WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study we investigated the role of WASP in the differentiation of CD34-positive (CD34+) cells isolated from the bone marrow of patients with WAS (n = 5) or with XLT (n = 4). Megakaryocyte colony formation was significantly decreased in patients with WAS when compared with normal controls. The formation of granulocyte-macrophage colonies and erythroid bursts were also decreased in WAS patinets. In contrast, in XLT patients, formation of all these colonies was normal. However, in vitro proplatelet formation of megakaryocytes induced by thrombopoietin was markedly decreased in both XLT and WAS. Electron microscopic examination revealed that megakaryocytes obtained from WAS or XLT patients grown in vitro had abnormal morphologic features, which seemed to be caused by defective actin cytoskeletal organization, including labyrinth-like structures of the demarcation membrane system and deviated distribution of the alpha-granules and demarcation membrane system. These observations indicate that WASP is involved in the proliferation and differentiation of CD34+ haemopoietic progenitor cells probably by its participation in signal transduction and in the regulation of the cytoskeleton.     

Simultaneous signalling through c-mpl, c-kit and CXCR4 enhances the proliferation and differentiation of human megakaryocyte progenitors: possible roles of the PI3-K, PKC and MAPK pathways

We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU-Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood-derived CD34(+)IL-6R(-) cells as a target. TPO alone induced a significant number of CFU-Meg colonies. Although stromal cell-derived factor-1 (SDF-1) or SCF alone did not support CFU-Meg colony formation, these factors had a synergistic effect on CFU-Meg colony formation in the presence of TPO. The combination of SDF-1, SCF and TPO induced twice as many CFU-Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU-Meg colony formation. LY294002 and GF109203X (inhibitors of PI3-K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU-Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF-1, and TPO + SCF + SDF-1, whereas it abolished the effect of SDF-1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c-mpl, c-kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU-Meg, and SDF-1 is a potentiator of human megakaryocytopoiesis.     

A combination of megakaryocyte growth and development factor and interleukin-1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes

Chemotherapy-induced thrombocytopenia is a major risk factor in cancer treatment. The transfusion of autologous ex vivo expanded megakaryocytes could be a new therapy to shorten the period of thrombocytopenia. Therefore we investigated, in a liquid culture system, the effect of various cytokine combinations composed of pegylated megakaryocyte growth and development factor (PEG-rHuMGDF), interleukin-1 (IL-1), IL-3, IL-6, IL-11 and stem cell factor (SCF) on the proliferation and differentiation of CD34+ cells, in order to define the most optimal and minimum levels of cytokine combinations for megakaryocyte expansion. Besides PEG-rHuMGDF, IL-1 was found to be important for optimal megakaryocyte expansion. Depletion of either SCF, IL-6 or IL-11 did not exert a large effect, but the absence of IL-1 strongly diminished the number of megakaryocytic cells. Addition of IL-3 to the combination PEG-rHuMGDF, IL-1, IL-6, IL-11 and SCF significantly reduced the number of megakaryocyte progenitors (CD34+CD41+ cells) and the number of CFU-Meg. Furthermore, we found a strong correlation between the number of CD34+CD41+ cells and the number of CFU-Meg obtained after 8 d culture. Our study shows that optimal ex vivo expansion of megakaryocytes is achieved by the combination of PEG-rHuMGDF and IL-1. The numbers of megakaryocytes and megakaryocyte progenitors (CD34+CD41+) obtained in our liquid culture system with the growth factor combination PEG-rHuMGDF and IL-1 are suitable for transfusion purposes.     

Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system

In serum-free cultures of human CD34 cells, recombinant human thrombopoietin (TPO) induced megakaryocyte colony formation in a dose-dependent fashion that was further enhanced by the presence of interleukin-3 (IL-3) and stem cell factor (SCF), but not by IL-6, IL-11 or erythropoietin. TPO gave rise to much smaller colonies and at an earlier time than IL-3, indicating that TPO affects predominantly more mature megakaryocytic progenitors. In liquid cultures, TPO increased the percentage and the absolute number of ≥8N megakaryocytes, but it did not shift their modal ploidy from 2N. TPO-induced endomitosis was totally inhibited by the presence of, or previous exposure of cells to, IL-3 and/or SCF. The mechanism by which TPO overcomes in vivo the negative effects of IL-3 and SCF on megakaryocyte ploidy remains unknown.     

Megakaryocyte progenitors in the bone marrow and peripheral blood of patients with myeloproliferative diseases

We studied the behavior in culture of megakaryocyte progenitor cells (CFU-Mk) from peripheral blood (PB) and bone marrow (BM) cells in eight patients with myeloproliferative diseases (MPD). In seven patients we observed megakaryocyte (Mk) colony formation from PB cells, which were generated in the absence of any added stimulator and which did not increase after the addition of a source of Mk-colony stimulating activity (CSA-Mk). The number of BM CFU-Mk was significantly higher in patients than in controls, and in seven out of eight patients the responsiveness to added CSA-Mk was retained. Plasma obtained from six patients did not stimulate normal donors' BM target cells to form Mk colonies. These data demonstrate an expansion of the CFU-Mk pool in MPD patients without increased plasma levels of CSA-Mk, and suggest that PB and BM CFU-Mk of MPD patients might have different kinetic properties.     

Wistar大鼠心肌梗死造模后SDF-1/CXCR4的表达

目的检测基质细胞来源因子-1（SDF-1）和其受体CXCR4在心肌梗死后不同部位心肌组织的表达，研究SDF-1／CXCR4轴在心肌梗死后血管新生中的作用，为近一步的研究进行基础研究。方法结扎Wistar大鼠左冠状动脉前降支复制的心肌梗死大鼠模型术后，分别于0．5h，7d，14d，28d处死后提取总RNA和总蛋白，分别用RT—PCR，PCR和Westem印迹检测心肌组织正常区，边缘区，梗死区在梗死后0．5h，7d，14d，28d时SDF-1、CXCR4在mRNA和蛋白水平表达变化情况。结果假手术组和对照组比较，SDF-1在心肌梗死后大鼠的心脏组织中表达有明显的增高（P〈0．01），一直持续到14d以后，在不同心肌部位的表达有明显的差异；CXCR4在心肌梗死后大鼠的心脏组织中表达有明显的增高（P〈0．01）。结论SDF-1／CXCR4轴在心肌梗死后修复中有着重要的作用。     

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.     

Serum albumin strongly influences SDF-1 dependent migration

Stem cell migration is largely regulated by the chemokine SDF-1 and its receptor CXCR4. In the present study, we analyzed the effect of protein on SDF-1 dependent chemotaxis using CXCR4 expressing primary CD34+ hematopoietic progenitor cells for transwell migration assays. We show that migration towards SDF-1 is abolished in the absence of protein, while addition of serum albumin rescues SDF-1 dependent migration. Acid hydrolyzation or tryptic digest of protein eliminates its migration supporting effect, showing that the intact protein is necessary. We demonstrate that gradients of human serum albumin (HSA) that are physiologically present in vivo between human plasma and interstitial fluid (bone marrow) greatly influence SDF-1 dependent migration of hematopoietic progenitor cells. While SDF-1 dependent migration is strongly enhanced in the presence of a HSA gradient from 4% (plasma) towards 1% (interstitial fluid), reversion of the protein concentrations inhibits SDF-1 dependent chemotaxis. Furthermore, migration is induced to lower serum albumin concentrations in the presence of equal SDF-1 concentration, while albumin gradients in the absence of SDF-1 have no effect. Our results suggest that physiological gradients of serum albumin between blood and bone marrow support SDF-1 dependent homing of hematopoietic progenitor cells to the stem cell niche.     

CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis)

Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell-stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28.7 +/- 12%vs 18.1 +/- 6.1% of input cells within 24 h, mean +/- SD, n = 8), whereas 9.4 +/- 12.6% (mean +/- SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks alpha4beta1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that alpha4beta1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment.     

基质细胞衍生因子-1/CXC趋化因子受体4在支气管哮喘大鼠气道炎症及气道重塑中的作用

目的 探讨基质细胞衍生因子-1(SDF-1)、CXC趋化因子受体4(CXCR4)在支气管哮喘(哮喘)大鼠气道炎症及气道重塑中的作用.方法 将18只SPF级SD雌性大鼠按随机数字表法随机分为对照组、哮喘4周组和哮喘8周组,每组6只.卵清白蛋白(OVA)致敏后,雾化吸人OVA制作哮喘模型.哮喘模型成功后,测定气道压力;通过HE染色、Image-Pro Plus图像分析软件分析大鼠气道平滑肌的嗜酸粒细胞浸润情况,测定支气管管腔的内周长、管壁面积、支气管平滑肌面积以及平滑肌细胞核数;RT-PCR、Western blot方法检测各组大鼠肺组织SDF-1、CXCR4的表达变化;免疫组织化学法检测各组大鼠气道壁SDF-1表达的变化;统计数据并分析SDF-1、CXCR4与哮喘气道重塑及气道炎症的关系.结果 哮喘4周组、哮喘8周组大鼠的气道反应性、气道壁嗜酸粒细胞计数、支气管管壁面积、支气管平滑肌面积、平滑肌细胞核数目均明显高于对照组,哮喘两组间上述指标差异均有统计学意义(均P＜0.01);RT-PCR检测结果显示,哮喘4周组、哮喘8周组大鼠肺组织SDF-1(分别为0.583±0.004和0.724±0.008)、CXCR4(分别为0.467±0.003和0.655±0.002)的表达明显高于对照组(SDF-1为0.146 ±0.003、CXCR4为0.281±0.002),哮喘8周组的SDF-1及CXCR4的表达亦明显高于哮喘4周组,差异均有统计学意义Western blot检测结果显示,(均P＜0.01).Western blot检测结果显示,哮喘4周组、哮喘8周组大鼠气道壁SDF-1的表达(分别为0.270 ±0.006和0.350±0.009)明显高于对照组(0.180±0.009),哮喘8周组的SDF-1的表达量亦高于哮喘4周组,差异均有统计学意义(均P＜0.01);各组大鼠肺组织、气道壁SDF-1、CXCR4mRNA及蛋白的表达与气道反应性、嗜酸粒细胞浸润数、支气管壁面积、支气管平滑肌厚度及支气管平滑肌细胞核数均呈正相关(均P＜0.01).结论 SDF-1/CXCR4信号轴可能在哮喘气道炎症及气道重塑病理过程中起重要作用.     

Characteristics of circulating megakaryocyte progenitors (CFU-MK) in patients with primary myelofibrosis

Characteristics of circulating CFU-MK and the effect of serum and plasma on CFU-MK growth were studied in 14 patients with primary myelofibrosis (MF) using short- and long-term culture methods. The number of CFU-MK in short-term cultures was significantly increased in the non-splenectomized patient group (p < 0.01). Without added PHA-LCM and normal serum, spontaneous colony formation was found in 9 out of 10 patients. In long-term blood cultures from 6 MF patients, 3 untreated patients formed confluent adherent layers and produced in suspension an equal number or an even greater number of nucleated cells, megakaryocytes and CFU-MK than those obtained in long-term bone marrow culture from normal individuals. 2 splenectomized patients showed neither an increased numbers of CFU-MK nor the capacity to develop an adherent layer. The serum and plasma of MF patients failed to stimulate megakaryocyte colony formation by normal bone marrow in a normal fashion. These findings indicate a megakaryocytopoietic abnormality, and a central role of the spleen in extramedullary haematopoiesis in MF.     

Thrombopoietin-independent effect of interferon-γ on the proliferation of human megakaryocyte progenitors

Flow cytometric study revealed that almost all CD34⁺ cells in human umbilical cord blood expressed interferon-γ receptor (IFN-γR). To clarify the precise functional roles of IFN-γR in human CD34⁺ cells, we examined the effect of IFN-γ alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34⁺ cells using a serum-free clonal culture. Surprisingly, IFN-γ alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-γ. IFN-γ at 1000 U/ml induced 10 ± 1.2 MK colonies from 1 × 10³ CD34⁺ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 ± 4.0, 22 ± 4.2, 4 ± 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-γ monoclonal antibody (mAb) to the IFN-γ culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-γ, suggesting that the observed effect of IFN-γ on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-γ to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-γ induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-γ may have regulatory roles in human megakaryocytopoiesis.     

Ex vivo expansion of megakaryocyte progenitor cells from normal bone marrow and peripheral blood and from patients with haematological malignancies

A number of haematological and non-haematological malignancies can be successfully treated using high-dose chemotherapy +/- irradiation followed by haematopoietic progenitor cell transplantation. Post transplant, thrombocytopenia and neutropenia always occur and patients require platelet transfusions. It may be possible to reduce the period of thrombocytopenia by re-infusion of ex vivo expanded megakaryocyte progenitors (MP), derived from the progenitor cell graft. We have investigated the expansion of MP from CD34+ enriched cells from normal bone marrow (NBM) and peripheral blood (PB) and remission BM or PB samples from patients with haematological malignancies. CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO), interleukin 1 (IL-1), IL-6 and stem cell factor (SCF) for 7 d, then cell proliferation was assessed by flow cytometry using lineage-specific markers. It was possible to significantly expand the number of MP cells from all sources. There were no major differences in yields of MP from normal BM or PB, or BM from multiple myeloma and non-Hodgkin's lymphoma patients. However, expansion of MP in acute myeloid leukaemia samples was lower than all other samples and the number of megakaryocyte colony-forming units was reduced. Several cytokine combinations were evaluated to optimize MP expansion from NBM. Equivalent yields of MP were obtained using TPO and one of IL-1, IL-3, granulocyte-macrophage colony-stimulating factor or SCF, suggesting that large cytokine combinations are not necessary for this procedure. It should be possible to scale up the culture conditions described to produce effective MP doses for clinical transplantation.     

SDF-1／CXCR4在老年卵巢癌组织中的表达及意义

目的探讨趋化因子SDF-1及其受体CXCR4在老年人上皮性卵巢癌组织中的表达及意义。方法应用westernblotting方法定量检测43例老年患者上皮性卵巢癌组织中SDF-1和CXCR4的蛋白表达情况。结果老年人上皮性卵巢癌组织中SDF-1／CXCR4蛋白表达较卵巢良性肿瘤对照组明显增加,其相对表达量分别为（2．38±0．20）和（3．32±0．26）,差异具有统计学意义（P〈0．05）。结论SDF-1／CXCR4生物学轴可能在老年人卵巢癌的发生与转移过程中起着重要作用。     

Effects of anoxia on megakaryocyte progenitors derived from cord blood CD34pos cells

BACKGROUND: Severe hypoxic insults to the fetus and neonate are associated with the development of thrombocytopenia. The thrombocytopenia in some cases is the result of disseminated intravascular coagulation, but that mechanism fails to account for all, perhaps the majority, of cases. OBJECTIVE: We hypothesized that human fetal megakaryocyte (Mk) progenitors are directly adversely affected by transient anoxia. DESIGN AND METHODS: To test this, we isolated CD34pos cells from the umbilical cord blood of 10 healthy term neonates, and exposed these to 0% or 20% O2 for 24 h, with or without recombinant thrombopoietin (rTpo, 50 ng/mL). After 24 h, a portion of the CD34pos cells were harvested for flow cytometric evaluation of apoptosis. The remaining cells were cultured for an additional 10-12 days, under normoxic conditions, in a collagen-based serum-free system containing rTpo, IL-3, and IL-6. In this way, we sought to determine the effect of transient anoxia on clonogenic capacity of Mk progenitors. RESULTS: Contrary to our hypothesis, anoxia did not increase either apoptosis or cell death of the CD34pos cells. The addition of rTpo was protective, with a significant decrease in apoptosis and cell death (P < 0.0001), and an increase in the number of Mk colonies cultured (P = 0.04). There was no difference between the normoxic and anoxic groups in proliferative potential of the Mk progenitor cells. CONCLUSIONS: The thrombocytopenia observed in neonates following an acute hypoxic event is not likely due to a direct deleterious effect of hypoxia on Mk progenitors.     

人巨细胞病毒对脐血巨核系祖细胞集落生长的影响

目的：探讨人巨细胞病毒（HCMV）对巨核系祖细胞（CFU－MK）的分化和增殖的影响。方法：收集20例脐血标本,采用体外甲基纤维素半固体培养技术,观察3种不同浓度 HCMV AD169株对CFU－MK集落形成的影响。结果：3个感染组的CFU－MK集落数均减少,与灭活病毒组比较,分别为24．0％、35．8％和48．6％（P＜0．05）,显示抑制程度与病毒感染滴定呈剂量－浓度依赖关系。结论：HCMV在体外能抑制CFU－MK的分化和增殖,提示巨细胞病毒感染引起的血小板减少与CFU－MK受该病毒抑制有关。