Replication and cytopathology of human parvovirus B19 in human umbilical cord blood erythroid progenitor cells.

Human parvovirus B19 productively infected erythroid progenitor (EP) cells from umbilical cord blood, in vitro as shown by an increase of viral DNA in supernatant fluid assayed by dot blot hybridization and liquid scintillation counting. Progeny virus was released into the supernatant fluid of CD34+ EP cells which had been purified by immunomagnetic separation. This supernatant fluid was infectious for bone marrow cells. Erythroid bursts infected with virus showed characteristic cytopathic effect by electron microscopy consisting of cytoplasmic vacuolization, marginated chromatin, and nuclear inclusions of lattice or crystalline arrays. Cultures of umbilical cord blood EP cells may be useful for the propagation of parvovirus B19 serological testing reagents and the study of virus-host cell interactions.     

脐血浆分离培养人脐带间充质干细胞

目的:利用脐带血血浆分离、培养人脐带间充质干细胞(HUCMSCs).方法:将脐血浆经盐析、透析等处理后,按10％体积成分分离、培养扩增HUCMSCs,观察细胞的形态,流式细胞仪检测细胞表面标志,MTT法检测细胞生长曲线、增殖能力,ELISA检测培养液中碱性成纤维细胞生长因子(bFGF)、noggin的分泌浓度.结果:利用含10％脐带血血浆的培养液能分离、扩增HUCMSCs,分离纯化的HUCMSCs成梭形、增殖能力强、具有分化为成骨细胞、脂肪细胞及神经干细胞的能力,高表达CD29、CD44,低表达或不表达CD34、CD45及HLA-DR.而且在含10％脐带血血浆的培养液中生长的HUCMSCs还能分泌人胚胎干细胞(hESC)生长所需的的bFGF及noggin.结论:脐带血血浆经简单处理后能分离培养HUCMSCs,而且其分泌的细胞因子更适合hESC培养需要.     

人胎盘血间质干细胞分离培养

目的：分离培养人胎盘血间质干细胞（hMSC），为hMSC探寻新来源。方法：采用羟乙基淀粉（HES）方法分离、富集胎盘血有核细胞；DMEM培养液体外培养、纯化、扩增hMSC；流式细胞仪检测细胞表面标记；地塞米松、IBMX、胰岛素和吲哚美辛定向诱导hMSC样细胞向脂肪细胞分化。结果：胎盘血来源有核细胞,在DMEM体外培养条件下，生长出具有塑料粘附特性的梭形细胞，阳性获得率29.17%（7/24），该细胞传代培养达6个月以上；流式细胞仪检测结果显示CD29、CD44、CD59、CD90、CD105、CD166表达阳性，CD14、CD34、CD45、CD80、CD86表达阴性；加入脂肪细胞诱导剂，细胞在形态上向脂肪细胞转化，胞内出现脂滴、脂泡，油红O阳性。结论：人胎盘血可以分离培养出hMSC，是hMSC的重要来源。     

Hemogenic endothelial progenitor cells isolated from human umbilical cord blood

Hemogenic endothelium has been identified in embryonic dorsal aorta and in tissues generated from mouse embryonic stem cells, but to date there is no evidence for such bipotential cells in postnatal tissues or blood. Here we identify a cell population from human umbilical cord blood that gives rise to both endothelial cells and hematopoietic progenitors in vitro. Cord blood CD34+/CD133+ cells plated at high density in an endothelial basal medium formed an endothelial monolayer and a nonadherent cell population after 14-21 days. AML-1, a factor required for definitive hematopoiesis, was detected at low levels in adherent cells and at high levels in nonadherent cells. Nonadherent cells coexpressed the endothelial marker vascular endothelial (VE)-cadherin and the hematopoietic marker CD45, whereas adherent cells were composed primarily of VE-cadherin+/CD45- cells and a smaller fraction of VE-cadherin+/CD45+ cells. Both nonadherent and adherent cells produced hematopoietic colonies in methylcellulose, with the adherent cells yielding more colony-forming units (CFU)-GEMM compared with the nonadherent cells. To determine whether the adherent endothelial cells were producing hematopoietic progenitors, single cells from the adherent population were expanded in 96-well dishes for 14 days. The clonal populations expressed VE-cadherin, and a subset expressed AML-1, epsilon-globin, and gamma-globin. Three of 17 clonal cell populations gave rise to early CFU-GEMM hematopoietic progenitors and burst-forming unit-erythroid progenitors. These results provide evidence for hemogenic endothelial cells in human umbilical cord blood.     

Parvovirus B19 infection in pregnancy.

Parvovirus B19 is a small single-stranded DNA virus and a potent inhibitor of erythropoiesis, due to its cytotoxicity to erythroid progenitor cells. Infection with parvovirus B19 during pregnancy can cause several serious complications in the fetus, such as fetal anemia, neurological anomalies, hydrops fetalis, and fetal death. Early diagnosis and treatment of intrauterine parvovirus B19 infection is essential in preventing these fetal complications. Testing maternal serum for IgM antibodies against parvovirus B19 and DNA detection by PCR can confirm maternal infection. If maternal infection has occurred, ultrasound investigation of the fetus and measurement of the peak systolic flow velocity of the middle cerebral artery are sensitive non-invasive procedures to diagnose fetal anemia and hydrops. Intrauterine transfusion is currently the only effective treatment to alleviate fetal anemia, but if the fetus is (near) term, induction of delivery should be considered. Most maternal infections with parvovirus B19 occur through contact with infected children at home. Individual counseling of susceptible pregnant women will reduce unnecessary fetal deaths.     

Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells

After successful invasion of susceptible hosts, systemic distribution of coxsackievirus B3 (CVB3) most likely requires interactions with the endothelial system. Thereby, infection of endothelial cells occurs directly or viruses and/or virus-infected leukocytes migrate through the endothelial barrier. Many of these processes have not been studied so far. In order to analyze viral replication in the endothelium, human umbilical vein endothelial cells (HUVEC) were isolated and infected with CVB3. Time-course experiments revealed maximal viral replication at 10–24 h and viral RNA persistence up to 120 h post-infection (p. i.) without the induction of obvious general cytopathic effects or the loss of cellular viability. However, the application of the EGFP-expressing recombinant virus variant CVB3/EGFP revealed shrinkage and death of individual cells. Using infectious center assays, a noticeable CVB3 replication occurred on an average of 20 % of HUVEC at 10 h p. i. This may be in part due to a higher coxsackievirus/adenovirus receptor expression in a small subgroup of HUVEC (5–7 %) as analyzed by flow cytometry. Interestingly, CVB3 replication escalated and cellular susceptibility increased significantly after reversal of cell cycle arrest caused by serum deprivation indicating that reactivation of cellular metabolism may help to promote CVB3 replication. Finally, CVB3-infected HUVEC cultures revealed increased DNA fragmentation, and inhibition of caspase activity caused an accumulation of intracellular virus particles indicating that apoptotic processes are involved in virus release mechanisms. Based on these observations, it is assumed that CVB3 replicates efficiently in human endothelial cells. But how this specific infection of the endothelium may influence viral spread in the infected host needs to be investigated in the future.     

人脐血单个核细胞向神经细胞分化

目的：观察培养前后人脐血单个核细胞(MNCs)的形态学及免疫反应性变化,探讨其能否向神经细胞的分化及机制。方法：密度梯度离心脐血中单个核细胞,接种并用。EGF和bFGF刺激细胞生长,倒置显微镜下观察培养前后细胞形态变化,并行免疫细胞化学鉴定。结果：培养前脐血MNCs胞体小呈圆形,nestin阳性细胞、AP2阳性细胞散在分布(阳性率为1．5％和3．4％)无GFAP阳性细胞着色。培养14d后,细胞群中相邻细胞突起连成网状;AP2、GFAP染色阳性细胞成片状分布(阳性率33．5％和24．6％),未见nestin阳性细胞。结论：脐血细胞中可能有多能干细胞,经体外培养后能分化为具有一定形态的神经细胞。     

Transplantation of human umbilical cord blood cells benefits an animal model of Sanfilippo syndrome type B

Sanfilippo syndrome type B is caused by alpha-N-acetylglucosaminidase (Naglu) enzyme deficiency leading to an accumulation of undegraded heparan sulfate, a glycosaminoglycan (GAG). Cell therapy is a promising new treatment and human umbilical cord blood (hUCB) cell transplantation may be preferred for delivery of the missing enzyme. We investigated the ability of mononuclear hUCB cells administered into the lateral cerebral ventricle to ameliorate/prevent histopathological changes in mice modeling Sanfilippo syndrome type B. These are the first results supporting enzyme replacement by administered hUCB cells. In vivo, transplanted hUCB cells survived long-term (7 months), migrated into the parenchyma of the brain and peripheral organs, expressed neural antigens, and exhibited neuron and astrocyte-like morphology. Transplant benefits were also demonstrated by stable cytoarchitecture in the hippocampus and cerebellum, and by reduced GAGs in the livers of treated mutant mice. A hUCB cell transplant may be an effective therapeutic strategy for enzyme delivery in Sanfilippo syndrome type B.     

人脐血来源MSCs的主要生物学特性的研究

目的：探讨人脐血间充质干细胞（MSCs）分离、纯化、扩增、表面标志、免疫表型及其造血生长因子（HGFs）分泌等生物学特性方法：(1) Ficoll法分离、纯化人脐血、成人骨髓和胎儿骨髓MSCs，动态观察不同来源MSCs的生长状况。(2) 流式细胞仪检测脐血和骨髓MSCs表面CD34、CD45、CD14、CD29、CD44、CD105、CD166、CD80、CD86、CD40和CD40L的表达。(3) ELISA法检测脐血和骨髓MSCs培养上清中SCF、TPO、FLT-3L和IL-6的分泌水平。结果：(1) 所有骨髓标本中均可分离纯化出MSCs，而15份脐血中仅4份分离纯化出MSCs，脐血MSCs和骨髓MSCs的细胞形态无明显差异,脐血MSCs原代培养所需要的单个核细胞（MNC）量为骨髓MSCs的3倍，且其原代培养的增殖速度较慢，但脐血MSCs传代培养的增殖速度和骨髓相似。(2) 脐血MSCs和骨髓MSCs表面标志无差异，均不表达造血细胞表面标志以及与免疫识别有关的表面抗原。(3) 脐血和骨髓MSCs分泌SCF、TPO、FLT-3L和IL-6的水平相似。结论： (1)脐血和骨髓同样可以分离、纯化MSCs，但脐血中MSCs的含量少，且大部分脐血中不含MSCs。(2)纯化的脐血MSCs扩增能力、表面标志、免疫学表型及HGFs分泌水平和骨髓MSCs相似。     

Some characteristics of mast cells cultured from human umbilical cord blood

Inflammation Research -     

Parvovirus B19 infection

Human parvovirus B19, discovered in 1974, is a single-stranded DNA virus which causes erythema infectiosum, arthralgia, aplastic crisis in patients with red cell defects, chronic anaemia in immunocompromised patients, and fetal hydrops. Seroprevalence in developed countries is 2–10% in children less than 5 years, 40–60% in adults more than 20 years, and 85% or more in those over 70 years. The virus may be transmitted by the respiratory route and by transfusion of infected blood and blood products. After an incubation period of six to eight days, viraemia occurs, during which reticulocyte numbers fall dramatically resulting in a temporary drop in haemoglobin of 1 g/dl in a normal person. Clearance of viraemia is dependent on development of specific antibody to the B19 structural proteins, VP1 and VP2. The red cell receptor for the virus is blood group P antigen. Diagnosis in immunocompetent persons depends on detection of specific IgM in serum. Diagnosis in immunocompromised persons depends on detection of B19 antigen or DNA in serum. There is no specific treatment for B19 infection; however, human normal immunoglobulin may be used as a source of specific antibody in chronically infected persons. A recombinant parvovirus B19 vaccine is under development.     

Biological and mechanical quality of red blood cells cultured from human umbilical cord blood stem cells

Human umbilical cord blood (CB) has moved from the status of biological waste to that of a valuable source of haematopoietic stem (HS) cells. There are potentially three major clinical applications for HS cells andex vivo-expanded HS cells: reconstitution of haematopoiesis in patients undergoing chemotherapy; gene therapy (e.g. in thalassaemia, sickle cell anaemia); and large-scale production of mature blood cells. Erythropoiesis is accomplished by highly complex interactions of haematopoietic progenitor cells, stromal cells and cytokines in the bone marrow. Among them, erythropoietin is the principal regulator.Ex vivo cell culture experiments to obtain mature red blood cells were the focus of this study. Attempts to elucidate appropriate medium components and amounts of haematopoietic growth factors were successful: enucleated and haemoglobin-filled erythroid cells were obtained from primitive HS cells. Dimethylsulphoxide (DMSO) was found to be of particular importance as an efficient differentiation inducer. The differentiation process was followed microscopically and by fluorescence-activated cell sorting (FACS). Using the micropipette aspiration technique, the elastic properties of erythroid cells were evaluated as erythropoiesis progressed. Discocyte-like cells, comprising reticulocytes and finally differentiated red blood cells, showed an about ten-fold higher membrane shear modulus compared with control cells.     

High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells.

Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to introduce foreign genes into human cord blood CD34+ cells and evaluate gene expression in CD34+/CD38(dim) and committed myeloid progenitors (CD33+, CD11b+). CD34+ cells were cultured in X-VIVO 10 supplemented with thrombopoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency and cell viability measured by flow cytometry using enhanced green fluorescent protein (EGFP) as a reporter indicated 31% +/- 2% EGFP+ /CD34+ efficiency and 77% +/- 3% viability as determined 48 hours post-electroporation. The addition of allogeneic cord blood plasma increased the efficiency to 44% +/- 5% with no effect on viability. Of the total CD34+ cells 48 hours post-electroporation, 20% were CD38(dim)/EGFP+. CD34+ cells exposed to interleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into CD33+ and CD11b+ cells, and 9% +/- 3% and 8% +/- 7% were expressing the reporter gene, respectively. We show that electroporation can be used to introduce foreign genes into early hematopoietic stem cells (CD34+/CD38(dim)), and that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33+, CD11b+) in response to corresponding cytokines. Further investigation is needed to determine the transgene expression in functional terminal cells derived from the genetically modified CD34+ cells, such as T cells and dendritic cells.     

Co-culture of umbilical cord blood CD34+ cells with human mesenchymal stem cells

Insufficient numbers of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) sometimes limit allogenic transplantation of umbilical cord blood (UCB). Ex vivo expansion may overcome this limitation. Mesenchymal stem cells (MSCs), as non-hematopoietic, well-characterized skeletal and connective-tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSCs/HPCs. MSCs are attractive for the rich environmental signals that they provide and for immunological compatibility in transplantation. Thus far, HSC/MSC co-cultures have mainly been performed in 2-dimensional (2D) configuration. We postulate that a 3-dimensional (3D) culture environment that resembles the natural in vivo hematopoietic compartment might be more conducive for regulating HSC expansion. In this study, we compared the co-culture of HSCs and MSCs in 2D and 3D configurations. The results demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. Direct contact between MSCs and HSCs in 3D cultures led to statistically significantly higher expansion of cord blood CD34+ cells than in 2D cultures (891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in colony-forming cell assay [CFC]). Engraftment assays in non-obese diabetic/severe combined immunodeficiency mice also indicated a high success rate of hematopoiesis reconstruction with these expanded cells.