Understanding the hematopoietic microenvironment in chronic myeloid leukemia: A concise review.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that results from the BCR-ABL gene-induced transformation of a primitive hematopoietic cell. This disease has been extensively studied, and, as a result, a very effective therapy has been developed: the tyrosine kinase inhibitors. Although, there is a significant knowledge about the intrinsic biology of CML cells, alterations in their bone marrow microenvironment are not yet completely understood. In this concise review, we summarized recent findings on the composition and function of the bone marrow microenvironment in CML, and their importance in the progression of the disease and treatment resistance.

Mesenchymal Stromal Cells from the Epidermis and Dermis of Psoriasis Patients: Morphology, Immunophenotype, Differentiation Patterns, and Regulation of T Cell Proliferation.

Psoriasis is a skin disease characterized by hyperproliferation of keratinocytes and chronic inflammation. Mesenchymal stem/stromal cells (MSCs) exhibit an immunoregulatory function that can be altered in the skin of these patients. However, to date, the presence and functional capacity of MSCs in the dermis and epidermis of patients with psoriasis have not been fully established. In the present study, we evaluated the presence of MSCs in the skin of patients by obtaining adherent cells from the dermis and epidermis of lesional and nonlesional areas and characterizing them in a comparative manner with corresponding cells obtained from the dermis (HD-MSCs) and epidermis (HE-MSCs) of healthy donors. We determined whether the adherent cells had immunophenotypic profiles and differentiation potentials that were characteristic of MSCs. In addition, we analyzed their immunosuppression function by evaluating their capacity to decrease T cell proliferation. Our results indicate the presence of MSCs in the dermis and epidermis of healthy donors and patients with psoriasis; adherent cells from all skin sources exhibited MSC characteristics, such as expression of CD73, CD90, and CD105 markers and a lack of hematopoietic and endothelial marker expression. However, the cell populations obtained showed differences in differentiation potential toward adipogenic, osteogenic, and chondrogenic lineages. In addition, we observed a low MSC obtention frequency in nonlesional epidermal samples (NLE-MSCs), which also showed alterations in morphology and proliferation rate. Interestingly, MSCs from both the nonlesional dermis (NLD-MSCs) and lesional dermis (LD-MSCs) showed higher HLA class I antigen (HLA-I) expression than HD-MSCs. Moreover, NLD-MSCs showed a low T cell proliferation suppression capacity. In summary, this study demonstrates the presence of MSCs in the epidermis and dermis of patients with psoriasis and suggests that such cells may favor the inflammatory process and thus psoriatic lesion development through high HLA-I expression and low immunosuppression capacity.

Decreased frequency, but normal functional integrity of mesenchymal stromal cells derived from untreated and Imatinib-treated chronic myeloid leukemia patients.

In vitro, Imatinib inhibits the proliferation and stimulates the osteogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). However, it is unknown whether Imatinib affects the biology of MSC in vivo. We asked whether MSC from long-term Imatinib-treated CML patients were affected by the in vivo treatment. MSC from untreated and Imatinib-treated patients displayed normal functional properties (i.e. proliferation, immunophenotype, differentiation and hematopoietic supportive capacity) - but a decreased frequency. In vitro, Imatinib lost its effect when discontinued; which suggest that it has a reversible effect on MSC. Therefore it might lose its effect on MSC after discontinuation in vivo.

Human mesenchymal stromal cells from adult and neonatal sources: comparative analysis of their morphology, immunophenotype, differentiation patterns and neural protein expression.

BACKGROUND: Bone marrow (BM) has been recognized as the main source of mesenchymal stromal cells (MSC); however, MSC have also been detected in umbilical cord blood (UCB) and placenta (PL). In the present study, we obtained MSC from these three sources and characterized them in a comparative manner. METHODS: MSC were obtained from BM, UCB and PL samples and analyzed to determine their morphology, cell-surface antigen (Ag) expression and differentiation potential. Particular emphasis was placed on the expression of neural markers. RESULTS: MSC were detected in 9/9, 11/104 and 5/5 samples from BM, UCB and PL, respectively. MSC populations comprised several morphologically distinct cell types, including neural-like cells. MSC were positive for 'mesenchymal' Ag (CD105, CD73 and CD90), although CD90 expression was very heterogeneous. Interestingly, CD13 expression was high in all three sources. In all cases, MSC showed osteogenic and chondrogenic differentiation; however, UCB MSC showed no adipogenic potential. Furthermore, MSC from UCB produced a different type of cartilage compared with MSC from BM and PL. It is noteworthy that in all three sources we detected the expression of neural proteins without any neural differentiation stimuli. A significant increase in the proportion of neural marker-positive MSC was observed in the presence of neural inducers. DISCUSSION: Our results indicate that PL may prove to be a more appropriate source for obtaining MSC than UCB, and suggest the possibility that a subpopulation of MSC may possess neural potential, which is favored by neural inducers.

Multiple myeloma-associated amyloidosis is an independent high-risk prognostic factor.

Several prognostic factors have been recognized in patients with multiple myeloma (MM). Among the most important are: the serum levels of beta2-microglobulin, albumin, and LDH; the labeling index; and an abnormal karyotype. Patients with amyloidosis (AL) have poor prognosis; however, little is known concerning the prognostic significance of AL associated to MM. In 201 consecutive patients with de novo MM, we performed a fat-pad biopsy needle aspiration (FPBNA) that was stained with Congo red. Sixty eight (34%) patients had AL and a poorer prognosis disease: lower performance status, presence of B symptoms, higher LDH and calcium values, and worse response to chemotherapy. Cox regression model for overall survival detected three variables having independent prognostic significance: the presence of AL (RR = 3.4, P < 0.004), serum albumin levels <3.5 g/dl (RR 3.2, p < 0.005), and patients not achieving complete remission or very good partial remission (RR 2.9, p < 0.02). In 28% of patients with de novo MM, FPBNA was useful to detect incidental amyloidosis. During follow-up, 69% of these patients had symptoms of AL. Excluding 16 patients with obvious symptoms of AL at diagnosis, overall survival was worse in patients who developed later symptoms of AL. MM-associated AL represents a poorer prognosis disease even in the absence of symptoms at diagnosis, and this specific association may be considered as an independent high-risk prognostic factor. The routine study of periumbilical fat-pad tissue should be mandatory in all patients with MM.

In vitro leukemic cell differentiation and WT1 gene expression.

Cell differentiation and four WT1 isoforms were assessed in CD34(+) cells from patients with acute myelogenous leukemia in presence or absence of recombinant human GM-CSF and G-CSF, on days 0, 10 and 20 of culture. We found that WT1 isoforms expression was consistently higher in AML-derived CD34+ cell-enriched cell fractions, as compared to their normal counterparts, and interestingly, in both cases, cells had differentiation towards the myeloid lineage with WT1 expression different patterns. This data suggest that WT1 expression seems to be modulated by the presence of cytokines, especially on day 20 of culture.

mRNA expression of MAGE-A3 gene in leukemia cells.

Leukemia-associated antigens such as proteins encoded by MAGE genes might provide tools for immunotherapy of leukemia. Positive and negative results of MAGE-A gene expression in hematological malignancies have been reported. This led us to study MAGE-A gene expression in human leukemias using RT-PCR. Among 115 leukemias from various subtypes, 14/34 (41.17%) AML were positive for one of the three genes analyzed (MAGE-A1 1/32; MAGE-A3 10/32; MAGE-B2 3/12). Expression was also detected in 23/76 (30.26%) B-cell ALL patients (MAGE-A1 2/53; MAGE-A3 20/53; MAGE-B2 1/32). One of these patients expressed both MAGE-A1 (weak signal) and -A3 (strong signal) genes. Other patient with CML were positive for MAGE-B2 (1/5, 20%). MAGE-A3 expression data were corroborated by real time RT-PCR through determination of MAGE-A3 transcript levels. We concluded that the MAGE-A3 gene is expressed at the mRNA level in a proportion of human leukemias.

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.

Peripheral blood mobilization of different lymphocyte and dendritic cell subsets with the use of intermediate doses of G-CSF in patients with non-Hodgkin's lymphoma and multiple myeloma.

Between June 2003 and November 2004, we collected mobilized peripheral blood units from 29 patients with non-Hodgkin's lymphoma and multiple myeloma for autologous peripheral blood stem cell transplantation. They received granulocyte colony-stimulating factor (G-CSF) (16 micro g/kg/day) for a total of 5 days. Immediately before and 3 h after the fourth and fifth dose of G-CSF, we performed flow cytometry analysis to quantify: T cells (CD3+CD4+, CD3+CD8+), B cells (CD19+), NK cells (CD3-CD16+CD56+), NKT cells (CD3+CD16+CD56+), type 1 dendritic cells (DC1) (lin-HLA-DR+CD11c+), type 2 dendritic cells (DC2) (lin-HLA-DR+CD123+), regulatory T cells (Tregs) (CD4+CD25+), and activated T cells (CD3+HLA-DR+). All cell subsets were mobilized after G-CSF treatment with the exception of B, NK, and NKT lymphocytes. The median number of Treg cells before and after G-CSF was statistically different (29+/-14.9x10(6)/l vs 70.1+/-46.1x10(6)/l, P<0.02). DCs were mobilized significantly with a 5.9-fold increase in DC2 (15.1+/-30.3x10(6)/l vs 89.8+/-81.0x10(6)/l, P<0.02) and a 2.6-fold increase for DC1 (41+/-42.5x10(6)/l vs 109.5+/-58.0x10(6)/l, P<0.04). Patients received a mean of 3.1+/-1.2x10(7)/kg NK cells, 1.3+/-0.9x10(7)/kg NKT cells, 0.41+/-0.29x10(7)/kg DC1, 0.2+/-0.22x10(7)/kg DC2, and 1.8+/-1.9x10(7)/kg Tregs. In conclusion, intermediate doses of G-CSF induce mobilization of different lymphocyte subsets, with the exception of B, NK, and NKT cells. The mobilization of certain suppressive populations (DC2 and Treg) could be in theory deleterious, at least in patients with cancer.

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 6x10(6)/kg CD34+ cells. In univariate analysis, variables associated with better survival were as follows: a dose of less than 1.5x10(7)/kg NKT cells and less than 1.7x10(6)/kg DC2 for disease-free survival (DFS), and a dose of less than 3x10(7)/kg NK cells, less than 1.5x10(7)/kg NKT cells, less than 3x10(6)/kg DC1, and less than 1.7x10(6)/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 8x10(6)/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.

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.

CD4+ CD25+ lymphocyte and dendritic cell mobilization with intermediate doses of recombinant human granulocyte colony-stimulating factor in healthy donors.

We prospectively conducted a quantitative and phenotypic analysis of T, B, natural killer (NK), NKT, type 1 and 2 dendritic cells (DC), and regulatory T cells, before and after mobilization with intermediate doses of granulocyte colony-stimulating factor (G-CSF) (16 microg/kg per day). Between November, 2003, and December, 2004, we collected stem cells from 25 HLA identical sibling donors for allogeneic hematopoietic stem cell transplantation. Before mobilization and 3 h after the fourth and fifth doses of G-CSF, blood samples were taken for blood counts and flow cytometry. The median number of regulatory T cells before and after G-CSF was statistically different (69 +/- 41 x 10(6)/L versus 161 +/- 159 x 10(6)/L, p < 0.01). We observed a 1.7-fold increase in NK and NKT cells (p < 0.009 and p < 0.02, respectively). DC were mobilized with a 11.5-fold increase in type 2 (p < 0.004) and a 8.5-fold increase in type 1 DC (p < 0.003). The patients received a mean of: 2.2 x 10(7)/kg +/- 1.4 x 10(7)/kg of NK cells, 0.95 x 10(7)/kg +/- 0.81 x 107/kg of NKT cells, 0.43 x 107/kg +/- 0.53 x 10(7)/kg of type 1 DC, 0.3 v 10(7)/kg +/- 0.45 x 10(7)/kg of type 2 DC and 1.4 x 10(7)/kg +/- 1.2 x 10(7)/kg of regulatory T cells. Using intermediate doses of G-CSF, we have demonstrated the mobilization of different lymphocyte subsets, in particular regulatory T cells and DC, which can be expanded later and used in the treatment of cancer and autoimmune diseases.

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.

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.

Tumor necrosis factor-alpha levels in long-term marrow cultures from patients with aplastic anemia: modulation by granulocyte-macrophage colony-stimulating factor.

We have previously shown that the levels of hematopoietic progenitors in long-term marrow cultures (LTMC) from patients with aplastic anemia (AA) are drastically reduced, as compared to normal LTMC. We have also reported that when LTMC from AA patients are supplemented with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) there is an increase in colony-forming cell (CFC) levels. However, such a stimulation is only transient and it is followed by an inhibition in CFC growth. Based on these observations, in the present study we have tested the hypothesis that the levels of tumor necrosis factor-alpha (TNF-alpha), an inhibitor of hematopoiesis, are increased in AA LTMC and that such levels are further increased after rhGM-CSF has been added to the cultures for several weeks. Accordingly, we have determined the levels of TNF-alpha in the supernatant of LTMC established from normal (n = 8) and AA (n = 6) bone marrow and in AA LTMC supplemented with rhGM-CSF (n = 6). At the time of culture initiation, TNF-alpha levels were below detection in all the samples analyzed. After 5 weeks of culture, TNF-alpha levels in normal LTMC were very low, with a median of 7.3 pg/mL. In contrast, AA LTMC contained higher levels of TNF-alpha (median of 49.6 pg/mL). In keeping with our hypothesis, addition of rhGM-CSF to AA LTMC resulted in a significant further increase of TNF-alpha levels (median of 135.4 pg/mL). Our results demonstrate an inverse correlation between reduced hematopoiesis in AA LTMC and increased levels of TNF-alpha in this culture system. Based on the results presented here, together with previous reports indicating that TNF-alpha is a potent inducer of apoptosis in hematopoietic progenitor cells, it seems reasonable to suggest that TNF-alpha is implicated in the pathophysiology of AA.

Long-term proliferation in vitro of hematopoietic progenitor cells from children with congenital bone marrow failure: effect of rhGM-CSF and rhEPO.

We have characterized the proliferation kinetics of hematopoietic cells in long-term marrow cultures (LTMC) from five normal children and seven children with congenital bone marrow failure (four with Fanconi anemia [FA] and three with congenital pure red cell aplasia [PRCA]). Total nonadherent and adherent cells, as well as nonadherent progenitors, were determined weekly in the presence or in the absence of rhGM-CSF (10 ng/ml) or rhEPO (3 U/ml). As compared to normal LTMC, hematopoiesis was drastically reduced in cultures from FA patients. Myeloid and erythroid progenitor cells reached undetectable levels after only 3 and 1 weeks of culture, respectively. This was observed even in cultures supplemented with rhGM-CSF, in which no response to this cytokine occurred. In LTMC from PRCA children, the growth of erythroid and multipotent progenitors was also drastically reduced. Myelopoiesis, on the other hand, showed normal levels during the first three weeks of culture; however, from week 4, there was a significant decrease in the levels of both progenitor and mature cells, reaching undetectable levels several weeks before normal cells did. Response to rhGM-CSF and rhEPO was transient and deficient. Our results suggest that in FA, alterations at the level of primitive progenitor cells are so severe that myeloid, erythroid and multipotent progenitors are unable to proliferate in LTMC, even in the presence of rhGM-CSF. In patients with PRCA the erythroid arm of hematopoiesis is preferentially affected and addition of rhGM-CSF and/or rhEPO to these cultures had little or no effect on erythroid cell production. Interestingly, myelopoiesis in this culture system was deficient as well and response to rhGM-CSF was defective, suggesting that the myeloid lineage is also altered in congenital PRCA.

Characterization of the adherent cells developed in Dexter-type long-term cultures from human umbilical cord blood.

We have previously shown that when human umbilical cord blood (UCB) cells are cultured in standard Dexter-type long-term cultures (D-LTC), adherent cells develop forming a discrete net on the bottom of the culture flask. The identity of such cells, however, has not been defined. Accordingly, the major goal of the present study was to characterize the adherent cells developed in standard UCB D-LTC. Cultures were established from 14 UCB samples and from nine bone marrow (BM) samples, as controls. Both UCB and BM cultures were initiated with the same number of mononuclear cells (MNC) (2.5 x 10(6) MNC/ml). After three weeks in culture, adherent cell numbers in UCB D-LTC were 24%-30% of the numbers found in BM cultures. More than 90% of the adherent cells in UCB D-LTC expressed the acid phosphatase enzyme, whereas no alkaline phosphatase-positive cells were observed. This was in contrast to BM D-LTC, in which alkaline and acid phosphatase were expressed by 60%-75% and 20%-45% of the adherent cells, respectively. Immunochemical analysis showed that CD61 (osteoclast marker) and Factor VIII (endothelial cell marker) were not expressed by the adherent cells developed in UCB cultures. Interestingly, the majority of such cells expressed CD1a (dendritic cell marker), CD14, CD68 and CD115 (antigens mainly expressed by macrophagic cells). When the cultures were supplemented with the recombinant cytokines epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor or granulocyte-macrophage colony-stimulating factor (GM-CSF), only GM-CSF had a significant positive effect on adherent cell number. In order to test for some functional properties of the adherent cells developed in culture, production of stem cell factor (SCF), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was assessed. IL-6 and TNF-alpha showed elevated levels in UCB D-LTC, whereas SCF levels were always below detection. Finally, analysis of fibroblast progenitors (fibroblast colony-forming units [CFU-F]) showed that these cells were present in BM samples (6 CFU-F/10(5) MNC) and were totally absent in UCB samples. Taken together, the results of the present study indicate that the vast majority of the adherent cells developed in standard UCB D-LTC belong to the macrophage lineage and that fibroblasts seem to be absent. Interestingly, the high proportion of CD1a+ cells suggests that dendritic cells are also present in these cultures.

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.