一种新的人胚胎干细胞自身来源的滋养层支持其体外培养

摘要: 通过人胚胎干细胞(Human embryonic stem cells, hESCs)经体内分化获取间充质干细胞(Mesenchymal stem cells, MSCs)为人胚胎干细胞提供一种新的滋养层。将约5×106个hESCs注射入重症免疫联合缺陷小鼠形成畸胎瘤, 8周后再从畸胎瘤中分离MSCs并鉴定, 将MSCs作为hESCs的滋养层细胞, 并检测和观察hESCs的生长情况、细胞特性和分化能力。从畸胎瘤中获得了纯度较高的具有类似骨髓来源的MSC特性的细胞群, 其形态相似、表面抗原标志相似(CD34和CD45阴性, CD29、CD49b、CD105、CD73和CD90阳性), 经诱导可以向成骨细胞和成脂细胞分化。将hESCs在MSCs滋养层细胞上传代培养10代以上, hESCs依然具有正常的细胞形态, 反转录PCR证实其特异转录因子Oct4、Nanog的表达, 干细胞表面标记SSEA-1显示为阴性, SSEA-4、TRA-1-60、TRA-1-81显示为阳性, 碱性磷酸酶染色显示为阳性, 并且核型正常。体外EB形成和体内畸胎瘤形成证明了其全能性。因此来源于hESCs本身的MSCs可以被用来作为支持胚胎干细胞生长并维持其未分化状态的滋养层细胞。     

一种新的培养人胚胎干细胞的包被基质

目的开发一种新的培养人胚胎干细胞(hESCs)的包被基质,使hESCs的培养更加简便。方法用甲醇固定的小鼠胚胎成纤维细胞(MEF)作为包被基质,人胚胎干细胞系X-01在该基质上生长,每隔5~6 d传代一次,培养10代后,对人胚胎干细胞特性进行检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达和分化能力。结果 hESCs在新的基质上生长良好,经10次传代后仍能保持典型的hESCs克隆形态。碱性磷酸酶染色阳性,免疫荧光染色Oct4、SSEA4、Tra-1-60为阳性,体外分化可形成拟胚体。结论此种固定的基质可以大量制备,长期保存,并可以长期维持hESCs的未分化状态,为人胚胎干细胞的体外扩增探索出了一个新的途径。     

人胚胎干细胞传代培养及细胞化学染色特性

目的 体外建立人胚胎干细胞传代培养方法,研究人胚胎干细胞细胞化学染色特性.方法 以小鼠胚胎成纤维细胞作为饲养层传代培养人胚胎干细胞,检测人胚胎干细胞、自发分化克隆及拟胚体的细胞化学染色特性.结果 人胚胎干细胞在小鼠胚胎成纤维细胞饲养层上传30代以上其形态保持不变;人胚胎十细胞碱性磷酸酶、过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性,自发分化克隆细胞阳性程度明显减弱;人胚胎干细胞形成的拟胚体碱性磷酸酶染色弱阳性,过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性.结论 小鼠胚胎成纤维细胞能支持人胚胎干细胞传代培养,细胞化学染色结果能初步鉴别人胚胎干细胞未分化特性.     

无饲养层培养人胚胎干细胞方法的建立

人胚胎干细胞(human embryonic stem cell,hES细胞)是当前医学研究的热点之一．然而hES细胞培养条件苛刻,通常需要采用鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEFs)饲养层来维持其未分化状态,成为目前hES细胞研究的瓶颈之一、本实验成功地将hES细胞接种在细胞外基质包被的六孔板上培养,传代20次后细胞仍然保持良好的未分化状态,各种hES细胞生物学特性(如表面标志物SSEA-3、SSEA-4、TRA-1-60和TRA-1-8l,OCT-4,碱性磷酸酶及体内外分化潜能等)均无改变;其冻存、复苏效果与生长在饲养层上的hES细胞无明显差异．因此,该无饲养层培养体系可以用于培养hES细胞,并为hES细胞转基因研究及大规模培养打下良好的基础．     

人脐静脉间充质干细胞的分离培养及生物学特性鉴定

为了对人脐静脉间充质干细胞(MSC)进行分离培养及其生物学特性鉴定。采用胶原酶分步消化法获得人脐静脉间充质干细胞(hUVMSC2)并对其进行体外培养、形态学观察及绘制生长曲线;利用条件培养基诱导法分析该细胞分别向成骨细胞和脂肪细胞分化能力;流式细胞术检测细胞表面标志物CD54、CD105、CD29、CD166、CD44、CD31、CD34、CD49、CD106等表达情况。结果该细胞形态为梭形或成纤维样,不表达内皮来源的vWF因子。在不同诱导条件下,该细胞可分别向成骨细胞和脂肪细胞分化。hUVMSC2细胞表面表达CD54、CD105、CD29、CD166、CD44等间质细胞黏附分子,不表达CD31、CD34、CD49、CD106等内皮或造血细胞相关标志物。该细胞指数生长期倍增时间约为26h,在添加bFGF条件下可迅速增殖,指数生长期倍增时间缩短为16h。研究证实人脐静脉内皮层下存在间充质干细胞,采用分步酶消化法可同时分别获得单根脐静脉的内皮细胞和间充质干细胞。hUVMSC2间充质干细胞具有向脂肪细胞和成骨细胞分化潜能并表达多种黏附分子。     

人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

脐带间充质干细胞成脂分化的研究

目的:探讨脐带间充质干细胞向成脂细胞方向分化的潜能,为脂肪组织工程提供一种来源丰富的干细胞来源。方法:采用胰酶和胶原酶Ⅰ型联合消化法获得脐带间充质干细胞,通过免疫细胞化学法对其表面标志物进行鉴定,化学诱导方法诱导脐带间充质干细胞向成脂细胞方向分化,倒置显微镜观察其形态变化,油红O染色对诱导后的细胞进行染色。结果:胰酶和胶原酶Ⅰ联合消化法分离的细胞贴壁生长,呈现成纤维细胞形态,免疫细胞化学显示脐带间充质干细胞表达CD29、CD44和CD105,但不表达CD31、CD34和CD105,脐带间充质干细胞在成脂诱导培养基中细胞生长速度明显减慢,细胞形态转变为肥大、扁平、含有大量脂滴的脂肪细胞,油红O染色示胞浆充满了油滴空泡。结论:脐带间充质干细胞具有向成脂细胞方向分化的潜能,为脂肪组织工程提供了一种来源丰富、免疫力低和低分化的种子细胞。     

人尿源性干细胞及人脐带间充质干细胞的生物学性状比较

该研究探讨人尿源性干细胞(human urine-derived stem cells,hUSCs)及人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状差异。分离培养hUSCs及hUC-MSCs,显微镜下观察细胞形态,流式细胞术检测干细胞表面标记物,锥虫蓝拒染实验及克隆形成实验检测细胞增殖能力,划痕实验及Transwell迁移实验检测细胞迁移能力,碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色、油红O染色及阿利新蓝染色评估多向分化潜能。hUSCs为米粒状贴壁生长细胞,hUC-MSCs为长梭形贴壁细胞,呈旋涡状排列生长,两种细胞表型分析相似,均表达多种间充质干细胞标志物,但CD24在hUC-MSCs表达阳性,而CD105在hUSCs表达阳性。hUC-MSCs的增殖及迁移能力优于hUSCs,但后者的克隆形成能力更强。hUSCs及hUCMSCs都具有成骨、成脂、成软骨分化能力,hUC-MSCs的成骨能力强而hUSCs的成脂能力强。该研究成功分离培养出增殖能力强并具有多向分化潜能的hUSCs,该细胞与hUC-MSCs相比具有相似的生物学性状,可作为再生医学自体移植的理想种子细胞来源。     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

人羊膜上皮细胞和胚胎干细胞

人胚胎干细胞有着巨大的医学应用前景,但人胚胎干细胞要求的生长条件很高,体外很难模拟其生长的体内环境,因此控制人胚胎干细胞的生长常不理想,而使用鼠胚胎成纤维细胞(MEF)作为滋养层则存在动物源性污染的问题。该文阐述人羊膜上皮细胞(HAEC)的特点及其作为滋养层培养胚胎干细胞的现状,并探讨基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用,为建立更优化的培养系统提供依据。     

The comparison of different protocols for expansion of umbilical-cord blood hematopoietic stem cells

Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34(+) cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96-fold increase of CD34(+)CD71(-) and a 3.13-fold increase of CD34(+)HLA-DR(-) was observed. The total number of colony forming cells (35 +/- 2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67 +/- 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

Proliferative feeder cells support prolonged expansion of human embryonic stem cells

Long-term proliferation of human embryonic stem (hES) cells is currently achieved by co-culturing with mitotically inactive primary mouse embryonic fibroblasts (mEFs), which serve as feeder cells. This study explores the possibility that proliferative mEFs can be used as feeder cells to maintain the prolonged expansion of hES cells. All undifferentiated hES cell clumps were re-plated on six different densities of proliferative mEFs. hES colonies cultured on 1 x 10(5) - 5 x 10(5) proliferative mEFs amplified over 130 days of continuous culture and remained undifferentiated, as did those cultured on mitotically inactive mEFs. This suggests that certain densities of proliferative mEFs can maintain the propagation of hES cells, which may be helpful for identifying the cytokines and adhesion molecules that are required for their self-renewal.     

人脐带间充质干细胞在急性肾小管坏死模型犬的体内分布及趋化性

目的 观察人脐带间充质干细胞在家犬急性肾小管坏死模型的体内分布及归巢.方法 健康家犬18 只随机分为3 组.模型1 组:肌注新鲜配制的0.2﹪二氯化汞溶液7 ml/kg建立急性肾小管坏死模型,采用经外周静脉注射法输注体外分离培养并用4',6- 二脒基-2- 苯基吲哚(DAPI)标记的人脐带间充质干细胞.模型2 组:造模...     

A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells

Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.