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.     

Derivation of endothelial cells from CD34- umbilical cord blood

CD34 is a transmembrane glycoprotein constitutively expressed on endothelial cells and hematopoietic stem cells. Use of CD34-recognizing antibodies has helped in the identification and isolation of CD34+ endothelial precursors from embryonic and adult tissues. However, CD34-null mice display no vascular abnormalities, demonstrating that CD34 antigen expression is not required for normal vascular development. Here we show that a CD34- cell population that includes endothelial cell precursors can be isolated from cord blood. In the presence of angiogenic factors, these cells mature to express the endothelial cell markers vascular endothelial-cadherin, vascular endothelial growth factor receptor-1 and -2, Tie-1 and -2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains), von Willebrand factor, and CD31 while maintaining their CD34- status, and can be expanded in vitro for over 20 passages. Moreover, in functional studies, these cells can undergo extracellular matrix-dependent morphogenic changes into capillary-like tubular structures. When transplanted into immunodeficient mice in conjunction with tumor cells or with the proangiogenic factor basic fibroblast growth factor, these cells can form functional microvessels arising along with host blood cells. These studies provide strong evidence for the existence of CD34- endothelial cell precursors in cord blood and suggest the use of ex vivo-expanded cord blood CD34- cells as a unique tool for the investigation of postnatal lineage diversification.     

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。     

人外周血与脐血内皮祖细胞生物学特性的比较

背景：内皮祖细胞可从外周血与脐血中获取,是修复各种疾病所致损伤的血管内皮细胞不可或缺的细胞来源。 目的：比较人外周血与脐血来源的内皮祖细胞经体外培养后生物学特性的差异。 方法：通过密度梯度离心法和6%羟乙基淀粉结合密度梯度离心法分别分离人外周血与脐血中的单个核细胞,分别设为脐血源组和外周血源组,计数各组单个核细胞数量,按1.0×106/cm2接种于大鼠尾胶包被的培养皿中,用内皮细胞培养基进行诱导,共培养7 d。 结果与结论：外周血与脐血体外培养分离出内皮祖细胞具有类似的形态学特征。光学显微镜下观察,随着培养天数的增加,大多数细胞由早期的贴壁圆形转变为梭形。外周血源组内皮祖细胞有细胞集落形成,脐血源组可见梭形细胞自行排列生长为典型的线样结构。锥虫蓝染色及绘制细胞生长曲线后发现,外周血源组单个核细胞及内皮祖细胞数量、内皮祖细胞活率及增殖能力均低于脐血源组(P < 0.05)。外周血源组和脐血源组内皮祖细胞在接种后第3天增殖速度达到峰值,在随后的培养中细胞增殖呈衰减态。流式细胞仪及免疫荧光染色检测结果显示,外周血源组和脐血源组内皮祖细胞均可表达具有内皮祖细胞表型的CD133、CD34和血管内皮细胞因子受体2表面标志物,两组既摄取Dil标记乙酰化低密度脂蛋白,也能标记体外内皮祖细胞的标志物荆豆凝集素Ⅰ。结果证实,脐血来源的内皮祖细胞与外周血来源的内皮祖细胞生物学特性相近,脐血来源的内皮祖细胞增殖能力更强。     

Phenotypic and functional heterogeneity of natural killer cells from umbilical cord blood mononuclear cells

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-gamma and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the na?ve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56(hi)CD16- NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-gamma expression by CD56(hi)CD16- and CD56(lo)CD16- subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.     

人脐血多种细胞因子的含量检测及植物血凝素和脂多糖的影响

目的:通过检测人脐血血清多种细胞因子含量,观察植物血凝素(PHA)和脂多糖(LPS)的诱生影响,探讨这些细胞因子的免疫功能及移植物抗宿主病(GVHD)的发病机制。方法:采用双抗体夹心酶联免疫吸附法(ELISA),测定26份正常足月新生儿脐血及16例正常儿童外周血血清中白细胞介素4(IL-4)、IL-10、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)、IL-12、IL-15、IL-18水平,以及PHA和LPS刺激后单个核细胞分泌上述细胞因子的诱生水平。结果:人脐血血清中IL-4的水平与正常外周血血清水平无显著差异(P＞0.05),脐血血清中IL-12的水平显著高于外周血血清(P＜0.05),其余5种细胞因子脐血血清水平均明显低于正常外周血血清水平(P＜0.05);PHA和LPS刺激后脐血单个核细胞分泌IL-4、IL-10、TNF-α、IFN-γ、IL-12、IL-15和IL-18的水平明显低于正常外周血(P＜0.05),尤以IL-4、TNF-α、IFN-γ、IL-15和IL-18非常明显。结论:脐血血清中上述细胞因子水平普遍低于正常外周血,以及脐血单个核细胞刺激后产生IL-4、IL-10、TNF-α、IFN-γ、IL-12、IL-15和IL-18等细胞因子水平的不足,表明脐血细胞免疫功能不成熟,是脐血移植后GVHD发生率低及程度轻的主要原因之一。     

人外周血单个核细胞来源内皮祖细胞的生物学性状

目的 探讨从成人外周血单个核细胞中分离、培养内皮祖细胞（EPCs）的方法以及EPCs的生物学特征。方法 密度梯度离心法获得单个核细胞（MNCs），种植于纤连蛋白包被的培养板中。每2h去除1次未黏附细胞共2次，然后隔日换液1次，7d后计数早期集落。每例血样均分为2等份，1份在获得早期集落后进行实验；另1份持续培养至晚期集落出现进行相同实验。流式细胞技术检测细胞表面抗原表达，ELISA法测定上清液中血管内皮生长因子（VEGF）浓度。胶原凝胶细胞种植实验测定体外血管生成功能。结果 早期集落中心为圆形细胞，周边是放射状排列的纺锤形细胞，再种植不能形成第二代集落且无体外血管形成功能，细胞表面主要表达CD14和CD45，培养上清液中可测到高浓度的VEGF。晚期集落在培养21至28d间出现，再种植可形成第二代内皮细胞集落，并能在胶原凝胶中形成管腔样结构。其构成细胞与早期集落相比CD45、CD14表达显著减少（P<0.001）而CD146表达明显增加（P<0.01）。结论 人外周血单个核细胞在内皮培养条件下可形成早期和晚期集落，构成早期集落的绝大多数细胞属于单核细胞系列，可分泌VEGF但不能分化成内皮细胞，晚期集落具有内皮祖细胞的形态和生物学特征。     

Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10⁶ cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10⁶ cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.     

Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood

The isolation of undifferentiated adult stem/progenitor cells remains a challenging task primarily due to the rare quantity of these cells in biological samples and the lack of unique markers. Herein, we report a relatively straightforward method for isolation of human mesenchymal stem cells (MSCs) based on their unusual resistance to osmotic lysis, which we term "osmotic selection" (OS). MSCs can remarkably withstand significant exposure to hypotonic conditions (> 30 min) with only a reversible impairment in cell proliferation and with no loss of stem cell potential after exposure. Comparison of MSCs to other circulating nonhematopoietic cells revealed a time regime, by which purification of these cells would be attainable without considerable cell loss. OS showed a 50-fold enrichment of fibroblast colony-forming units from umbilical cord blood samples when compared to commonly employed techniques. After upstream processing, isolated cells using OS were immunophenotyped to be CD14-, CD34-, CD45-, CD44+, CD105+, and CD106+, and displayed multipotent differentiation. Preliminary investigations to determine mechanisms responsible for osmolytic resistance revealed MSCs to have an ineffective volume of 59%, with the ability to double cell volume at infinite dilution. Disruption of filamentous actin polymerization by cytochalasin D sensitized MSCs to osmotic lysis, which suggests a cytoskeletal element involved in osmolytic resistance.     

Validation of endothelial progenitor cells in human umbilical veins and the isolated endothelial cells.

To detect endothelial progenitor cells in human umbilical veins and isolated endothelial cells, the authors examined protein and mRNA expression levels of cell surface markers for endothelial progenitor cells in human umbilical veins before and after trypsin treatment and at different passages of the isolated endothelial cells. CD133(+) (2.14 +/- 0.57 per mm) and KDR(+) (35.74 +/- 8.28 per mm) cells were observed in the intima of umbilical veins. The amounts of CD133(+), KDR(+), CD34(+), and CD105(+) cells decreased in the intima after trypsin treatment, whereas the percent of CD133(+) and KDR(+)cells in the media did not change significantly. Moreover, similar protein and mRNA expression levels of CD133 and KDR were detected in the umbilical veins before and after trypsin treatment. In the isolated cells from umbilical veins, the percent of CD133(+) and CD34(+) cells in P1 was 3.43% +/- 3.85%, which was higher than those in P3 (0.17% +/- 0.21%, p = 0.005) and P6 (0.14% +/- 0.18%, p = .001). The mRNA expression levels of CD133 and CD105 were down-regulated in later passages compared to those in P1, whereas the expression level of KDR was up-regulated in late passages. Thus it is suggested that endothelial progenitor cells reside in the distinct zone (e.g., initma and media) of human umbilical veins, and retain the capacity of differentiation to endothelial cells in vitro.     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

人脐血源性内皮祖细胞血管发生活性及其参与人恶性胶质瘤细胞异种移植瘤血管新生的作用

目的 观察人脐血源性内皮祖细胞(EPC)血管发生能力和在恶性胶质瘤血管新生过程中的作用.方法 应用密度梯度离心法分离新鲜人脐血的单个核细胞,接种于EGM-2培养液中培养获得EPC.取生长到第7～10天的细胞进行CD34和VEGFR-2免疫荧光双标染色.检测血管内皮生长因子(VEGF)刺激下EPC增殖活性、迁移能力和体外形成小管样结构的能力.采用人恶性胶质瘤细胞系U87在免疫缺陷小鼠进行皮下移植,于接种肿瘤后第7天经尾静脉注射EPC(每只5×103),并于接种肿瘤后第28天取材检测肿瘤微血管和EPC组织分布及定位,采用抗人CD31和抗鼠CD31免疫荧光双标记肿瘤微血管,计算人源性EPC来源的血管占肿瘤血管网的比例.结果 培养的细胞在第7～10天时可见条索样结构,生长并逐渐融合形成铺路石样排列的单层细胞,表达内皮细胞标记物CD34和VEGFR-2.在VEGF刺激下EPC具有较强的增殖活性、迁移能力和体外形成小管样结构的能力.外源性EPC能特异性归巢到异种移植瘤组织并形成新生血管,占肿瘤血管网的(18.68±1.32)%.结论 EPC在体内外具有形成血管能力,并参与异种移植瘤血管新生,提示其在恶性肿瘤血管新生过程中具有重要作用,并可能参与肿瘤微血管构筑表型异质性.     

人脐带血中内皮样前体细胞的初步研究

目的 观察脐带血中内皮细胞的前体细胞，并研究该前体细胞分化成熟所需条件。方法 采用常规分离方法，分离脐带血中的单个核细胞，培养于含小牛下丘脑提取液和地塞米松的MCDB131培养液中，以获得具有粘附性的细胞，于倒置相差显微镜下每日观察两次一并记录。结果 初步的实验结果表明脐带血中存在内皮细胞的前体细胞，小牛下丘脑提取物对该细胞的生成有促进作用，而地塞米松对其生成有抑制作用。结论 初步证实脐带血中有内皮细胞的前体细胞存在，并对其分化发育条件有了初步认识，为对其进一步的研究及应用打下了基础。     

人脐带内皮祖细胞的分离、鉴定及转染EGFP质粒的实验研究

目的 从脐带中分离内皮祖细胞(EPCs),考察其体外增殖、基因转染绿色荧光蛋白质粒的行为.方法 以酶消化法从脐带中分离出内皮祖细胞,并通过流式细胞仪和共聚焦显微镜对内皮祖细胞进行鉴定,以Lipofectamine 2000为转染试剂考察了内皮祖细胞转染绿色荧光蛋白质粒的行为.结果 从脐带中分离培养的内皮祖细胞在第9天形成了典型的内皮细胞集落,流式细胞仪分析结果显示CD133和激酶插入区受体(KDR)的含量均有所提高,并具有内皮祖细胞结合异硫氰酸荧光素(FITC)标记的荆豆凝聚素1(FITC-UEA-1)和吞噬DiI标记的低密度脂蛋白(DiI-ac-LDL)的功能,能较好地表达绿色荧光蛋白.结论 从脐带中分离的内皮祖细胞体外在适当的培养条件下,可增殖、诱导分化为内皮细胞,并能较好地表达外源基因,是基因与细胞治疗理想的载体.     

人脐血和外周血内皮祖细胞的分离培养和鉴定

背景：国内外有关内皮祖细胞的分离培养和鉴定方面的文章很多,但是人脐血和外周血内皮祖细胞的鉴别方面文章不多。 目的：从人脐血和外周血中分离出内皮祖细胞,并对其进行培养和鉴定。 方法：选取密度梯度离心法从脐血和外周血中分离获得单个核细胞,按照1× 10⁶/cm²的浓度种植于预先铺有纤维连接蛋白的培养板中,用含血管内皮生长因子的M199培养基进行诱导培养。 结果与结论：人脐血和外周血中存在内皮祖细胞,浓度梯度离心联合贴壁筛选获得的单个核细胞在血管内皮生长因子的诱导培养下可分化成内皮祖细胞,内皮祖细胞表达CD34、CD133、CD105、KDR和CD31,能吞噬乙酰化低密度脂蛋白,结合荆豆凝集素1,它们可作为体外分选内皮祖细胞的标志。     

人外周血内皮祖细胞培养方法的建立

背景：成人外周血来源丰富,但内皮祖细胞含量较少,为使其能够更好的应用于组织工程及细胞治疗,有必要建立外周血内皮祖细胞成熟、稳定的体外扩增体系。 目的：建立稳定的人外周血分离、培养和体外扩增血管内皮祖细胞的方法。 方法：应用密度梯度离心法,获取外周血单个核细胞,将分选后细胞接种于预先包埋了人纤维连接蛋白的培养板上,加入内皮祖细胞专用培养基中培养3 d后,洗掉非贴壁细胞,培养至第6天,收集贴壁细胞,应用倒置显微镜和苏木精-伊红染色进行细胞形态学观察;采用MTT法和细胞计数测定第1,3代细胞生长曲线;应用流式细胞仪测定祖细胞和内皮细胞系标志,对培养的细胞进行鉴定。 结果与结论：细胞生长曲线测定表明接种后第3天细胞进入指数增生期,至第6天进入平台期,随着传代次数的增加,细胞增殖速度变慢,同时表达干细胞表面标志CD34、CD133和内皮细胞表面标志血管性血友病因子、血管内皮生长因子受体2。证明人外周血可以分离培养内皮祖细胞。     

Human umbilical cord blood as a source of transplantable hepatic progenitor cells

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21-day cultures expressed ALB, and the ALB(+) cells coexpressed hepatocyte lineage markers. The ALB-expressing cells were able to proliferate in the culture system. Moreover, in the cell-transplantation model into liver-injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB-derived cell transplantation as a novel therapeutic option for liver failure.     

人脐血单个核细胞中CD133+细胞移植治疗心力衰竭的评价

BACKGROUND:Cell purification can eliminate the biological variability of cells, providing new insight into cell regeneration therapy. OBJECTIVE:To study the Influence of CD133⁺ cells on human umbilical cord blood mononuclear cell transplantation for treatment of heart failure. METHODS:Human cord blood mononuclear cells were isolated using lymphocyte separation medium method, and CD133⁺ and CD133^- cells were sorted using immunomagnetic beads at a cell density of 1×10⁸/L. Forty Sprague-Dawley rats were randomized into five groups: sham group, model group, CD133⁺ cell group, CD133^- cell group and mononuclear cell group. Animal models of heart failure were made using intraperitoneal injection of isoproterenol in all the groups except for the sham group. Rats in the CD133⁺ cell group and CD133^- cell group were given 1 mL CD133⁺ cells plus 1 mL PBS and 1 mL CD133^- cells plus 1 mL PBS via the tail vein, respectively. Rats in the mononuclear cell group were given 1 mL CD133⁺ cells plus 1 mL CD133^- cells via the tail vein, and those in the sham and model groups given 2 mL PBS via the tail vein. After 4 weeks, cardiac pathology, degree of myocardial fibrosis and colonization of CD133⁺ cells in myocardial tissues were observed. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that myocardial tissues arranged disorderly in the model group, but regularly in the sham group; myocardial disorders were mildest in the CD133⁺ cell group, successively followed by the mononuclear cell group, and severest in the CD133^- cell group and model group. Masson staining showed that in the model group, collagen fibers were proliferated, arranged irregularly and even broken, while in the sham group, the collagen fibers were less in number and arranged in order. Additionally, there was less reduction in collagen fibers and milder myocardial disorders in the CD133⁺ cell group compared with the other groups. Area of collagen fibers was increased significantly in all the groups except for the sham group (P < 0.05), but this increment was the minimal in the CD133⁺ cell group. Findings from immunohistochemistry and immunofluorescence staining showed that there were no CD133⁺ cells in the myocardial tissues of rats. Therefore, our data indicate that compared with the mononuclear cell transplantation, CD133⁺ cell transplantation exerts superiorities in relieving myocardial damage and reducing myocardial fibrosis. However, CD133⁺ cells are not colonized in the myocardial tissue after transplantation.