Thrombopoietin enhances the production of myeloid cells, but not megakaryocytes, in juvenile chronic myelogenous leukemia

We previously reported the aberrant growth of granulocyte-macrophage (GM) progenitors induced by a combination of stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in juvenile chronic myelogenous leukemia (JCML). We examined here the effects of thrombopoietin (TPO) on the proliferation and differentiation of hematopoietic progenitors in JCML. In serum-deprived single-cell cultures of normal bone marrow (BM) CD34+CD38high cells, the addition of TPO to the culture containing SCF + GM-CSF resulted in an increase in the number and size of GM colonies. In the JCML cultures, in contrast, the number of SCF + GM-CSF-dependent GM colonies was not increased by the addition of TPO. However, the TPO addition caused an enlargement of GM colonies in cultures from the JCML patients to a significantly greater extent compared with the normal controls. There was no difference in the type of the constituent cells of GM colonies with or without TPO grown by JCML BM cells. A flow cytometric analysis showed that the c-Mpl expression was found on CD13+ myeloid cells generated by CD34+CD38high BM cells from JCML patients, but was at an undetectable level in normal controls. The addition of TPO to the culture containing SCF or SCF + GM-CSF caused a significant increase in the production of GM colony-forming cells by JCML CD34+CD38neg/low population, indicating the stimulatory effects of TPO on JCML primitive hematopoietic progenitors. Normal BM cells yielded a significant number of megakaryocytes as well as myeloid cells in response to a combination of SCF, GM-CSF, and/or TPO. In contrast, megakaryocytic cells were barely produced by the JCML progenitors. Our results may provide a fundamental insight that the administration of TPO enhances the aberrant growth of GM progenitors rather than the recovery of megakaryocytopoiesis.     

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, G-CSF, and IL-3 were found to be the most important HGF for production of neutrophilic cells. The 4-HGF combination of IL-3, IL-6, G-CSF, and SCF was optimized by performing dose-response experiments and shown to be as potent as 6 HGF for production of nascent CFU-GM and neutrophilic precursors.     

Influence of cytokines on the growth kinetics and immunophenotype of daughter cells resulting from the first division of single CD34(+)Thy-1(+)lin- cells

There is a need to determine whether culture conditions may exist for ex vivo expansion of hematopoeitic stem cells (HSC), which favor solely proliferative self-renewal of HSC as opposed to proliferation with differentiation. Using single cells, we studied the effects of individual and combinations of cytokines in serum-free medium on the kinetics of the first cell doubling and the resulting phenotype of each of individual daughter cell. CD34(+)Thy-1(+)lin- cells were plated 1 cell per well in Terasaki plates in serum-free medium containing cytokines. Each well containing a single cell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype of the daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with an undivided single cell, wells in which the cell had divided, and wells in which the cell had died were scored. The number of doublets with conserved phenotype (CD34(+)lin-) was compared to those wells with one or more differentiated daughter cells (CD34(+)lin+). Over 7 days, cells cultured in single factors showed that between 13% (interleukin-6 [IL-6]) and 29% (thrombopoietin [TPO]) of the cells were undivided, between 13% (IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) and greater than 60% (IL-11, IL-1, or hepatocyte growth factor [HGF]) died. When combinations of cytokines were used over 7 days, between 5% (FLT-3 ligand [FLT-3L], stem cell factor [SCF], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], beta nerve growth factor [betaNGF]) and 22% (FLT-3L + HGF) of the cells remained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68% (SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64. 6%) of cells with conserved phenotype (percent conserved doublets + percent with 1 cell conserved), followed by SCF + TPO, (50%) and the combination of FLT-3L, SCF, IL-3, IL-6, G-CSF, betaNGF (53%). These combinations also produced the highest yield of cells with conserved phenotype after one division (FLT-3L + TPO - 81 cells/100 initial cells, SCF + TPO - 68 cells/100 initial cells) (P =.01). Observation of the time of the initial cell division and phenotype of the daughter cells allowed us to identify candidate combinations of cytokines that promote maintenance of lin- cells (TPO), or recruit the primitive cells to divide and undergo phenotypic self-renewal (FLT-3L + TPO, SCF + TPO).     

Responses of circulating urea cycle and branched-chain amino acids to feeding in adult and aged Fischer-344 rats

The goal of our study was to identify cytokine combinations that would result in simultaneous ex vivo expansion of both the megakaryocyte (Mk) and granulocyte lineages, since these cell types have the potential to reduce the periods of thrombocytopenia and neutropenia following chemotherapy. We investigated the effects of cytokine combinations on expansion of the Mk (CD41a+ cells and colony forming unit [CFU]-Mk) and granulocyte (CD15+ cells and CFU-granulocyte/monocyte [GM]) lineages. Peripheral blood CD34+ cells were cultured in serum-free medium with interleukin 3 (IL-3), stem cell factor (SCF), and various combinations of thrombopoietin (TPO), IL-6, GM-CSF, and/or G-CSF. The Mk lineage was primarily influenced by TPO in our cultures, although Mk and CFU-Mk numbers were increased when TPO was combined with IL-6. The primary stimulator of the granulocyte lineage was G-CSF, although many synergistic and additive effects were observed with addition of other factors. Expansion of CFU-GM increased upon addition of more cytokines. The cytokine combination of IL-3, SCF, TPO, IL-6, GM-CSF and G-CSF produced the greatest number of granulocytes and CFU-GM. The minimum cytokines necessary for expansion of both the Mk and granulocyte lineages included TPO and G-CSF, since no other factors examined could increase Mk and granulocyte numbers to the same extent. The number of hematopoietic progenitors produced in our culture system should be sufficient for successful engraftment following myelosuppressive therapy if produced on a scale of about one liter.     

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Effects of stem cell factor (SCF) on human marrow neutrophil, neutrophil/macrophage mixed, macrophage and eosinophil progenitor cell growth

The effects of stem cell factor (SCF) on the subpopulations of granulocyte/macrophage colony-forming units (CFU-GM) were examined. Hematopoietic progenitor cells were enriched from normal adult bone marrow specimens by immunomagnetic beads using an anti-CD34 antibody and lineage marker antibodies for positive selection and negative selection, respectively. SCF enabled neutrophil and neutrophil/macrophage mixed progenitors to respond to granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3) and to develop the colony and further cluster formation. The neutrophil colonies stimulated by GM-CSF or IL-3 consisted mainly of immature cells, while the colonies stimulated by granulocyte colony-stimulating factor (G-CSF) consisted of mature neutrophils irrespective of the addition of SCF. In macrophage and eosinophil lineages, SCF augmented the colony formation in the presence of GM-CSF or IL-3, whereas the enhancement of total progenitor cell growth (colonies plus clusters) was not so marked as compared with the neutrophil lineage. Time-course observation revealed that SCF could stimulate macrophage and eosinophil progenitors to form colonies rapidly. These findings indicate that SCF acts on late myeloid progenitor cells in manners different from the lineages of commitment.     

Effect of thrombopoietin on proliferation of blasts from CD7-positive acute myelogenous leukaemia

We investigated the effect of thrombopoietin (TPO) on the growth of leukaemic blasts from 30 acute myelogenous leukaemia (AML) patients according to the surface expression of CD7 and CD34: 10 patients were CD7 positive (CD7+), nine were CD7 negative/CD34+ (CD7-/CD34+) and the remaining 11 were CD7-/CD34-. Significant growth response of leukaemic blasts to TPO was observed in 10/10 CD7+, 5/9 CD7-/CD34+ and 2/11 CD7-/CD34- AML cases using 3H-thymidine incorporation. Synergistic stimulatory effects of TPO with stem cell factor (SCF), interleukin-3 (IL-3), granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor were observed in both TPO-responding cases (9/17) and TPO-non-responding cases (8/13). In a leukaemic blast colony assay. significant growth response to TPO was observed in 5/6 CD7+ and 4/17 CD7-AML cases examined. However, the effect of TPO on the growth of CD7+ leukaemic blasts was not so potent as that of IL-3 and SCF, both of which support the proliferation of primitive haemopoietic progenitors. Expression of c-mpl (TPO receptor) was significantly higher in CD7+ AML cases than in CD7- cases, suggesting a relationship between expression of c-mpl and proliferative response to TPO. These data indicate that CD7+ leukaemic blasts express functional TPO receptors and proliferate in response to TPO. These observations also imply that CD7 expression on AML blasts may indicate involvement of leukaemic progenitors at an early stage of multipotent haemopoietic stem cells.     

Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture

Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive- or negative-acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma-stem cell interactions. We have used a sensitive bioassay to investigate production of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and stem cell growth factor (SCF), by normal and AA stroma in long-term bone marrow culture (LTBMC). LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension. These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34+ cells, in clonogenic assays, in the absence of exogenous cytokines. Cytokines responsible for the colony-stimulating activity (CSA) and burst-promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines. All normal stroma populations produced G-CSF and GM-CSF, 93% produced IL-3, 80% produced IL-6, and 70% produced SCF. Similarly, all AA stroma produced G-CSF and GM-CSF, and 71% produced SCF. In contrast, only 71% of AA stroma produced IL-3 and 36% produced IL-6. Target cell stimulation was not dependent on direct stroma-target cell contact, suggesting production of soluble cytokines. However, although both IL-6 and G-CSF were detected in LTBMC supernatants by enzyme-linked immunoassay (ELISA), IL-3 and GM-CSF were undetectable, perhaps indicating low-level local production of these factors.     

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 10(3)-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface IgM (sIgM) after 5 weeks of co-culture. CD34+CD19- cells also showed a similar development of CD19+ cells and CD19+sigM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19-CD13- CD33- cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19-CD13-CD33- progenitors require the cell-cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.     

The effects of Flk-2/flt3 ligand as compared with c-kit ligand on short-term and long-term proliferation of CD34+ hematopoietic progenitors elicited from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood

The Flk-2/flt3 ligand (FL) was evaluated and compared with c-kit ligand (KL) for its in vitro proliferative effects on CD34+ cells from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood. Using a 7-day liquid culture system, FL in combination with interleukin-3 (IL-3), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF) was comparable with KL in combination with IL-3, IL-6, and G-CSF for the expansion of hematopoietic progenitors. When FL-containing cultures were assayed after 21 or 28 days, a greater number of progenitors were generated as compared with KL-containing cultures. Using bone marrow microvascular endothelial cells as support stroma, cultures supplemented with FL generated a greater number of progenitors in both the nonadherent and adherent layers at day 35. These data suggest that FL ligand, in combination with other cytokines, can be used for short-term ex vivo expansion of hematopoietic progenitors and facilitates the preservation and possible expansion of primitive cells capable of long-term generation of progenitors.     

Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.     

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.     

Abnormalities of nasal potential difference measurement in Liddle''s syndrome

Vascular endothelial growth factor (VEGF) production was analysed in megakaryocytic cell lines and CD34+ haematopoietic progenitors following treatment with thrombopoietin (TPO). In CMK cells TPO caused a time- and dose-dependent increase in the levels of VEGF released into the medium. A similar effect was observed in UT-7/mpl cells transfected with the TPO receptor c-Mpl, but not in parental UT-7 cells. In CD34+ haematopoietic progenitor cell cultures TPO stimulated VEGF mRNA expression and VEGF protein release. Production of VEGF in CD34+ cultures increased with TPO-induced megakaryocytic differentiation, but not with erythroid or myelomonocytic differentiation induced respectively by erythropoietin and granulocyte-macrophage colony-stimulating factor. These results demonstrate that TPO stimulates VEGF release in c-Mpl-expressing cells and suggest that this process is an integral feature of the megakaryocytic differentiation programme.     

Circumferential forearm fasciocutaneous free flap reconstruction of forequarter amputation/chest wall resection using simultaneous extra-anatomic revascularization (SEAR)

In recent years, many cytokines have been defined and some of them used clinically. In hematological malignancies, cytokines, including granulocyte colony-stimulating factor (G-CSF), have been widely used for leukopenia after chemotherapy. However, in acute myelogenous leukemia (AML), some leukemic cells may be induced to proliferate by these cytokines and they must be used with care. In this study, we have investigated cell reactivity and proliferation with G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CMF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF) and thrombopoietin (TPO) in cases of AML. We have also investigated the reactivity of some myeloid leukemia cell lines to TPO. G-CSF, GM-CSF, M-CSF, SCF and TPO caused proliferation of leukemic cells in 25%, 58.3%, 8.3%, 21.1% and 0% of cases, respectively. Because of this result, the use of G-CSF in AML should be regarded as potentially hazardous. TPO did not cause proliferation of leukemic cells in any case of AML, or in cell lines except MO7E, which is a megakaryocytic cell line. This result suggests that TPO might cause proliferation of some megakaryocytic leukemia cells. We cannot conclude that TPO does not cause proliferation of other AML cells, as the number of cases was small and it has been reported elsewhere that leukemia cells may proliferate when exposed to TPO in 50% of AML cases. Reactivity of AM L cells to TPO is an important factor when deciding the indications of TPO in AML and myelodysplastic syndrome.     

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.     

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.     

The potential role of FLT3 ligand in progenitor and primitive hematopoietic cell expansion

We have previously defined the experimental conditions for hematopoietic cell expansion. CD34+ human marrow cells were maintained in a serum-free, stroma-free liquid culture system, at a concentration of 10(3) cells/ml, for 10 days at 37 degrees C, in the presence of various cytokine combinations. The basic combination of early cytokines SCF (100 ng/ml), IL3 (5 ng/ml), IL6 (10 ng/ml), has a modest stimulating effect on all compartments: the number of total cells increased 56-fold and CD34+ cells 1-fold; CFU-GM, BFU-E and CFU-MK, increased 6-fold, 5-fold and 3-fold respectively. As far as CD34+ cells are concerned, the subpopulation CD34+/CD38- was only maintained. Interestingly, the addition of 100 ng/ml of Flt3 ligand (FL) significantly enhanced the amplification of total cells (276-fold), CFU-GM (54-fold) and BFU-E (15-fold). The number of CD34+ cells and the subpopulation CD34+/38- increased to 7-fold and 22-fold respectively. Moreover, long term culture-initiating cells (LTC-ICs) in limiting dilution assay (LDA) were found to increase 3-fold. Further addition of MGDF (10 ng/ml), G-CSF (10 ng/ml) and Epo (0.5 U/ml), in various combinations, acted synergically with the previous cytokine combination to support the formation of multiple types of hematopoietic colonies. As expected, the addition of MGDF increased the number of CFU-MK up to 5-fold expansion. Interestingly, MGDF addition was synergistic also for BFU-E and CFU-GM expansion. In the combination of SCF+ IL3+ IL6+ FL + MGDF, CFU-GM expanded to 73-fold and BFU-E to 17-fold. G-CSF in SCF + IL3 + IL6 + FL conditions stressed the expansion of the granulopoietic compartment doubling the number of CFU-GM and CD33+ cells, with no consequence on LTC-IC or BFU-E. Surprisingly, G-CSF induced the expansion of the megakaryocytic lineage up to 6-fold, in a similar way as MGDF. Epo in presence of SCF+ IL3+ IL6+/-FL dramatically increased total cell expansion (2300-2800-fold), mainly erythroblastic (70% glycoA) without exhaustion of all other compartments. The simultaneous use of these three cytokines (MGDF + G-CSF + Epo) in presence of four early cytokines (SCF + IL3 + IL6 + FL) clearly allows a significant expansion of all hematopoietic compartments, precursors, progenitors, and primitive stem cells. In conclusion, these data show the ability of a stroma-free, serum-free liquid system to expand all myeloid lineages, including CFU-MK and LTC-IC which are critical for clinical application of ex vivo expanded cells.     

Recombinant human c-Mpl ligand (thrombopoietin) not only acts on megakaryocyte progenitors, but also on erythroid and multipotential progenitors in vitro

To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma-containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU-Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst-promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.     

A double-blind comparison of the efficacy and safely of paroxetine and imipramine in the treatment of depression with dementia

Marrow stromal cells of patients treated by autologous bone marrow transplantation (ABMT) for malignancies have been assessed for their ability to secrete granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), transforming growth factor beta1 (TGFbeta1) and macrophage inflammatory protein-1alpha. (MIP-1alpha). Long-term marrow cultures were established from 10 patients prior to and 3 months after ABMT, from 7 patients 1 yr after ABMT and from 11 controls. Cytokines in culture supernatants of stromal layers (SL) were evaluated by enzyme-linked immunosorbent assay (ELISA). Significant differences between patient groups and controls were apparent in baseline production of GM-CSF, SCF, MIP-1alpha and TGFbeta1. After IL-1beta addition in cultures, G-CSF production was reduced in pretransplant and post-transplant patients compared to controls. The production of TGFbeta1, LIF, IL-6 and more particularly SCF were reduced in post-transplant patients, while elevated levels of GM-CSF and MIP-1alpha were observed in these patients only when the values were corrected for the number of cells growing in the SL. These results indicate a prolonged stromal defect in growth factor production following ABMT for the early-stage acting cytokines IL-6, LIF and SCF as well as for G-CSF, but not for GM-CSF, while the production of the 2 inhibitors shows different pathways.