人CD34^＋和CD34^-细胞在NOD／SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型,比较了新鲜及培养后的CD34 和CD34-细胞在体内植入及重建造血能力.从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34 和CD34-细胞,经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内,6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测.经检测,输注CD34' 细胞和混合细胞的小鼠,其体内CD45 细胞及人源各系血细胞的含量相近,两者均远远高于输注CD34-细胞的小鼠.输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34 细胞的小鼠存活率约为66.7%,而输注培养后混合细胞的小鼠全部存活,且在两组存活的小鼠体内均能检测到CD45 细胞及人源各系血细胞.结果表明:无论是新鲜还是培养后的CD34 细胞均具有在NOD/SCID小鼠体内植入和重建造血能力,而CD34-细胞不具有该能力,但CD34-细胞与CD34 细胞同时输注有助于提高小鼠的存活率,说明其对CD34 细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用.     

CD34+和CD34-细胞在NOD/SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型, 比较了新鲜及培养后的CD34+和CD34-细胞在体内植入及重建造血能力。从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34+和CD34-细胞, 经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内, 6周后处死存活的小鼠, 取其骨髓、脾脏和外周血细胞, 分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测。经检测, 输注CD34+细胞和混合细胞的小鼠, 其体内CD45+细胞及人源各系血细胞的含量相近, 两者均远远高于输注CD34-细胞的小鼠。输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34+细胞的小鼠存活率约为66.7%, 而输注培养后混合细胞的小鼠全部存活, 且在两组存活的小鼠体内均能检测到CD45+细胞及人源各系血细胞。结果表明: 无论是新鲜还是培养后的CD34+细胞均具有在NOD/SCID小鼠体内植入和重建造血能力, 而CD34-细胞不具有该能力, 但CD34-细胞与CD34+细胞同时输注有助于提高小鼠的存活率, 说明其对CD34+细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用。     

Human Cytomegalovirus miRNAs Regulate TGF-β to Mediate Myelosuppression while Maintaining Viral Latency in CD34+ Hematopoietic Progenitor Cells

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Differential Gene Expression of Human CD34+ Hematopoietic Stem and Progenitor Cells Before and After Culture

Ex vivo expanded CD34⁺ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34⁺ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34⁺ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background

Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34⁺ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.

Methodology/Principal Findings

Using commercially available G-CSF mobilized peripheral blood (PB) CD34⁺ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.

Conclusions/Significance

This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34⁺ cells.     

Gene Expression Profiling Identifies HOXB4 as a Direct Downstream Target of GATA-2 in Human CD34+ Hematopoietic Cells

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.     

Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34Cells

Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34⁺pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34⁺cells. CB CD34⁺cells were cryopreserved at −80°C for 2 days. Cryopreserved CB CD34⁺cells as well as freshly isolated CB CD34⁺cells cultured with granulocyte—macrophage colony-stimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-α (TNF-α) for 14 days gave rise to CD1a⁺/CD4⁺/CD11c⁺/CD14⁻/CD40⁺/CD80⁺/CD83⁺/CD86⁺/HLA-DR⁺cells with dendritic morphology. DCs derived from cryopreserved CB CD34⁺cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34⁺cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34⁺cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34⁺cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34⁺cells cultured with GM-CSF/TNF-α/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34⁺cells.     

Nonproductive Infection of Guinea Pig Cells with Human Cytomegalovirus

Human cytomegalovirus was capable of adsorbing to and penetrating guinea pig cells, but was unable to replicate new virus. Cultures infected with virus inoculum of high titer showed a cytopathic effect (CPE) characterized by cell rounding. This CPE depended upon the presence of infectious virus, and its extent was directly related to the multiplicity of infection. Staining by indirect immunofluorescence by using human convalescent sera was positive as early as 4 h postinfection. Maximal fluorescence was observed 24 h postinfection when 50% of the cells contained fluorescent antigens both in nuclei and cytoplasm. No evidence for viral replication was found, and no defective particles were detected by electron microscopy. Treatment with actinomycin D or with cycloheximide strongly inhibited both the fluorescent antigens and the CPE, whereas 5-fluorodeoxyuridine and bromodeoxyuridine were ineffective.     

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8⁺ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8⁺ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8⁺ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8⁺ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8⁺ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.     

Pre-Transplantation Serum Parathyroid Hormone Influences the Number of Mobilized CD34+ Hematopoietic Stem Cells in Autologous Hematopoietic Stem Cell Transplantation

Background:Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation.Methods:Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment.Results:High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH.Conclusion:We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.Key Words: Auto-HSCT, CD34+ Cell, Pre- transplant PTH     

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.     

Elevation of Survivin Levels by Hematopoietic Growth Factors Occurs in Quiescent CD34+ Hematopoietic Stem and Progenitor Cells Before Cell Cycle Entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is over-expressed during G2/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34+ hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34+ cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34+ cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G0/G1. Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67negative and Cyclin Dnegative CD34+ cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G0 CD34+ cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G0 CD34+ cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSEbright G0 CD34+ cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34+ cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.

Key Words:

Survivin, Cord blood, CD34+ cells, Cell cycle     

促红细胞生成素基因修饰的胎肝基质细胞促进脐血CD34 +细胞向红系分化

通过重组慢病毒系统感染人胎肝基质细胞(fetal liver stromal cells,FLSCs),建立了能够稳定高效表达促红细胞生成素(erythropoietin,EPO)的细胞株EPO/FLSCs.从胎儿肝脏克隆EPO基因,构建重组慢病毒EPO的表达载体,感染FLSCs,根据荧光表达强弱进行流式分选,获得能够继续稳定传代的高表达EPO基因的FLSCs,RT-PCR和ELISA结果证实,细胞株中的EPO基因稳定表达.RT-PCR结果显示,FLSCs的EPO在mRNA水平的表达分别是未转染FLSCs和转染空载体FLSCs的5.63倍和5.71倍.ELISA法检测了转染重组慢病毒EPO表达载体的FLSCs EPO蛋白表达水平,结果显示EPO蛋白的表达水平也明显升高.收集EPO/FLSCs的条件培养基,体外诱导脐血CD34+细胞向造血细胞分化,结果显示向红系定向分化的细胞比例明显居多,有可能为临床细胞治疗提供稳定、高质量的细胞来源.