Megakaryocyte Progenitor Cells Research Articles

CD34+ cells derived from fetal liver contained a high proportion of immature megakaryocytic progenitor cells.

Endoreplication and maturation of the megakaryocyte (MK) may be retarded or delayed during ontogenesis. In this study, CD34+ cells were isolated from both human fetal liver and adult bone marrow and incubated with thrombopoietin (TPO). The cell number, morphological characteristics, platelet-associated antigen phenotype, maturation stage and DNA ploidy of CD41+ cells were examined from day 0 to day 12 in culture. 1) TPO stimulated the proliferation of fetal liver (FL)-derived CD34+ cells with a mean 73.14-fold increase of CD41+ cells after 12 d in culture. Adult BM-derived CD34+ cells increased only slightly, with a mean 8.18-fold increase of CD41+ cells. 2) Although the membrane phenotype of both FL CD34+-derived MKs and BM CD34+ -derived MKs analyzed with CD41a, CD42a, CD61 and CD34 were similar, all FL CD34+-derived MKs were in maturation stage I and II and in low ploidy (<4N) class. By comparison, BM CD34+ MKs possessed 15% MKs in maturation stage III and IV and with 23% MKs in high ploidy class ( > 4N). 3) Most of cultured FL-derived CD34+ cells did not have a well developed demarcation system (DM) and numerous alpha-granules after 12 d incubation. von Willebrand factor (vWF) appeared earlier on the cultured BM-derived CD34+ cells than on FL-derived CD34+ cells. 4) The expression of both cyclin E and cyclin B1 progressively increased in FL CD34+ cells induced by TPO during 12 d in culture. 5) The expression of cyclin D1 gradually decreased in FL CD34+ cells induced by TPO over 12 d incubation. 6) Immunocytochemical analysis showed that cyclin D3 was detected only in cytoplasm of cultured FL-derived CD34+ cells, whereas in both cytoplasm and nuclei of cultured BM-derived CD34+ cells. These data suggest that FL-derived CD34+ cells contain a high proportion of immature megakaryocytic progenitor cells. It further suggests that TPO can push these progenitor cells into proliferation by upregulating the expression of cyclins B1 and E, and drive a high proportion of cells into megakaryocytic lineage.

Regulation of megakaryocytopoiesis in an in vitro stroma model: Preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation

Objective Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. Materials and Methods Human CD34 + cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. Results After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the β 1 integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. Conclusion In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.

Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK)

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that acts as a stimulator of pre-B lymphocyte cell growth and as a chemoattractant for T cells, monocytes, and hematopoietic stem cells. More recent studies also suggest that megakaryocytes migrate in response to SDF-1. Because genetic elimination of SDF-1 or its receptor lead to marrow aplasia, we investigated the effect of SDF-1 on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-MK]). We report that SDF-1 augments the growth of CFU-MK from whole murine bone marrow cells when combined with thrombopoietin (TPO). The addition of SDF-1 to interleukin-3 (IL-3) or stem cell factor (SCF) had no effect. Specific antagonists for CXCR4 (the sole receptor for SDF-1), T22, and 1-9 (P2G) SDF-1 reduced megakaryocyte colony growth induced by TPO alone, suggesting that many culture systems contain endogenous levels of the chemokine that contributes to the TPO effect. To examine whether SDF-1 has direct effects on CFU-MK, we developed a new protocol to purify megakaryocyte progenitors. CFU-MK were highly enriched in CD41(high) c-kit(high) cells generated from lineage-depleted TPO-primed marrow cells. Because the growth-promoting effects of SDF-1 were also observed when highly purified populations of CFU-MK were tested in serum-free cultures, these results suggest that SDF-1 directly promotes the proliferation of megakaryocytic progenitors in the presence of TPO, and in this way contributes to the favorable effects of the bone marrow microenvironment on megakaryocyte development.

The residual megakaryocyte and platelet production in c-Mpl–deficient mice is not dependent on the actions of interleukin-6, interleukin-11, or leukemia inhibitory factor

Mice lacking thrombopoietin (TPO) or its receptor c-Mpl are severely thrombocytopenic, consistent with a dominant physiological role for this cytokine in megakaryocytopoiesis. However, these mice remain healthy and show no signs of spontaneous hemorrhage, implying that TPO-independent mechanisms for platelet production exist and are sufficient for hemostasis. To investigate the roles of cytokines that act through the gp130 signaling chain in the residual platelet production of mpl (-/-) mice, mpl (-/-)IL-6(-/-), mpl(-/-)LIF(-/-), and mpl(-/-)IL-11Ralpha(-/-) double-mutant mice were generated. In each of these compound mutants, the number of circulating platelets was no lower than that observed in mice lacking only the c-mpl gene. Moreover, the deficits in the numbers of megakaryocytes and megakaryocyte progenitor cells in the bone marrow and spleen were no further exacerbated in mpl(-/-)IL-6(-/-), mpl(-/-)LIF(-/-), or mpl(-/-)IL-11Ralpha(-/-) double-mutant mice compared with those in Mpl-deficient animals. In single IL-6(-/-), LIF(-/-), and IL-11Ralpha(-/-) mutant mice, platelet production was normal. These data establish that, as single regulators, IL-6, IL-11, and LIF have no essential role in normal steady-state megakaryocytopoiesis, and are not required for the residual megakaryocyte and platelet production seen in the c-mpl(-/-) mouse. (Blood. 2000;95:528-534)

Defective Megakaryocytic Development in Myelodysplastic Syndromes

Megakaryocytic proliferation and differentiation is typically abnormal in patients with myelodysplastic syndromes (MDS). The underlying mechanisms for this finding are not known, but may involve defects at the level of the thrombopoietin-receptor (c-mpl) or post-receptor signaling pathways in megakaryocyte progenitor cells. Premature apoptosis of the bone marrow cells and inhibitory effects of cytokines such as tumor necrosis factor alpha have been implicated as contributing to altered megakaryopoiesis in MDS, but their significance remains unclear. The availability of thrombopoietin (TPO) has facilitated more detailed analysis of megakaryocytic biology using several experimental in-vitro systems. However numerous studies have shown that the developmental abnormalities of MDS megakaryocytes could not be corrected by TPO. Increasing investigations are being extended to the evaluation of signal transduction pathways of c-mpl both in cell lines and human hematopoietic cells in order to identify the molecular mechanisms responsible for the defective megakaryocytic development in MDS.

Effects of recombinant thrombopoietin on the growth of murine primitive and committed hematopoietic progenitors in serum-free culture.

The aim of the present study was to determine the effect of thrombopoietin (Tpo) in combination with other cytokines on the growth of murine megakaryocytic, granulocyte-macrophage, erythroid and primitive hematopoietic progenitor cells, excluding possibilities of synergistic effects by serum factor(s). Serum-free clonogenic assay systems were used for assay of colony forming units in megakaryocytes (CFU-Mk), colony forming units in granulocytes-macrophages (CFU-GM), burst-forming units in erythrocytes (BFU-E) in marrow of normal mice and of high proliferative potential colony forming cells in 5-fluorouracil-treated marrow. Serum-free culture enabled megakaryocyte colony growth by recombinant murine (rm) Tpo and this was synergistically supported with rm interleukin (IL)-3, rm stem cell factor (SCF) or recombinant human (rh) erythropoietin (Epo). Recombinant human IL-6, rhIL-11 and rm granulocyte-macrophage colony stimulating factor (GM-CSF) showed synergistic effects with rmTpo only in the presence of serum. Recombinant murine IL-3 or rmSCF increased the large colonies and mixed-type colonies containing other populations, suggesting that these cytokines promoted the proliferation of immature populations of CFU-Mk. The rmTpo enhanced the growth of granulocyte-macrophage (GM) colonies stimulated by rmGM-CSF or rmIL-3 and erythroid bursts by rhEpo, with or without rmIL-3. The rmTpo also significantly increased HPP colonies in synergy with rmIL-3 only in the presence of serum or rmSCF. Serum-free culture is valuable for evaluating synergistic effects of cytokines and Tpo acts not only on megakaryocytic progenitors but also on granulocyte-macrophage, erythroid and primitive progenitor cells in combination with other cytokines, such as rmIL-3 and rmSCF. However, serum or SCF was required for enhancement of the colony growth of primitive progenitors by Tpo.

Expression and characterization of bioactive human thrombopoietin in the milk of transgenic mice.

Human thrombopoietin (hTPO) is the primary physiological regulator of platelet production and plays a pivotal role in promoting the proliferation and maturation of megakaryocytic progenitor cells and megakaryocytes. In this study, transgenic mice were produced harboring either full-length or the erythropoietin (EPO)-like amino-terminal domain of hTPO cDNA sequences fused to the regulatory elements of the bovine beta-casein gene. The transgene RNA was expressed exclusively in the mammary glands of eight transgenic mice, and a trace amount of the transgene was also found in the lungs of one mouse. The full-length form induced efficient expression of the protein with the highest expression level of 1500 microg/ml; however, the EPO-like domain alone expressed the protein at <0.1 microg/ml. The proteins from the two recombinant cDNAs have apparent molecular weights of about 74 and 17 kDa, due to glycosylation in the case of the full-length cDNA. Cell proliferation assay in vitro indicated that both of the recombinant forms stimulated proliferation of the TPO-dependent BaF3-Mpl cells. A positive correlation appeared between the amount of TPO in the milk of lactating animals and their blood platelet levels. About a twofold increase in platelet numbers in the blood was observed after direct subcutaneous injection of the recombinant hTPO at the level of 30 microg/kg of body weight. On the basis of these results, we anticipate that the recombinant hTPO produced efficiently in milk of transgenic mice will have the same activities as the native hTPO in a few in vivo as well as in vitro biochemical aspects.

Interaction of thrombopoietin with the platelet c-mpl receptor in plasma: binding, internalization, stability and pharmacokinetics.

Thrombopoietin (TPO) is the primary regulator of platelet production and acts through binding its receptor, c-mpl, found on megakaryocyte progenitor cells, megakaryocytes and platelets. Circulating levels of TPO are regulated primarily by the clearance of TPO after it binds to c-mpl receptors on circulating platelets. In this study the interaction of TPO with the platelet c-mpl receptor has been analysed under physiological conditions using radiochemical and pharmacokinetic approaches. 125I-rHuTPO was prepared using a novel method of gentle iodination that preserved its biological activity and used to demonstrate that platelets, but not endothelial cells, have a single class of binding sites (56 +/- 17 binding sites/platelet) with high affinity (Kd = 163 +/- 31 pM). Cross-linking experiments confirmed that TPO, but not erythropoietin (EPO), specifically associated with the 95 kD platelet c-mpl receptor. Upon addition of TPO to platelets, 80% of the TPO binding sites were internalized within an hour and were not recycled. TPO that was not bound by platelets was stable for up to 6 d in both platelet-poor and platelet-rich plasma. Using unlabelled recombinant human TPO (rHuTPO), standard pharmacokinetic analysis demonstrated that platelets have an average TPO clearance of 1.24 +/- 0.38 ml/h/109 platelets and that TPO clearance was reduced by low temperature but not by a number of drugs or metabolic inhibitors. The maximal amount of TPO removed by platelets in vitro was identical to that predicted by the total number of TPO binding sites. These results provide a biochemical and pharmacokinetic basis for the clinical use of TPO and for understanding possible disorders of platelet production.

Developmental Expression of Plasminogen Activator Inhibitor-1 Associated With Thrombopoietin-Dependent Megakaryocytic Differentiation

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet -granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34+/CD41− cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-typec-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor- (TNF-), and interleukin-1β (IL-1β) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34+/CD41− cells to CD34−/CD41+ cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.

Developmental Expression of Plasminogen Activator Inhibitor-1 Associated With Thrombopoietin-Dependent Megakaryocytic Differentiation

AbstractPlasminogen activator inhibitor-1 (PAI-1) is present in the platelet α-granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34+/CD41− cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-typec-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34+/CD41− cells to CD34−/CD41+ cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts.

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.

Circulating haemopoietic progenitor cells in primary and secondary myelofibrosis: relation to collagen and reticulin fibrosis.

The relationship between the extent of bone marrow reticulin and collagen fibrosis and the concentration of granulocytic (CFU-GM), erythroid (BFU-E) and megakaryocyte (CFU-Mk) progenitor cells in the peripheral blood of patients with primary agnogenic myeloid metaplasia (AMM) and secondary myelofibrosis (sMF) has not been definitively correlated. We studied 23 patients with established diagnosis of AMM and 12 patients with sMF for the extent of reticulin and collagen bone marrow fibrosis and for the spontaneous colony (sCFU-GM, sBFU-E and sCFU-Mk) formation. The control group consisted of 11 healthy volunteers. Trephine biopsy of the posterior iliac crest was performed in all individuals studied to determine the type and degree of reticulin and collagen fibrosis. Gomori's silver impregnation technique was used. sCFU-GM, sBFU-E and sCFU-Mk colony formation was related positively to spleen size, the white blood cell counts and the degree of collagen fibrosis in AMM (p < 0.01). Stimulated CFU-GM were also significantly correlated with the degree of bone marrow reticulin and collagen fibrosis. There was no correlation between the extent of peripheral blood progenitor concentration and the degree of bone marrow reticulin or collagen fibrosis in sMF and in control individuals. In conclusion, the extent of bone marrow fibrosis is significantly correlated with the peripheral blood progenitor colony formation in AMM but not in sMF.

New concepts in the aetiology and management of neonatal thrombocytopenia

Platelets are derived from haemopoietic stem cells via megakaryocyte progenitor cells and megakaryocytes. This process is controlled by haemopoietic growth factors, particularly thrombopoietin (Tpo). The healthy fetus possesses all of these elements of megakaryocytopoiesis. Despite this, thrombocytopenia is the commonest haematological abnormality in newborns, occurring mostly in preterm babies with impaired fetal megakaryocytopoiesis and platelet production following placental insufficiency/fetal hypoxia. The precise mechanism of this is unknown. Many neonatal complications then exacerbate this thrombocytopenic potential, and 20% of thrombocytopenias in neonatal intensive care (NICU) patients are severe (platelets < 50 × 10⁹/l). The available evidence suggests that avoiding severe thrombocytopenia by platelet transfusion in NICU patients reduces the risk of significant haemorrhage. As platelet underproduction underlies the majority of neonatal thrombocytopenias, recombinant haemopoietic cytokines, including Tpo, may be useful in ameliorating thrombocytopenia in NICU patients.

Cord blood is better than bone marrow for generating megakaryocytic progenitor cells

Thrombocytopenia remains an important problem for patients post high-dose chemotherapy and hematopoietic stem cell transplantation. The study of megakaryocytes, the direct precursors of platelets, has been hampered by their relatively low frequency in hematopoietic tissues. In an attempt to obtain a large number of functional megakaryocytic cells, we established a serum-free culture system to grow megakaryocytic progenitor cells derived from normal human bone marrow (BM) and cord blood (CB). Highly purified (purity >95%) CD34 + cells were obtained using magnetic cell sorting (MACS) followed by fluorescence activated cell sorting (FACS). The cells were cultured in a serum-free culture system for 3 weeks in the presence of a single dose of MGDF (50 ng/mL). On days 0, 5, 8, 12, 14, 18, and 21 of culture, the cellularity and morphology were examined. Megakaryocytic cells were monitored by detecting the expression of GPIIIa (CD61), GPIIb/IIIa (CD41) and GPIb (CD42b), and the distribution of megakaryocyte (MK) ploidy was analyzed by two-color flow cytometry. MGDF alone induced maximal nucleated cell expansion at day 14, resulting in a 38.20 ± 10.47-fold increase in cell number for CB and a 5.08 ± 1.30-fold increase in cell number for BM. On day 14 of the culture, the percentage of CD41 + /CD14 − cells derived from CB reached 73.54% ± 6.01% giving an absolute number of CD41 + /CD14 − cells of 27.25 ± 2.23 × 10 4/mL (27,250-fold increase), whilst the percentage of CD41 + /CD14 − cells derived from BM was only 29.21% ± 5.63% with an absolute number of 1.36 ± 0.26 × 10 4/mL (680-fold increase). Increased expression of GPIIIa occurred the earliest in culture, followed by GPIIb/IIIa, and then GPIb. The majority (81.6%–92.6%) of megakaryocytes (CD41 + cells) on day 14 of culture were 2N, although we did detect some 4N, 8N and greater ploidy cells. In conclusion, CD34 + cells stimulated by MGDF alone generated highly enriched MK progenitor cells at day 14 of serum-free culture. CB stem and progenitor cells have a greater proliferative response to MGDF alone than those derived from BM and may, therefore, prove to be a better source of cells for MK expansion.

Downregulation of c-kit (Stem Cell Factor Receptor) in Transformed Hematopoietic Precursor Cells by Stroma Cells

AbstractWe show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.

Downregulation of c-kit (Stem Cell Factor Receptor) in Transformed Hematopoietic Precursor Cells by Stroma Cells

We show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.

Extracellular Matrix Receptors and the Differentiation of Human Megakaryocytes in vitro

We investigated the expression and functions of extracellular matrix receptors (or integrins) in the course of the differentiation of human megakaryocytes (Mks) leading to the formation of platelets. Integrins β1 or Very Late Antigens (VLA) are specialized transmembrane receptors allowing the attachment of the cells to collagen (VLA-2), fibronectin (VLA-4 and-5) and laminin (VLA-6). A proportion of committed megakaryocytic progenitor cells (CFU-MK) adhere to fibronectin but not to collagen or laminin. The early immature Mks are retained on fibronectin (30%) and laminin (12%) but not on collagen whereas large mature Mks are still adherent to fibronectin and laminin and also acquired the capacity to adhere to collagen. The expression of the different VLA in the maturation of Mks correlates well with their adhesive properties. Hence, VLA-2 is not expressed on immature Mks but is present on the mature polyploid cells. VLA-4 is detected only on immature Mks which do not seem to bear VLA-5, while this last integrin appears on late Mks. VLA-6 showed a broad distribution from the early to late stages of Mks differentiation.Integrins β3 of the cytoadhesin family are represented by αIIbβ3 that is the receptor for fibrinogen and αVβ3 which mediates adhesion to vitronectin. αIIbβ3 is present on the CFU-MK and highly expressed throughout the Mks maturation stages while αVβ3 expression is much lower and seems to be detected only on the late Mks. The regulation of the expression of these receptors by cytokines and their respective roles in the maturation of Mks and the final production of platelets, are discussed. The development of efficient culture systems of human Mks in the presence of the recently cloned thrombopoietin will undoubtly help to shed more light on the molecular mechanisms of their interactions via integrins with the BM microenvironment.

Serum levels of thrombopoietin and stem cell factor in acute leukemia patients with chemotherapy-induced cytopenia and complicating infections

Thrombopoietin (Tpo) and stem cell factor (SCF) are growth factors for megakaryocyte progenitor cells and can also modulate platelet function. We have characterized variations in serum levels of these two cytokines in acute leukemia patients undergoing intensive chemotherapy. Compared with healthy controls, serum Tpo levels were significantly increased prior to consolidation chemotherapy, and serum levels were correlated to peripheral blood platelet counts. Serum Tpo levels increased when the patients developed chemotherapyinduced cytopenia, and a further increase was observed during complicating bacterial infections. In contrast to Tpo, SCF serum levels in leukemia patients did not differ from healthy controls neither before chemotherapy nor during the period of chemotherapy-induced cytopenia. Serum levels of Tpo (and possibly SCF) may influence thrombopoiesis and/or platelet functions in patients undergoing intensive chemotherapy for acute leukemia.

Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two-step separation procedure.

Cord blood (CB) transplantation is primarily performed in children, rather than in adults, due to the low number of haemopoietic progenitor cells obtained from the small volume of a single CB collection. Prolonged thrombocytopenia is a major problem following CB transplantation. Efforts are currently underway to expand the number of CB progenitor cells ex vivo, in order to enable transplantation in adults and to decrease the period of thrombocytopenia. In this study we investigated different techniques for enrichment and expansion of megakaryocyte (Mk) progenitor cells and haemopoietic stem cells from CB. CBs from 20 normal deliveries were depleted of red blood cells (RBC) by dividing each sample and testing cell separation on 3% gelatin, Hespan, Ficoll-Paque or a two-step 3% gelatin followed by Ficoll-Paque separation. The two-step procedure was found to be superior to the other methods in enrichment of the Mk progenitor cells (CFU-Mk) (34.3-fold), while at the same time retaining the number of myeloid and erythroid progenitors, CD34+ and CD41+ cells. In short-term (14d) liquid culture of non-adherent nucleated cells isolated by gelatin and Ficoll-Paque, a 40-fold expansion of clonable Mk progenitor cells was obtained in the presence of thrombopoietin (r-hu-TPO) and stem cell factor (r-hu-sCF. In similar cultures of isolated CD34+ cells, a 100-fold clonable Mk progenitor was obtained at day 14. Therefore this new technique may facilitate the ex vivo expansion of Mk progenitor cells and be adopted for future use in CB transplantation.

Bromocriptine markedly inhibits progression of murine retrovirus-induced immunodeficiency

Lithium salts have been demonstrated to induce the production of hematopoietic cells following administration in vivo and to minimize the reduction of these cells following treatment with either radiation, chemotherapeutic or antiviral drugs. We have previously demonstrated that lithium, when administered in vivo to immunodeficient mice infected with LP-BM5 MuLV (MAIDS) significantly reduced the development of lymphadenopathy, splenomegaly, and the lymphoma associated with late-stage immunodeficiency disease in this model, and increased the survival of these animals compared to virus-infected controls not receiving lithium. We report here the results of in vivo studies in the MAIDS model that determined the effect of lithium on peripheral blood indices and the number of myeloid (CFU-GM), erythroid (BFU-E) and megakaryocyte (CFU-Meg) hematopoietic progenitors from bone marrow and spleen harvested from immunodeficient mice receiving lithium carbonate (1 mM) placed in their drinking water compared to virus-infected controls not receiving lithium. Time-points evaluated were at weeks 1, 5, 9, 13, 17, and 21 postviral infection. Virus-control mice not receiving lithium demonstrated all the signs that are characteristic of MAIDS, i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, reduced hematopoiesis, and death. Infected mice receiving lithium demonstrated diminished presence of splenomegaly, lymphadenopathy, hypergammaglobulinemia, no suppression of hematopoiesis nor mortality. Enhanced hematopoiesis was demonstrated by neutrophilia, lymphocytosis, thrombocytosis, and erythrocytosis that was evident by increased myeloid, erythroid, and megakaryocyte progenitor cells cultured from bone marrow and spleen. These studies further demonstrate that lithium influences the disease process in the MAIDS model and restricts the development of hematopoietic suppression that develops in this retroviral animal model of immunodeficiency.