Granulocyte colony-stimulating factor mobilizes dormant hematopoietic stem cells without proliferation in mice.

Granulocyte colony-stimulating factor (G-CSF) is used clinically to treat leukopenia and to enforce hematopoietic stem cell (HSC) mobilization to the peripheral blood (PB). However, G-CSF is also produced in response to infection, and excessive exposure reduces HSC repopulation capacity. Previous work has shown that dormant HSCs contain all the long-term repopulation potential in the bone marrow (BM), and that as HSCs accumulate a divisional history, they progressively lose regenerative potential. As G-CSF treatment also induces HSC proliferation, we sought to examine whether G-CSF-mediated repopulation defects are a result of increased proliferative history. To do so, we used an established H2BGFP label retaining system to track HSC divisions in response to G-CSF. Our results show that dormant HSCs are preferentially mobilized to the PB on G-CSF treatment. We find that this mobilization does not result in H2BGFP label dilution of dormant HSCs, suggesting that G-CSF does not stimulate dormant HSC proliferation. Instead, we find that proliferation within the HSC compartment is restricted to CD41-expressing cells that function with short-term, and primarily myeloid, regenerative potential. Finally, we show CD41 expression is up-regulated within the BM HSC compartment in response to G-CSF treatment. This emergent CD41Hi HSC fraction demonstrates no observable engraftment potential, but directly matures into megakaryocytes when placed in culture. Together, our results demonstrate that dormant HSCs mobilize in response to G-CSF treatment without dividing, and that G-CSF-mediated proliferation is restricted to cells with limited regenerative potential found within the HSC compartment.

Superior integrin activating capacity and higher adhesion to fibrinogen matrix in buffy coat-derived platelet concentrates (PCs) compared to PRP-PCs.

BACKGROUND: Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study. METHODS: PRP- and BC-platelet concentrates (PCs) were subjected to flowcytometry analysis to examine the expression of platelet activation marker, P-selectin as well as active confirmation of the GPIIb/IIIa (αIIbß3) on day 0, 1, 3 and 5 post-storage. Platelet adhesion to fibrinogen matrix was evaluated by fluorescence microscopy. Glucose concentration and LDH activity were also measured by colorimetric methods. RESULTS: The increasing P-selectin expression during storage was in a reverse correlation with PAC-1 binding (r = -0.67; p = .001). PRP-PCs showed the higher level of P-selectin expression than BC-PCs, whereas the levels of PAC-1 binding and platelet adhesion to fibrinogen matrix were significantly lower in PRP-PCs. Higher levels of active confirmation of the GPIIb/IIIa in BC-PCs were also associated with greater concentration of glucose in these products. CONCLUSION: We demonstrated the superior capacities of integrin activation and adhesion to fibrinogen for BC-PCs compared to those of PRP-PCs. These findings may provide more advantages for BC method of platelet preparation.

Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.

UNLABELLED: Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. KEY POINTS: Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

CD41 is developmentally regulated and differentially expressed on mouse hematopoietic stem cells.

CD41 expression is associated with the earliest stages of mouse hematopoiesis. It is notably expressed on some cells of the intra-aortic hematopoietic clusters, an area where the first adult-repopulating hematopoietic stem cells (HSCs) are generated. Although it is generally accepted that CD41 expression marks the onset of primitive/definitive hematopoiesis, there are few published data concerning its expression on HSCs. It is as yet uncertain whether HSCs express CD41 throughout development, and if so, to what level. We performed a complete in vivo transplantation analysis with yolk sac, aorta, placenta, and fetal liver cells, sorted based on CD41 expression level. Our data show that the earliest emerging HSCs in the aorta express CD41 in a time-dependent manner. In contrast, placenta and liver HSCs are CD41⁻. Thus, differential and temporal expression of CD41 by HSCs in the distinct hematopoietic territories suggests a developmental/dynamic regulation of this marker throughout development.

Platelets induce endothelial tissue factor expression in a mouse model of acid-induced lung injury.

Although the lung expresses procoagulant proteins under inflammatory conditions, underlying mechanisms remain unclear. Here, we addressed lung endothelial expression of tissue factor (TF), which initiates the coagulation cascade and expression of which signifies development of a procoagulant phenotype in the vasculature. To establish the model of acid-induced acute lung injury (ALI), we intranasally instilled anesthetized mice with saline or acid. Then 2 h later, we isolated pulmonary vascular cells for flow cytometry and confocal microscopy to detect the leukocyte antigen, CD45 and the endothelial markers VE-cadherin and von Willebrand factor (vWf). Acid increased both the number of vWf-expressing cells as well as TF and P-selectin expressions on these cells. All of these effects were markedly inhibited by treating mice with antiplatelet serum, suggesting the involvement of platelets. The increased expressions of TF, vWf, and P-selectin in response to acid also occurred in platelets. Moreover, the effects were replicated in endothelial cells derived from isolated, blood-perfused lungs. However, the effect was inhibited completely in lungs perfused with platelet-depleted and, to a lesser extent, with leukocyte-depleted blood. Acid injury increased endothelial expressions of the platelet proteins, CD41 and CD42b, providing evidence that platelet proteins were transferred to the vascular surface. Reactive oxygen species (ROS) were implicated in these responses, in that the endothelial and platelet protein expressions were inhibited. We conclude that acid-induced ALI causes NOX2-mediated ROS generation that activates platelets, which then generate a procoagulant endothelial surface.

Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation.

Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain. Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1(W84X/W84X) embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval "bloodless" phase and develop to fertile adults with multilineage hematopoiesis. Using cd41-green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1(W84X/W84X) embryos developed cd41(+) HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1.

miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53.

The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a is strongly up-regulated during phorbol ester-induced megakaryocyte differentiation, but not during hemin-induced erythrocyte differentiation. Enforced expression of miR-34a in K562 cells inhibits cell proliferation, induces cell-cycle arrest in G(1) phase, and promotes megakaryocyte differentiation as measured by CD41 induction. miR-34a expression is also up-regulated during thrombopoietin-induced differentiation of CD34(+) hematopoietic precursors, and its enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates expression of MYB, facilitating megakaryocyte differentiation, and of CDK4 and CDK6, to inhibit the G(1)/S transition. However, these miR-34a target genes are down-regulated rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This suggests that miR-34a is not responsible for the initial down-regulation but may contribute to maintaining their suppression later on. Previous studies have implicated miR-34a as a tumor suppressor gene whose transcription is activated by p53. However, in p53-null K562 cells, phorbol esters induce miR-34a expression independently of p53 by activating an alternative phorbol ester-responsive promoter to produce a longer pri-miR-34a transcript.

Classification of Iranian patients with Glanzmann's Thrombasthenia using a flow cytometric method.

Glanzmann's Thrombasthenia (GT) is a rare inherited autosomal recessive platelet disorder caused by a deficiency or dysfunction of the GPIIb-IIIa receptor on platelets, which is characterized by a lack of platelet aggregation in response to multiple physiologic agonists and a life-long bleeding disorder. Flow cytometry is a rapid and highly sensitive method that can detect reduced levels of receptors, as well as absolute deficiency. The aim of this study was to classify Iranian GT patients by a flow cytometric method, and to correlate these findings with the severity of clinical bleeding. The expression of GPIIb-IIIa on the platelet surface was assessed in 123 GT patients using quantitative flow cytometry to determine the most common subtype among these patients. We used a panel of antibodies to detect the expression of glycoproteins GPIb, GPIIb, GPIIIa, as well as Integrin αv. Patients were also interviewed with regard to the severity and frequency of bleeding, according to history and gender, in order to evaluate the nature of their bleeding phenotype, and classify them as mild, moderate or severe bleeders, in accordance with the Glanzmann's Thrombasthenia Italian Team (GLATIT) protocol. In the detailed analysis of the results of our investigation, 95 out of 123 (77.5%) were classified as type I; 20 (16%) as type II with residual GPIIb-IIIa, and eight (6.5%) as GT variants. The variant type was diagnosed by the inability of GPIIb-IIIa to bind fibrinogen, as evidenced by the absence of platelet aggregation in response to physiologic agonists. There was no significant correlation between bleeding severity and different subtypes of GT. This study demonstrates that GT type I is the most common subtype among Iranian patients. There was no correlation between severity of symptoms and cytometric phenotype of the disease. The identification of families at risk may significantly decrease the incidence of the severe form of the disorder if genetic counseling is provided.

Cholinergic drugs inhibit in vitro megakaryopoiesis via the alpha7-nicotinic acetylcholine receptor.

Besides thrombopoietin several additional factors (i.e neurotransmitters and receptors) are known to be involved in the regulation of megakaryopoiesis at different stages. Recently, we identified functional α7 nicotinic acetylcholine receptors (nAChRα7) on platelets and megakaryocytic precursors. In platelets nAChRα7 form functional Ca(2+) channels and are involved in fibrinogen receptor activation and aggregation. Here, we investigated the impact of nAChRα7 on the differentiation of the human megakaryoblastic cell line MEG-01. In vitro differentiation of MEG-01 cells was induced by the phorbol ester TPA for 5 days in the absence or presence of nicotine or the nAChRα7-selective antagonist methyllycaconitine (MLA), and this was monitored by the expression of the megakaryocytic antigens CD41 and CD61. In the presence of the cholinergic drugs (nicotine or MLA) CD41 and CD61 expression was significantly reduced, both at RNA and protein level. We postulate that the nAChRα7 receptor is involved in megakaryopoietic signal transduction and gene regulation. This could affect the generation of platelets in vivo and contribute to the development of novel therapeutic drugs that regulate platelet formation.

Lnk inhibits Tpo-mpl signaling and Tpo-mediated megakaryocytopoiesis.

Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor, c-mpl, activates multiple pathways including signal transducer and activator of transcription (STAT)3, STAT5, phosphoinositide 3-kinase-Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here, we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl, and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)-derived megakaryocyte culture. Consistent with this result, we found that in both BM and spleen, Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice. In addition, Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3, STAT5, Akt, and MAPK signaling pathways in CD41+ megakaryocytes. Furthermore, the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast, the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to, but is not essential for, inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus, Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo.

Downregulation of signal transducer and activator of transcription 5 (STAT5) in CD34+ cells promotes megakaryocytic development, whereas activation of STAT5 drives erythropoiesis.

Although it has been proposed that the common myeloid progenitor gives rise to granulocyte/monocyte progenitors and megakaryocyte/erythroid progenitors (MEP), little is known about molecular switches that determine whether MEPs develop into either erythrocytes or megakaryocytes. We used the thrombopoietin receptor c-Mpl, as well as the megakaryocytic marker CD41, to optimize progenitor sorting procedures to further subfractionate the MEP (CD34(+)CD110(+)CD45RA(-)) into erythroid progenitors (CD34(+)CD110(+)CD45RA(-)CD41(-)) and megakaryocytic progenitors (CD34(+)CD110(+)CD45RA(-)CD41(+)) from peripheral blood. We have identified signal transducer and activator of transcription 5 (STAT5) as a critical denominator that determined lineage commitment between erythroid and megakaryocytic cell fates. Depletion of STAT5 from CD34(+) cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in an increase in megakaryocytic progenitors (CFU-Mk), whereas erythroid progenitors (BFU-E) were decreased. Furthermore, an increase in cells expressing megakaryocytic markers CD41 and CD42b was observed in STAT5 RNAi cells, as was an increase in the percentage of polyploid cells. Reversely, overexpression of activated STAT5A(1*6) mutants severely impaired megakaryocyte development and induced a robust erythroid differentiation. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed changes in expression of a number of genes, including GATA1, which was downmodulated by STAT5 RNAi and upregulated by activated STAT5.

GATA1-mediated megakaryocyte differentiation and growth control can be uncoupled and mapped to different domains in GATA1.

The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.

The cdx-hox pathway in hematopoietic stem cell formation from embryonic stem cells.

Embryonic stem cells (ESCs) differentiated in vitro will yield a multitude of hematopoietic derivatives, yet progenitors displaying true stem cell activity remain difficult to obtain. Possible causes are a biased differentiation to primitive yolk sac-type hematopoiesis, and a variety of developmental or functional deficiencies. Recent studies in the zebrafish have identified the caudal homeobox transcription factors (cdx1/4) and posterior hox genes (hoxa9a, hoxb7a) as key regulators for blood formation during embryonic development. Activation of Cdx and Hox genes during the in vitro differentiation of mouse ESCs followed by co-culture on supportive stromal cells generates ESC-derived hematopoietic stem cells (HSCs) capable of multilineage repopulation of lethally irradiated adult mice. We show here that brief pulses of ectopic Cdx4 or HoxB4 expression are sufficient to enhance hematopoiesis during ESC differentiation, presumably by acting as developmental switches to activate posterior Hox genes. Insights into the role of the Cdx-Hox gene pathway during embryonic hematopoietic development in the zebrafish have allowed us to improve the derivation of repopulating HSCs from murine ESCs.

Expression of platelet surface receptors and early changes in platelet function in patients with STEMI treated with abciximab and clopidogrel versus clopidogrel alone.

BACKGROUND: Platelets play a crucial role in the pathogenesis of acute coronary syndromes (ACS). The efficacy of antiplatelet treatment is pivotal in the success of percutaneous coronary intervention (PCI) performed in patients with ACS. OBJECTIVE: The aim of the study was to investigate the effects of clopidogrel with or without abciximab on the expression of platelet surface receptors and platelet function in patients with ST-segment elevation myocardial infarction (STEMI) undergoing PCI. MATERIALS AND METHODS: Thirty patients with STEMI were included in the study. During acute primary coronary intervention, patients received aspirin (acetylsalicylic acid) and clopidogrel in a loading dose of 300mg. Clopidogrel was the only antiplatelet therapy used by nine patients (group B). Twenty-one patients (group A) received additional abciximab. Blood samples were collected and analyzed twice: before and up to 22 hours after administration of antiplatelet therapy. The platelet aggregation was established as primary platelet-related hemostasis (closure time [CT] assessed using the PFA100 system). The absolute number of platelet surface antigens as CD41a, CD42a, CD42b, CD61, and CD62P were determined by flow cytometry analysis. RESULTS: The study revealed a statistically significant increase in CT induced by adenosine diphosphate and adrenaline (epinephrine) +130 seconds (p < 0.0001) and +94 seconds (p < 0.0001), respectively, in group A patients post-therapy. While in group B the parameters of CT did not change after treatment. In addition, the absolute number of CD41a antigens (glycoprotein [GP] IIb/IIIa) increased significantly after treatment in group A. No significant changes were observed after treatment in the expression of CD62P (P-selectin) antigens in either treatment group. There was a significant reduction in the percentage of CD62P-positive platelets in group B after antiplatelet therapy. CONCLUSIONS: The absolute number of GP IIb/IIIa receptors increases and platelets are not activated up to 12 hours after cessation of abciximab therapy. Treatment of STEMI patients undergoing PCI with a loading dose of clopidogrel reduces the percentage of active platelets but does not influence the CT.

Cord blood in vitro expanded CD41 cells: identification of novel components of megakaryocytopoiesis.

BACKGROUND: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.

Systemic white blood and endothelial cell response after revascularization of critical limb ischemia is only influenced in case of ischemic ulcers.

AIM: The aim of this study was to study the inflammatory response to open revascularization of an ischemic leg in terms of activation of white blood cells (WBC), platelets and endothelial cells. DESIGN: prospective study. METHODS: Venous samples from 21 patients suffering critical limb ischemia (CLI) were drawn before, and 4 weeks after (20 patients) revascularization. Total WBC, differentiated WBC, and platelets were counted. Expression of CD11b/CD18 on granulocytes and monocytes and CD41 on platelets was measured by flow cytometry. Soluble endothelial markers (sICAM-1, sVCAM-1, sE-selectin and sP-selectin) were analysed with ELISA. RESULTS: WBC and granulocyte count decreased in the subgroup of patients with ulcer and gangrene but no change in activation of WBC was recorded. The endothelial marker sICAM-1 decreased while VCAM-1 increased following surgery, most evident in the subgroup with ulcers and gangrene. CONCLUSIONS: This study shows that revascularization of CLI does not significantly influence the inflammatory response in patients with rest pain only, but a limited response of down regulation was found in the ulcer/gangrene patients probably as an effect of healing ulcers.

Inefficient megakaryopoiesis in mouse hematopoietic stem-progenitor cells lacking T-bet.

Differentiation of hematopoietic stem-progenitor cells (HSPCs) into mature blood lineages results from the translation of extracellular signals into changes in the expression levels of transcription factors controlling cell fate decisions. Multiple transcription factor families are known to be involved in hematopoiesis. Although the T-box transcription factor family is known to be involved in the differentiation of multiple tissues, and expression of T-bet, a T-box family transcription factor, has been observed in HSPCs, T-box family transcription factors do not have a described role in HSPC differentiation. In the current study, we address the functional consequences of T-bet expression in mouse HSPCs. T-bet protein levels differed among HSPC subsets, with highest levels observed in megakaryo-erythroid progenitor cells (MEPs), the common precursor to megakaryocytes and erythrocytes. HSPCs from T-bet-deficient mice exhibited a defect in megakaryocytic differentiation when cultured in the presence of thrombopoietin. In contrast, erythroid differentiation in culture in the presence of erythropoietin was not substantially altered in T-bet-deficient HSPCs. Differences observed with respect to megakaryocyte number and maturity, as assessed by level of expression of CD41 and CD61, and megakaryocyte ploidy, in T-bet-deficient HSPCs were not associated with altered proliferation or survival in culture. Gene expression micro-array analysis of MEPs from T-bet-deficient mice exhibited diminished expression of multiple genes associated with the megakaryocyte lineage. These data advance our understanding of the transcriptional regulation of megakaryopoiesis by supporting a new role for T-bet in the differentiation of MEPs into megakaryocytes.

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood.

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.

Megakaryocytopoiesis and apoptosis in patients with myelodysplastic syndromes.

In order to investigate simultaneously the megakaryocytopoiesis and apoptotic characteristics in bone marrow in patients with myelodysplastic syndromes (MDS), we used CD41 immunoenzyme (alkaline phosphatase anti-alkaline phosphatase) and DNA in situ end-labeling techniques on plastic embedded bone marrow biopsy sections of 29 MDS patients. Fourteen patients with iron deficiency anemia served as controls. The results showed that CD41-positive cells in MDS marrow numbered 26.2 +/- 18.2/mm2 (mean +/- standard deviation) compared with 15.6 +/- 7.1/mm2 in controls (P < 0.05). Numbers of cells with the morphology of micro-megakaryocytes in MDS marrow were significantly higher than in controls (P < 0.01). Furthermore, megakaryocytes in MDS marrow were frequently distributed along trabeculae (in 27 cases) and formed clusters (in 25 cases). Apoptotic megakaryocytes in MDS marrow accounted for just 4.4 and 9.3% of all CD41-positive cells and all apoptotic cells, respectively (P > 0.05 compared with controls), but apoptosis occurred only in micro-megakaryocytes. Based on these observations, we conclude that megakaryocytosis and dysmegakaryocytosis are the features of dyshematopoiesis in MDS marrow. Decreased thrombocyte production and thrombocyte release coming from increased dys(micro)megakaryocytes and abnormally located megakaryocytes perhaps play a more important role in peripheral thrombocytopenia than megakaryocytic apoptosis itself. Apoptosis of micro-megakaryocytes may be a protective biological mechanism to remove useless megakaryocytes.

Exploring erythropoiesis of common carp (Cyprinus carpio) using an in vitro colony assay in the presence of recombinant carp kit ligand A and erythropoietin.

The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, ß-globin, epor and gata1. Recombinant carp Kitla alone induced limited colony formation, whereas a combination of Kitla and Epo dramatically enhanced erythroid colony formation and colony cell growth, as well as stimulated the formation of thrombocytic/erythroid colonies expressing not only erythroid markers but also thrombocytic markers, cd41 and c-mpl. Utilizing this colony assay to examine the distribution of distinct erythroid progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in erythropoiesis, while the spleen plays a secondary. Furthermore, we showed that presumably bi-potent thrombocytic/erythroid progenitor cells localize principally in the trunk kidney. Our results indicate that teleost fish possess mechanisms of Epo- and Kitla-dependent erythropoiesis similar to those in other vertebrates, and also help to demonstrate the diversity of erythropoietic sites among vertebrates.