherapy, possessed reduced proliferation and expansion potentials in vitro, as compared to their normal counterparts. Interestingly, in patients analyzed 18 months after treatment the proliferation and expansion levels were similar to those of normal HPC, indicating a complete restoration of the hematopoietic function. Although the reason for these observations is not clear, our results suggest the possibility that primitive CD34+ progenitor cells present in bone marrow, which show deficient proliferation and expansion potentials in vitro, are involved in the origin/progression of DLBCL.

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.

Hematopoietic changes during progression from Fanconi anemia into acute myeloid leukemia: case report and brief review of the literature.

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by bone marrow (BM) failure and a wide array of physical abnormalities. Around 9% of FA patients develop acute myeloid leukemia (AML), which makes FA a good genetic model to study leukemogenesis. To date, however, no information exists on the functional integrity of the hematopoietic system of FA patients during the period in which they develop AML. Herein, we report on the characterization of hematopoietic progenitor cells from a pediatric FA patient that developed AML. Our results show that significant changes occurred in the hematopoietic system of the patient from the time he presented with FA to the time he developed AML. Such changes included marrow cellularity, frequency of CD34(+) cells and CFC, as well as proliferation potential of progenitor cells in liquid cultures supplemented with recombinant cytokines. Interestingly, no significant changes in the karyotype of marrow cells were observed, indicating that progression from FA into AML may proceed without major chromosomal alterations (i.e. translocations and/or deletions). This study represents one of the first steps towards the cellular characterization of the hematopoietic system in FA patients that develop AML.

Sequential Variations in the Content of Bone Marrow Colony-forming Cells in Individual Patients with Aplastic Anemia Before and After Immunosuppressive Therapy; Hematopoiesis.

Previous studies have shown that the levels of hematopoietic progenitor cells (colony-forming cells; CFC) are drastically reduced in the vast majority of patients with aplastic anemia (AA). This has been observed both in patients before and after immunosuppressive therapy. In those studies, however, both groups of patients were usually formed by different individuals, thus it was not possible to follow the kinetics of such cells in each particular patient. In the present study, we have determined the content of myeloid and erythroid CFC in individual AA patients before and after therapy. Treated patients were studied at two different times (8-18 months apart) to detect any possible variations due to the ongoing treatment. At diagnosis, the levels of both myeloid and erythroid CFC were drastically reduced, as compared to normal bone marrow, in all the patients studied. This correlated with very low levels of leukocytes and hemoglobin in circulation. After the patients entered an immunosuppressive treatment, all of them showed significant increments in their CFC levels, and this correlated with increments in their hematological parameters in peripheral blood. However, in most patients CFC levels were still below the normal range. When the second sample after treatment was obtained, great variations in CFC numbers were observed. In terms of erythroid CFC levels, a further increase was seen in most patients, and this correlated with a further increase in hemoglobin levels. In contrasts, the levels of myeloid CFC were increased in only some of the patients, whereas in others, significant reductions were evident. Interestingly, in this latter group of patients, CFC never reached the levels observed before treatment. Our results indicate that, in a significant proportion of patients, a common pattern seems to exist. That is to say, low CFC numbers are present before treatment; an increase in the numbers of such cells results as an effect of the immunosuppressive therapy and further variations in CFC numbers (within individual limits that may differ significantly from one patient to another) take place as long as the treatment continues. Finally, we observed a correlation between CFC levels and the clinical status of the patients, i.e., those patients that showed a complete or a partial response to treatment showed higher levels of both myeloid and erythroid CFC than those patients that did not respond to therapy.