鸭胚胎干细胞分离与鉴定

采用药勺法分离仙湖3号肉鸭鸭胚的胚盘细胞,并以鸭胚成纤维细胞作为饲养层,培养鸭胚胎干细胞。在此基础上,通过对体外培养的鸭胚胎干细胞形态观察,碱性磷酸酶染色(AKP)以及胚胎阶段表面特异性抗原(SSEA-1)免疫组化等方法,分离与鉴定鸭胚胎干细胞。结果表明传至第3代的鸭胚胎干细胞经AKP和SSEA-1鉴定均为阳性,AKP染色为深蓝色,SSEA-1染色呈绿色荧光,表明培养至第3代的鸭胚胎干细胞仍保持干细胞未分化特性,具有胚胎干细胞的特征。结果提示本试验分离、培养的鸭胚盘细胞为鸭胚胎干细胞。     

人羊水来源干细胞的体外培养及其生物学性状分析

目的:体外分离培养人羊水来源干细胞(hAFSC),观察分析其基本的生物学性状。方法:取孕中期产前诊断所抽取的羊水,离心收集细胞,然后贴壁培养获得hAFSC。在细胞培养的基础上,观察hAFSC的形态;研究其增殖能力;用流式细胞术测定细胞周期及不同代次细胞表面阶段特异性胚胎抗原4(SSEA-4)表达率的变化;利用RT-PCR方法检测细胞干性基因的表达;利用诱导培养基诱导,检测其向肝样细胞与成骨细胞分化的潜能。结果:在体外培养条件下,hAFSC表现为成纤维细胞形态;具有良好的增殖能力,群体倍增时间约为36 h;细胞周期检测G0/G1期细胞约占86.7%,S+G2/M期细胞约占13.3%;流式细胞术检测结果表明,hAFSC表达SSEA-4,且SSEA-4阳性率随传代次数呈现先上升后下降的趋势;hAFSC在mRNA水平上表达Nanog、Oct4和Rex1等胚胎干细胞标志性基因;经诱导培养基诱导后,hAFSC可以向肝样细胞与成骨细胞分化。结论:hAFSC是一群呈成纤维细胞样,有良好增殖能力,且具有多向分化潜能的细胞,加之其来源方便,伦理学限制较少,因此在细胞治疗及组织工程等方面有着广泛的应用前景。     

山羊精原干细胞的鉴定及培养体系优化的研究

该研究优化了山羊精原干细胞(goat spermatogonial stem cells,g SSCs)培养体系,使山羊精原干细胞能在体外长期培养,维持自我更新的能力并保持未分化状态。取3~5月龄山羊睾丸,采用两步酶消法结合差速贴壁方法得到山羊精原干细胞悬液,分别通过形态学观察、碱性磷酸酶(alkaline phosphatase,AKP)染色、特异基因表达及蛋白质水平的分析对培养的细胞进行鉴定;并以山羊睾丸支持细胞(goat sertoli cells,g SCs)、小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)和层黏连蛋白(laminin,L)为饲养层,观察饲养层对山羊精原干细胞体外增殖的影响。结果表明,山羊精原干细胞体外增殖形成克隆簇,AKP染色呈阳性。经RT-PCR检测,Oct-4、C-myc、Cyclin D1、Ngn3和TERT等干细胞特异基因均有表达。细胞免疫组化结果显示,Oct-4、SSEA-1、α6-integrin、Vasa和Thy-1蛋白质呈阳性。克隆簇统计显示,在山羊睾丸支持细胞上形成的山羊精原干细胞(goat spermatogonial stem cells,g SSCs)克隆数与其他两组比较差异显著(P0.05)。山羊睾丸支持细胞饲养层上的精原干细胞可在体外传3~4代,培养时间为2个月。结果证明,通过两步酶消法和差速贴壁法可以分离获得山羊精原干细胞,且山羊睾丸支持细胞能够促进g SSCs的增殖。     

人-山羊异种核移植胚胎发育的初步研究

以体外分离培养的人胚胎成纤维细胞为核供体,经血清饥饿培养后,通过显微操作技术移入山羊去核卵母细胞中,采用化学方法激活重组胚.通过体外培养观察,2-细胞胚胎发育率可达51.33%,4-细胞发育率为31.42%,但发育至桑椹胚阶段的胚胎数目大大减少,仅为9.73%.虽然目前尚未能获得异种核移植囊胚,但实验结果说明山羊成熟卵母细胞可以支持人体细胞核完成重编程,人-山羊异种体细胞核移植重组胚可在体外完成其早期发育.     

限定因子诱导胎猪成纤维细胞重编程为多能性细胞

尝试运用限定因子融合蛋白建立猪的诱导多能性干细胞．试验采用Oct4、Sox2、Klf4、c-Myc四种限定因子经慢病毒表达载体系统介导感染猪胎儿成纤维细胞,对表达外源限定因子的猪胎儿成纤维细胞进行培养传代,逐步分离培养出集落边缘界限清晰的细胞克隆,细胞集落生长状态稳定、核型正常、碱性磷酸酶检测为阳性,免疫细胞化学检测显示,Oct4、Nanog、SSEA-1蛋白表达为阳性,体内能够分化形成含有三个胚层的畸胎瘤．结果证实分离培养的细胞克隆为猪诱导多能性干细胞,为进一步完善诱导方案和深入研究应用猪诱导多能性干细胞奠定了基础．     

不同培养条件对鸡胚胎生殖细胞Oct4启动子DNA甲基化的影响

旨在在体外通过胚胎生殖细胞(EGC)和细胞外基质共同构建一个EGC的小生境(niche)模型,通过比较Oct4DNA甲基化的变化,研究niche中特有的表观遗传效应对胚胎生殖细胞自我更新的影响.构建EGC细胞标准培养方案(对照组)和改良培养方案(试验组),采用甲基化特异性PCR( MS-PCR)技术、RT-PCR技术,对比分析两种培养方案中Oct4启动子的甲基化状态,判断基因表达与其CpG岛甲基化的关系,分析DNA甲基化模式与EGC细胞自我更新的关系.结果显示,试验组可扩增出Oct4的非甲基化扩增产物,而对照组为其甲基化扩增产物,试验组Oct4表达水平明显高于对照组.试验组EGC生长状态明显好于对照组.试验表明,在改良培养条件下,Oct4基因启动子DNA甲基化程度较低,且与基因表达水平呈负相关,更有利于维持胚胎生殖细胞的自我更新状态.     

胚胎干细胞在体外模拟心肌生长环境中向心肌分化

目的:探讨获得大量成熟同质有功能的心肌细胞(cardiomyocytes,CMs)的方法.方法:胚胎干细胞(embryonic stem cells,ESCs)经悬滴培养法形成拟胚体(embryoid bodies,EBs),将EBs用抗坏血酸处理后与天然心肌细胞共培养诱导ESCs的分化,采用免疫组化、流式细胞技术、RT-PCR、超微分析等方法分析分化细胞(ESC-derived CMs,ESCMs)的结构和功能.结果:共培养组,常规组和对照组中有(90.1±3.40)%,(29.6±6.03)%和(20.1±3.82)%的EBs表现节律性收缩,差异有统计学意义(P＜0.05).在体外模拟的心脏微环境中,不但跳动EBs的数目明显增加,且单个跳动EBs中含有功能CMs的百分比也明显增加.兴奋收缩的自律性能维持更长时间,在后期分化得到的有功能ESCMs在细胞形态上接近于天然的心肌细胞.结论:心脏微环境是促使ESCs特定地分化为CMs的关键因素,抗坏血酸处理和共培养诱导策略可使ESCs定向分化为CMs.     

通过形成类胚体诱导人羊水多能干细胞向心肌细胞分化

由人羊水中分离羊水多能性干细胞,通过形成类胚体诱导其向心肌细胞分化.取人羊水标本进行体外培养,分离得到人羊水干细胞,已连续传代培养至42代,采用免疫细胞化学、RT-PCR和流式细胞仪技术对羊水干细胞的生物学特性进行检测.取10～15代羊水干细胞,悬浮培养使其形成类胚体,进而向心肌细胞诱导分化.培养的羊水干细胞呈成纤维样,表达部分胚胎干细胞特异标志基因,悬浮培养可形成类胚体.类胚体碱性磷酸酶(AP)检测呈阳性,表达三胚层特异标志基因fgf5、ζ-globin和α-fetoprotein.羊水干细胞形成类胚体后进行诱导,得到α-actin阳性细胞,表达心肌细胞特异标志基因Tbx5、Nkx2.5、GATA4和α-MHC.试验结果表明,从人羊水标本中可分离得到具有胚胎干细胞特性的细胞,经初步检测确定为羊水干细胞,并能通过形成类胚体诱导其向心肌细胞分化.     

小鼠睾丸支持细胞的生物学特性研究

目的:获得高纯度的小鼠支持细胞,用以研究睾丸支持细胞在诱导胚胎干细胞向雄性生殖细胞分化过程中的作用,同时借助睾丸支持细胞减少进行体内诱导试验时可能产生的免疫排斥反应。方法:用胶原酶和胰蛋白酶组合消化结合选择性贴壁法从1周龄昆明白小鼠睾丸分离获得睾丸支持细胞,纯化后进行体外培养,观察其体外培养的生物学特性。结果:睾丸支持细胞体外培养3～4h即贴壁,贴壁后伸出3～4个突起,为成纤维型细胞,在体外培养2～3d即长满全瓶。油红Ο染色显示,其胞质含有大量脂滴。透射电镜观察结果表明,支持细胞核仁周围有卫星核小体。RT-PCR结果显示获得的细胞表达缪勒管抑制物,不表达促黄体素受体和小鼠VASA同源物。结论:获得了较高纯度的小鼠睾丸支持细胞,可以用于诱导小鼠胚胎干细胞向雄性生殖细胞分化的体内和体外试验。     

Isolation,culture, and characterization of primordial germ cells in Mongolian sheep

This study was performed to culture and preliminarily identify the primordial germ cells (PGCs) isolated from the genital ridge of the Mongolian sheep fetus. The growth characteristics of the sheep PGCs were detected in different culture systems such as culture media, resources, and state and passages of feeder cells. The obtained embryonic germ (EG) cells were identified by morphology, enzymology, and immunofluorescence. The results showed that the sheep EG cell colonies were ridgy, typically nest like, and compact, and had regular edges. Alkaline phosphatase staining reaction was weakly positive. EG cells expressed Kit, Rex-1, Nanog, and Oct-4. Immunofluorescence detection was weakly positive for Oct3/4, whereas positive for SSEA-1, SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80.     

Efficient derivation of human embryonic stem cell lines from discarded embryos through increases in the concentration of basic fibroblast growth factor

We describe the derivation and characterization of three novel human embryonic stem (hES) cell lines (YT1, YT2, YT3). One hES line (YT1) was obtained from six discarded blastocysts in a culture medium supplemented with 12 ng/ml basic fibroblast growth factor (bFGF), and two lines (YT2,YT3)were obtained from three discarded blastocysts in the same medium but supplemented with 16 ng/ml bFGF. These cell lines were derived by partial or whole embryo culture followed by further expansion after manual dissection of the passaged cells. These cells were passaged continuously for more than 6 or 8 months and possessed all of the typical features of pluripotent hES cell lines, such as typical morphological characteristics and the expression of hES-specific markers (TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, alkaline phosphatase, Oct4, Nanog) and pluripotency-related genes (Oct4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex1). The lines maintained normal karyotypes after long-term cultivation. The karyotype of YT1 and YT3 was 46,XX, and that of YT2 was 46, XY. Pluripotency was confirmed by in vitro and in vivo differentiation, and genetic identity was demonstrated by DNA fingerprinting.Our results indicate that higher concentrations of bFGF at the early culture stage support efficient the hES cell derivation.     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Cell growth arrest and apoptosis induced by Oct4 or Nanog knockdown in mouse embryonic stem cells: a possible role of Trp53

It has been clear that both Oct4 and Nanog play essential roles in maintaining embryonic stem cells (ESCs) undifferentiation. However, the roles of Oct4 and Nanog in ESCs growth and apoptosis have been much less explored. In this study, we systematically examined the effects of Oct4 or Nanog knockdown on mouse ESCs (mESCs) growth and apoptosis as well as potential mechanisms. Our results show that Oct4 or Nanog knockdown induces growth arrest and apoptosis in mESCs, indicating that the two genes also play important roles in mESCs survival and growth. Moreover, upregulation in Trp53 and its downstream genes expression was detected in Oct4 or Nanog knockdown mESCs, suggesting a possible role of Trp53 in Oct4 or Nanog knockdown induced mESCs growth arrest and apoptosis.     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog 基因 表达的比较

目的:比较通过慢病毒方法获得的人诱导多能性干细胞(iPSCs)与人胚胎干细胞(hESCs)分化过程中全能性基因Oct4、Nanog的表达变化。方法:收集分化不同时间点的拟胚体(EBs),检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果:iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷(SCID)小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论:通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

Effect of GSK-3 inhibitor on the proliferation of multipotent male germ line stem cells (mGSCs) derived from goat testis

The glycogen synthase kinase 3 (GSK3) inhibitor, 6-bromoindirubin-3′-oxime (BIO), is a key regulator of many signaling pathways to maintain pluripotency of human and mouse embryonic stem cells (ESCs). However, the effect of BIO on derivation of dairy goat male germline stem cells (mGSCs) remains unclear. The objectives of this study were to investigate whether BIO influences derivation of dairy goat mGSCs. Dairy goat mGSCs were cultured in mTeSR containing BIO medium and its effects on the proliferation ability of goat mGSCs (derived from goats ≤2 mo of age) were evaluated by 5-Bromo-2-deoxyuridine (BrdU) incorporation and alkaline phosphatase (AP) staining. Furthermore, its effects on maintenance of the undifferentiated state of mGSCs in late passages of cultures, as well as the capacity of mGSCs to differentiate into embryoid bodies (EBs) were examined. The presence of BIO increased the mitosis index and the number of AP positive colonies, as well as expression of pluripotent markers, Oct4, Nanog, Sox2, C-myc, Klf4, E-cadherin, and the proliferative markers, Pcna and C-myc. In contrast, there was no significant change in expression of apoptosis markers, P53, P21 and cyclin-related genes (Cyclin A, CDK2, Cyclin D1), as determined by RT-PCR analysis. When mGSCs were cultured in mTeSR medium containing BIO, EBs were formed, which were capable of further differentiating into various cell types found in the three embryonic germ layers, as determined by immunofluorescence and/or histologic staining. In conclusion, adding BIO to cultures BIO significantly promoted establishment of goat mGSC colonies and maintained their undifferentiated state.