In vitro hematopoiesis in patients with malignant lymphoma during active disease and at complete clinical remission after chemotherapy

Malignant lymphomas are neoplastic diseases of lymphoid cells, which usually originate in the lymph nodes. During the last two decades, significant progress has been made in the characterization of chromosomal and molecular alterations in these malignancies. To date, however, the composition and function of the hematopoietic system in this group of hematological disorders is still not fully understood. In the present study, we have determined the progenitor cell content in 10 patients with diffuse large-cell lymphoma (DLCL) and characterized the proliferation of these cells in long-term marrow cultures. We have also addressed some issues regarding the composition and function of the hematopoietic microenvironment in this malignancy. All the patients included in this study showed normal hematological parameters in peripheral blood, both before and after chemotherapy, however, significant hematopoietic alterations were consistently observed. As compared to normal subjects, lymphoma patients showed a 35% reduction in progenitor cell numbers, including myeloid, erythroid and multipotent progenitors. The in vitro proliferation of these cells was also deficient, since their levels in long-term marrow cultures were significantly lower than those observed in normal bone marrow cultures. Fibroblastic progenitors were reduced by >50% and this correlated with a deficient adherent cell layer development in culture. A reduction was also seen in the levels in culture supernatant of the stimulatory cytokines Stem Cell Factor and Interleukin-6. Interestingly, all the hematopoietic alterations mentioned above were still present in patients at complete clinical remission after chemotherapy. Thus, in the present study we have demonstrated significant in vitro deficiencies in the composition and function of the hematopoietic system in patients with diffuse large-cell lymphoma, both during active disease and at the time of complete clinical remission.     

Biology of the hemopoietic microenvironment.

In adult mammals, hemopoiesis takes place primarily in the bone marrow. The steady-state production of blood cells depends to a large extent on the interaction between hemopoietic stem/progenitor cells (HPC) and the different components of the microenvironment present in the medullary cavity. During the last three decades, in vivo and in vitro studies have allowed significant advances in understanding of the biology of such a hemopoietic microenvironment. Although not evident in histological sections, it is well known that the hemopoietic microenvironment is a highly organized structure that regulates the location and physiology of HPC. The hemopoietic microenvironment is composed of stromal cells (fibroblasts, macrophages, endothelial cells, adipocytes), accessory cells (T lymphocytes, monocytes), and their products (extracellular matrix and cytokines). Microenvironmental cells can regulate hemopoiesis by interacting directly (cell-to-cell contact) with HPC and/or by secreting regulatory molecules that influence, in a positive or negative manner, HPC growth. Recent in vitro studies suggest that functional abnormalities of the hemopoietic microenvironment may be implicated in the manifestation of certain hematological disorders such as aplastic anemia, and acute and chronic myelogenous leukemia. Thus, the characterization of the structure and function of the human hemopoietic microenvironment may have relevance in understanding and treating different hematological disorders.     

Establishing a cord blood banking and transplantation program in Mexico: a single institution experience

BACKGROUND: Over the past decade, umbilical cord blood (UCB) banking and transplantation have increased significantly worldwide. The experience in developing countries, however, is still limited. In January 2005 the Mexican Institute of Social Security (IMSS) initiated its UCB banking and transplantation program. This study reports on the experience generated at this institution during the first 2 years of activities. STUDY DESIGN AND METHODS: A public UCB bank was established at La Raza Medical Center, IMSS, in Mexico City. Good manufacturing practices and standard operating procedures were used to address donor selection, as well as UCB collection, processing, and cryopreservation. Based mainly on human leukocyte antigen (HLA) and total nucleated cell (TNC) content, specific UCB units were thawed, processed, and released for transplantation. RESULTS: Based on stringent selection criteria, 360 UCB units were collected from January 2005 to December 2006. A total of 201 (56%) units (minimum volume, 50 mL without anticoagulant) were processed and stored. Median values for specific parameters were as follows: volume, 89.9 mL; viability, 94.8%; TNCs, 0.91 x 10(9); CD34+ cells, 3.13 x 10(6); and colony-forming cells, 1.20 x 10(6). During this period, 10 units had been released for transplantation to seven patients (six children and one adult). Engraftment was observed in five patients; four of them were still in remission (114-293 days after transplant). In spite of showing sustained engraftment, one patient died on Day +88. Two patients showed no engraftment and died 29 to 30 days after transplant. CONCLUSION: The results obtained during this initial period are encouraging and indicate that the UCB banking and transplantation program at IMSS will help to improve already existing hematopoietic cell transplant programs in Mexico. The experience generated at IMSS may be helpful to other institutions, particularly those in developing countries.     

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.     

Enrichment of CD34 (My10)-positive myeloid and erythroid progenitors from human marrow and their growth in cultures supplemented with recombinant human granulocyte-macrophage colony-stimulating factor

Myeloid and erythroid progenitor cells were enriched from human marrow by selecting CD34-positive (CD34 + ve) cells, labeled with the My10 (HPCA-1) antibody, using a fluorescence-activated cell sorter. Seventy-one percent of CD34 + ve cells were blasts and most of these were too primitive to be identified by standard morphological criteria. On average, 9.5% of CD34 + ve cells formed clones after 14 days of culture in semisolid medium supplemented with erythropoietin and medium conditioned by 5637 bladder carcinoma cells. Over 2.5% of CD34 + ve cells were day-14 myeloid colony-forming cells and 2.4% were erythroid colony (burst)-forming progenitors. The remaining progenitors formed myeloid and erythroid clusters. A subpopulation of day-14 myeloid colony-forming cells failed to respond to recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) after accessory cells were removed during enrichment, so it appears that this factor can induce myeloid growth indirectly as well as directly. Recombinant human GM-CSF also supported erythroid colony-formation in cultures of CD34 + ve cells, which suggests that this hemopoietin may act directly on erythroid progenitors.     

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. STUDY DESIGN AND METHODS: By using immunomagnetic‐negative selection and cell sorting, five cell populations were obtained: unseparated mononuclear cells (MNCs; I); two lineage‐negative cell populations, one enriched for CD34+ CD38+ cells (II) and the other enriched for CD34+ CD38? cells (III); and two CD34+ cell fractions purified by fluorescence‐activated cell sorting, one containing CD34+ CD38+ cells (IV) and the other containing CD34+ CD38? cells (V). The kinetics of such populations were analyzed in both relative and absolute terms. RESULTS: No expansion was observed in Population I; in contrast, significant increments in the numbers of both progenitor and stem cells were observed in cultures of Populations II to V. Population V (reaching 12,800‐fold increase in total cells; 1280‐fold increase in CD34+ cells; 490‐fold increase in colony‐forming cells; and 12‐fold increase in long‐term culture–initiating cells) showed the highest proliferation and expansion potentials. CONCLUSION: Our study suggests that the cell fraction containing greater than 98% CD34+ CD38? cells would be the ideal one for large‐scale ex vivo expansion; however, based on our data, it seems that, except for MNCs, all other cell populations could also be used as input cell fractions.     

Functional integrity in vitro of hematopoietic progenitor cells from patients with chronic myeloid leukemia that have achieved hematological remission after different therapeutic procedures

In this study, we have assessed the in vitro growth of hematopoietic progenitor cells (HPC) from chronic myeloid leukemia (CML) patients that have recovered after different treatments. Bone marrow cells were obtained from 33 CML patients, including patients at diagnosis, before treatment (n=12), and patients that have achieved hematological remission (and in most cases a major cytogenetic response) after different therapeutic procedures (n=21), including patients treated with Interferon-alpha (IFN; n=5), imatinib mesylate (IMATINIB; n=8) and patients that received an allogeneic hematopoietic cell transplant (HCT; n=8). Marrow cells were enriched for CD34(+) cells and cultured in a serum- and stroma-free liquid culture system, supplemented with a combination of 8 recombinant cytokines. Normal samples were studied as controls. HPC from CML patients before therapy showed deficient proliferation and expansion potentials in culture (140-fold increase in nucleated cell number and 1.3-fold increase in colony-forming cell number) as compared to normal progenitors (1200-fold increase in nucleated cell number and 25-fold increase in colony-forming cell number). In contrast, HPC from patients treated with IMATINIB showed growth potentials similar to those of normal progenitors. Progenitors from patients after HCT also showed significant proliferation and expansion capacities. Interestingly, progenitors from IFN-treated patients showed proliferation and expansion kinetics similar to those of cells from untreated patients. These results indicate that, although treatment of CML patients with IFN, IMATINIB or HCT resulted in complete hematological remission (and a major cytogenetic response), only patients treated with IMATINIB and, to a lesser extent, with HCT showed a full hematopoietic recovery, as determined by the in vitro growth of HPC in our culture system.     

Clinical relevance of NK, NKT, and dendritic cell dose in patients receiving G-CSF-mobilized peripheral blood allogeneic stem cell transplantation

To analyze the relationship between the cellular composition of peripheral blood allografts and clinical outcome, we performed a prospective study in 45 adult patients who underwent allogeneic peripheral blood hematopoietic stem cell transplantation (HSCT) from a histocompatibility leukocyte antigen identical sibling donor for different hematological malignancies. The dose of CD34+, CD3+, CD4+, CD8+, and CD19+ lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, type 1 and type 2 dendritic cells (DC1 and DC2), as well as regulatory T (Treg) lymphocytes was analyzed. All patients were conditioned with busulphan and cyclophosphamide (BuCy2) ± VP-16 and received a short course of methotrexate and cyclosporin-A as graft-versus-host disease (GVHD) prophylaxis. Acute GVHD (aGVHD) was present in 9 of 43 (21%) patients, and chronic GVHD (cGVHD) developed in 18 of 39 (46%) patients. There was a significantly higher incidence of aGVHD in patients receiving more than 6×10⁶/kg CD34+ cells. In univariate analysis, variables associated with better survival were as follows: a dose of less than 1.5×10⁷/kg NKT cells and less than 1.7×10⁶/kg DC2 for disease-free survival (DFS), and a dose of less than 3×10⁷/kg NK cells, less than 1.5×10⁷/kg NKT cells, less than 3×10⁶/kg DC1, and less than 1.7×10⁶/kg DC2 for overall survival (OS). In the Cox regression analysis, the dose of NKT cells was the only variable associated with better DFS, while the doses of NK, NKT, and CD34+ cells (less than 8×10⁶/kg) were associated with better OS. In conclusion, different circulating cell populations, other than CD34+ cells, are also of relevance in predicting the clinical outcome after allogeneic peripheral blood HSCT.     

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.