Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34⁺ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2), fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM-CD34⁺ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation. The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.     

Physiologically significant effects of pH and oxygen tension on granulopoiesis

OBJECTIVE: Granulocyte differentiation in the bone marrow (BM) takes place in regions with lower pH and O(2) tension (pO(2)) than those in the BM sinuses. This suggests that granulopoiesis will be enhanced at subvascular pH and pO(2). MATERIALS AND METHODS: The effects of pH AND pO2 on granulocyte proliferation, differentiation, and granulocyte colony-stimulating factor receptor (G-CSFR) expression were evaluated using mobilized peripheral blood CD34(+) cells directed down the granulocytic pathway with stem cell factor, interleukin 3, interleukin 6, and G-CSF. RESULTS: Cell expansion was enhanced at subvascular pH, with twice as many total cells and CD15(bright)/CD11b(+) late neutrophil precursors (myelocytes, metamyelocytes, bands) produced at pH 7.07 to 7.21 as was produced at pH 7.38. Low pH accelerated the rate of differentiation concomitant with this increase in proliferation. Also, total, CD15(bright)/CD11b(-) (promyelocytes, early myelocytes), and CD15(bright)/CD11b(+) cell expansion was enhanced at lower pO(2), with twice as many of each cell type produced at 5% O(2) as at 20% O(2). The effects of low pH and low pO(2) were additive, such that generation of total, CD15(bright)/CD11b(-), and CD15(bright)/CD11b(+) cells was 3.5-, 2.4-, and 4.0-fold greater at pH 7.21 and 5% O(2) than at the standard hematopoietic culture conditions of pH 7.38 and 20% O(2). Low pH resulted in faster upregulation of G-CSFR surface expression, whereas pO(2) had no effect on G-CSFR expression. CONCLUSION: These data provide compelling evidence that pH and pO(2) gradients within the BM play significant roles in regulating hematopoiesis. More rapid granulocytic cell proliferation and differentiation at low pH may be explained in part by more rapid G-CSFR expression. The ability to alter cell development by manipulating pH and pO(2) has important implications for optimizing ex vivo production of neutrophil precursors.     

Soluble interleukin-6 (IL-6) receptor with IL-6 stimulates megakaryopoiesis from human CD34(+) cells through glycoprotein (gp)130 signaling

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.     

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.     

c-Kit expression and stem cell factor-induced hematopoietic cell proliferation are up-regulated in aged B6D2F1 mice

Expression of c-Kit (CD117) and stem cell factor/c-Kit-mediated cell proliferation were tested in vitro in young and old B6D2F1 mice to study the role of c-Kit signaling in hematopoietic stem cell (HSC) senescence. Increasing age is associated with a significant increase in bone marrow (BM) cells without affecting mature blood cells. The number of c-Kit-expressing BM cells increased significantly in old mice when compared to young controls, to 201% in total BM cells, 261% in Lin(-) cells, 517% in Lin(-)CD34(+)Sca1(+) progenitor cells, and 1272% in Lin(-)CD34(-)Sca1(+) HSCs. Sorted Lin(-)Sca1(+)CD117(+) BM cells from an old mouse expanded 5-fold when cultured in vitro for 72 hours with stem cell factor at 25 ng/ml, which was significantly higher than a 2.5-fold expansion of the same cells from a young donor. HSCs and progenitor cells from B6D2F1 mice maintain extremely high proliferative potentials and do not reach proliferative arrest at old age during a normal life span.     

The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors.

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However, little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb), 97A6, that specifically detects human Ba, MC (lung, skin), and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils, eosinophils, monocytes, lymphocytes, platelets) did not react with MoAb 97A6, and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (>98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40), Ba-eosinophil colonies (7 of 40), Ba-macrophage colonies (3 of 40), and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast, no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells, starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (>30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors, MC progenitors, Ba progenitors, as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors.     

Normal human bone marrow CD34(+)CD133(+) cells contain primitive cells able to produce different categories of colony-forming unit megakaryocytes in vitro

OBJECTIVE: To evaluate the megakaryocyte potential of normal bone marrow (NBM) CD34(+)CD133(+) cells, a subset offering a possible alternative for clinical CD34 immunoselection, we evaluated their colony-forming unit megakaryocyte (CFU-Mk) content and their ability to produce clonogenic Mk progenitors in comparison with the CD133(-) subset. MATERIALS AND METHODS: Sorted NBM CD34(+)CD133(+) and CD34(+)CD133(-) subsets were evaluated for Mk clonogenic capacity before and after in vitro proliferation in serum-free liquid culture containing kit ligand, Flt3 ligand, thrombopoietin, interleukin-3, and interleukin-6. The segregation of CFU-Mk according to the expression of CD34, CD133, and CD41 was compared between fresh BM cells and expanded cells. RESULTS: Although the fresh NBM CD133(-)CD34(+) subset included two thirds CFU-Mk, only the CD133(+) subset contained primitive cells able to produce all categories of CFU-Mk in vitro. Immunophenotyping confirmed that CD41 antigen is nonspecific for Mk lineage and showed that the usual CD34(+)CD41(+) subset does not specifically define a CFU-Mk population. The segregation of CFU-Mk before and after expansion according to CD34, CD41, or CD133 was modified in relation with down-regulation of CD34 and CD133 antigens and up-regulation of CD41 antigen. CONCLUSIONS: The NBM CD133(+) subset contains primitive cells able to generate CFU-Mk, a subset probably relevant to platelet recovery after infusion. The alteration of antigen expression during in vitro proliferation calls for caution in the identification of the different categories of Mk subsets produced and in the assessment of their predictivity for in vivo platelet production.     

CD34+ cell selection is required to assess HOXA9 expression levels in patients with myelodysplastic syndrome

Overexpression of HOXA9 is linked to the molecular pathogenesis of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS), conferring a poor prognosis. HOXA9 expression levels were analysed in the diagnostic bone marrow (BM) samples of 13 MDS patients. HOXA9 was expressed by CD34(+) BM cells at median levels 3.1-fold higher than in CD34(-) cells from the same patient and at median levels 4.3-fold higher than in CD34(+) cells from healthy donors. These results indicate that CD34(+) cell selection is required to accurately assess the expression levels of HOXA9 and related genes in the multipotential malignant progenitor cells of MDS patients.     

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 (14 d) 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.     

Pluripotent and myeloid-committed CD34+ subsets in hematopoietic stem cell allografts

The present study compared the contents of pluripotent and lineage-committed hematopoietic progenitor cells (HPCs) in various types of allografts. Bone marrow (BM) allografts and single leukapheresis products (LPs) collected from G-CSF-mobilized donors contained similar amounts of pluripotent HPCs (CD34(+)CD38(-)) and total CD34(+) cells. However, the content of late-myeloid HPCs (CD34(+)CD33(+)CD15(+)) were significantly higher in BM grafts compared to LPs (P>0.02), whereas the contents of early-myeloid HPCs (CD34(+)CD33(+)CD15-) were 2.5-fold higher in LPs (P<0.03). In comparison to grafts from adult donors, cord blood (CB) grafts contained 26-65-fold lower amounts of early-myeloid HPCs (P<0.001), but only 8-12-fold lower contents of pluripotent HPCs (P<0.04). Additional findings demonstrated that among all tested parameters the numbers of early-myeloid HPCs were the most accurate measure of the total colony-forming cell (CFC) numbers in allografts. Hence, the earlier engraftment observed after transplantation of LPs compared to BM grafts might be explained by the higher content of early-myeloid HPCs/CFCs in LPs. Moreover, the slow engraftment following CB transplantation might not be affected essentially by the low number of myeloid HPCs, but rather by pluripotent HPCs. Finally, this study reports a new gating strategy for the enumeration of pluripotent CD34(+)CD38(-) subsets.     

Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor

The expression/function of vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt1 and VEGFR2/KDR/Flk1) in hematopoiesis is under scrutiny. We have investigated the expression of Flt1 and kinase domain receptor (KDR) on hematopoietic precursors, as evaluated in liquid culture of CD34(+) hematopoietic progenitor cells (HPCs) induced to unilineage differentiation/maturation through the erythroid (E), megakaryocytic (Mk), granulocytic (G), or monocytic (Mo) lineage. KDR, expressed on 0.5% to 1.5% CD34(+) cells, is rapidly downmodulated on induction of differentiation. Similarly, Flt1 is present at very low levels in HPCs and is downmodulated in E and G lineages; however, Flt1 is induced in the precursors of both Mo and Mk series; ie, its level progressively increases during Mo maturation, and it peaks at the initial-intermediate culture stages in the Mk lineage. Functional experiments indicate that Mk and E, but not G and Mo, precursors release significant amounts of VEGF in the culture medium, particularly at low O(2) levels. The functional role of VEGF release on Mk maturation is indicated by 2 series of observations. (1) Molecules preventing the VEGF-Flt1 interaction on the precursor membrane (eg, soluble Flt1 receptors) significantly inhibit Mk polyploidization. (2) Addition of exogenous VEGF or placenta growth factor (PlGF) markedly potentiates Mk maturation. Conversely, VEGF does not modify Mo differentiation/maturation. Altogether, our results suggest that in the hematopoietic microenvironment an autocrine VEGF loop contributes to optimal Mk maturation through Flt1. A paracrine loop involving VEGF release by E precursors may also operate. Similarly, recent studies indicate that an autocrine loop involving VEGF and Flt1/Flk1 receptors mediates hematopoietic stem cell survival and differentiation.     

Quinine-induced thrombocytopenia: drug-dependent GPIb/IX antibodies inhibit megakaryocyte and proplatelet production in vitro

The development of immune cytopenias is a well-recognized side effect of many drugs. Quinine- and quinidine-dependent antibodies are classic examples of drug-induced effects that cause severe, life-threatening thrombocytopenia. Whereas the effects of drug-dependent antibodies on platelets have been well documented, their effects on megakaryocyte (Mk) biology are still unclear. We analyzed sera from several quinine-induced thrombocytopenia (QITP) patients on highly pure Mks (98% glycoprotein IIb-positive [GPIIb(+)]; 92% GPIX(+)) derived from human CD34(+) cells cultured with human thrombopoietin. We demonstrate by flow cytometry and confocal microscopy that QITP IgGs bind Mks efficiently in the presence of quinine. Incubation of day-4 Mks with QITP sera or purified IgG resulted in induction of apoptosis, a significant decrease in cell viability, and an increase in cell death. Furthermore, QITP sera preferentially reduced the number of late GPIX(+)/GPIbα(+) Mks and the number of receptors per cell in the surviving population. Ploidy distribution, lobularity, and average cell size of Mks remained unchanged after treatment. In addition, treated Mks showed a marked decrease in their proplatelet production capacity, suggesting that drug-dependent antibodies hinder platelet production. Therefore, QITP antibodies considerably reduce the proplatelet production capabilities of Mks despite undetectable effects on DNA content, morphology, and cell size.     

CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern

Gene expression patterns of CD34(+)CD38(-) cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic epsilon-globin expression were observed-but no adult beta-globin message-in CD34(+)CD38(-) cells from ESCs, whereas high levels of beta-globin expression-but no embryonic epsilon-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34(+)CD38(-) cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.     

Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38- cells and SCID-repopulating cells

The CD34(+)CD38- phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34(+) cells, HSC content is difficult to measure since committed CD34(+)CD38+ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/severe combined immunodeficiency (SCID)-repopulating cells (SRCs). Contact coculture of fluorescence-activated cell sorter (FACS)-sorted bone marrow (BM) CD34(+)CD38- cells with human brain endothelial cells (HUBECs) supported a 4.4-fold increase in CD34(+)CD38- cells with a concordant 3.6-fold increase in SRCs over 7 days. Noncontact HUBEC cultures and the addition of thrombopoietin, stem cell factor (SCF), and macrophage colony stimulating factor I receptor (Fms)-like tyrosine kinase 3 (Flt-3) ligand supported further increases in CD34(+)CD38- cells (6.4-fold and 13.1-fold), which correlated with significant increases in SRC activity. Moreover, cell-sorting studies performed on HUBEC-cultured populations demonstrated that SRCs were significantly enriched within the CD34(+)CD38- subset compared with the CD34(-)CD38- population after culture. These results indicate that human HSCs can be identified and characterized by phenotype following expansion culture. These studies also demonstrate that HUBEC-elaborated soluble factors mediate a unique and potent expansion of human HSCs.     

Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation.

Peripheral blood stem cell (PBSC) transplants from HLA-A, -B, and -DR compatible unrelated donors (n = 45) were compared with bone marrow (BM; BM group, n = 45). Eighteen patients received CD34-selected PBSC (CD34 group). The PBSCs contained more mononuclear cells, CD34(+), CD3(+), and CD56(+) cells compared with marrow (P <.001). Engraftment was achieved in all 45 patients in the BM group, in 43 of 45 (95%) in the PBSC group, and in 14 of 18 (78%) in the CD34 group (P <.01). In multivariate analysis, a short time to absolute neutrophil count (ANC) equal to 0.5 x 10(9)/L was associated with the PBSC/CD34 groups (P <.001) and granulocyte colony-stimulating factor (G-CSF) treatment (P =.017). A short time to platelets equal to 50 x 10(9)/L was associated with PBSC (P =. 003) and no methotrexate (P =.015). Grades II-IV acute graft-versus-host disease (GVHD) was 20% in the BM controls, 30% in the PBSC group, and 18% in the CD34 group (not significant [NS]). The probability of chronic GVHD was 85% in the BM group, 59% in the PBSC group, and 0% in the CD34 group (P <.01). One-year transplant-related mortality was 21% and 27% and survival was 53% and 54% in the BM and PBSC groups, respectively (NS). The 2-year relapse-free survival was 41% and 46% in the two groups, respectively.     

Erythroid differentiation and maturation from peripheral CD34+ cells in liquid culture: cellular and molecular characterization

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation from BFU-E to mature erythrocytes both in normal and pathological conditions. Most of the available in vitro liquid cultures are from cell lines or are limited by the production of few erythroid cells mixed with myeloid cells. Here we describe an erythroid liquid culture system starting from CD34(+)-enriched cells obtained from peripheral blood. CD34(+) cells were cultured for 21 days in different conditions. Precisely stem cell factor (SCF, 20 ng/mL) and IL-3 (10 ng/mL) were added at starting point plus Epo (3 U/mL) at day 0 or 7 of culture with or without cyclosporine A (Cy; 1 mg/mL). In all the conditions, the highest recovery was obtained at day 14 of culture. Epo and Cy added at day 0 produced the highest cell expansion (170-fold mean amplification of the initial cell input by day 14) and recovery of erythroid cell. Sixty seven percent of the cells were GP(+) at day 7 and 97% by day 14 respectively. Most of the cells were proerythroblasts at day 7 and mature erythroblasts at day 14 (>90% were benzidine(pos)). The presence of Cy favoured erythroid differentiation and maturation and reduced the percentage of non-erythroid CD45(+) cells (2% with Cy versus 5% without Cy). Cells cultured with Epo and Cy reproduced erythropoiesis also at the molecular level. The results suggest that in 14 days different steps of human erythropoiesis from peripheral CD34(+) cells could be reproduced, with high recovery of highly purified erythroid cells. The high number and purity of erythroid cells produced from a small amount of peripheral blood make this method useful for studying either normal or pathological erythropoiesis.     

The susceptibility to X4 and R5 human immunodeficiency virus-1 strains of dendritic cells derived in vitro from CD34(+) hematopoietic progenitor cells is primarily determined by their maturation stage.

Dendritic cells (DC) were sorted on day 8 from cultures of CD34(+) cells with stem cell factor/Flt-3 ligand/ granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor-alpha (TNF-alpha)/interleukin-4 (IL-4). Exposing immature CCR5(+)CXCR4(lo/-) DC to CCR5-dependent human immunodeficiency virus (HIV)-1Ba-L led to productive and cytopathic infection, whereas only low virus production occurred in CXCR4-dependent HIV-1LAI-exposed DC. PCR analysis of the DC 48 hours postinfection showed efficient entry of HIV-1Ba-L but not of HIV-1LAI. CD40 ligand- or monocyte-conditioned medium-induced maturation of HIV-1Ba-L-infected DC reduced virus production by about 1 Log, while cells became CCR5(-). However, HIV-1Ba-L-exposed mature DC harbored 15-fold more viral DNA than their immature counterparts, ruling out inhibition of virus entry. Simultaneously, CXCR4 upregulation by mature DC coincided with highly efficient entry of HIV-1LAI which, nonetheless, replicated at the same low level in mature as in immature DC. In line with these findings, coculture of HIV-1Ba-L-infected immature DC with CD3 monoclonal antibody-activated autologous CD4(+) T lymphocytes in the presence of AZT decreased virus production by the DC. Finally, whether they originated from CD1a+CD14(-) or CD1a-CD14(+) precursors, DC did not differ as regards permissivity to HIV, although CD1a+CD14(-) precursor-derived immature DC could produce higher HIV-1Ba-L amounts than their CD1a-CD14(+) counterparts. Thus, both DC permissivity to, and capacity to support replication of, HIV is primarily determined by their maturation stage.     

Removal of autologous activated CD4-positive T lymphocytes also results in increased colony-forming units in patients with low and intermediate-1 risk myelodysplastic syndromes

Autologous activated CD4(+) T lymphocytes (CD4(+) /CCR5(+) double-positive cells) that derived from BMNCs of patients with low and intermediate-1 risk myelodysplastic syndrome were depleted or added to in vitro cultures. The BMNCs depleted of CD4(+) /CCR5(+) T cells exhibited significantly increased numbers of colony-forming units (CFUs). Conversely, the bone marrow mononuclear cells cultures with a fourfold augmentation of CD4(+) /CCR5(+) T lymphocytes exhibited no colonies in cultures in vitro. The apoptotic index (AI) of colony cells was decreased compared with that of preculture counterparts. After depletion of CD4(+) /CCR5(+) in vitro cultures, the clonal cells increased in patients with chromosome 5q- or 20q- abnormalities but remained unchanged in patients with trisomy 8. In addition, after removal of CD4(+) /CCR5(+) T cells, the number of CFUs was increased in those patients with a higher number of BM Th1 (CD4(+) / IFN-γ(+) ) cells, hypocellularity, or bearing the DR15 allele. We concluded that the selective removal of autologous activated CD4(+) T cells can increase the generation of CFUs. However, whether the increased CFUs consisted of cells derived from residual normal hemopoiesis or clonal hemopoiesis remains unknown.