ES细胞体外定向分化为成熟肝细胞的实验研究

探讨了肝细胞在胚胎干细胞（ES cell）体外诱导分化系统中成熟分化的条件、机制及其鉴定方法.利用TGF, bFGF、HGF等细胞生长因子进行BALB/c小鼠ES细胞向肝细胞方向的定向诱导.利用反转录聚合酶链反应（RT-PCR）、免疫细胞化学（ICC）和放射免疫法（RIA）动态检测肝细胞特异性基因和蛋白AFP,ALB,G6P,TAT,CK8, CK18等在培养体系中的表达,并测定肝细胞的尿素合成功能,最后测定肝细胞分化率.结果,肝细胞特异基因AFP, ALB,G6P和TAT最早分别于第3、9、11、13天表达,肝细胞特异蛋白AFP,CK8,CK18和ALB最早分别于第7、9、9和11天开始表达.第12天开始检测到尿素出现,浓度为8.3 μmol/L,并随培养时间延长而浓度逐渐增加.最后,测得生长因子诱导组肝细胞的分化率为32％,对照组肝细胞分化率为8%.说明肝细胞可以在ES细胞体外诱导分化系统中出现并成熟分化,bFGF、HGF、OSM等可以明显提高细胞分化率和成熟度,有望成为解决肝功能替代疗法中细胞来源问题的新希望.     

小鼠胚胎干细胞向脂肪细胞分化过程中PPARγ2基因的表达模式

将PPARγ2基因启动子和报告基因荧光素酶相连接克隆于特定载体构建成表达质粒 ,电穿孔转染小鼠ES细胞 ,筛选阳性克隆。诱导ES细胞向脂肪细胞分化 ,通过定量检测荧光素酶活性跟踪PPARγ2基因的表达情况 ,以此研究脂肪细胞分化过程中该基因的表达模式。结果显示PPARγ2基因在未分化的ES细胞和EB形成的前两天中不表达 ,从EB形成的第 3天开始表达 ,直至脂肪细胞分化完成。该基因在已完成分化的脂肪细胞中的表达远强于在分化中的前脂肪细胞中的表达。首次报道了从小鼠ES细胞到脂肪细胞分化过程中PPARγ2基因的表达模式 ,支持了PPARγ2基因为脂肪组织特异性表达基因的已有报道 ,并为脂肪细胞分化机理研究提供了线索。     

人源诱导多能干细胞体外定向分化为功能性肝细胞的方法研究

摘要 目的：研究开发一种简易、快速在体外使多能诱导干细胞（induced pluripotent stem cells,iPSCs）定向分化为功能性肝样细胞的培养方法。方法：根据正常肝细胞在体内的发育规律,设计简化诱导方法使iPS细胞定向分化为内胚层细胞,应用qPCR和流式细胞术鉴定其纯度后进一步诱导分化为肝样细胞,并通过qPCR、ELISA、免疫荧光等技术鉴定肝细胞的性状和功能。结果：iPS细胞诱导7天后, OCT4和NANOG的表达水平显著下降,内胚层细胞相关基因CXCR4、FOXA2和HNF4A表达水平明显升高。内胚层细胞继续诱导培养15天后,肝细胞特异性标志基因ALB、TDO2、RBP4、G6PC和肝药酶基因CYPs等显著上调,同时产生高水平的白蛋白和尿素;PAS糖原染色为阳性,能主动摄取和释放吲哚菁绿,证实诱导成的肝样细胞具备正常肝细胞的部分功能。结论：该诱导方案能够在体外使iPS细胞遵循正常肝脏发育通路简易、高效地分化为功能性肝细胞。本研究为大量获得iPS来源的肝细胞及其在细胞疗法和药筛模型中的运用提供了可能性。     

细胞间相互作用影响iPSc来源肝细胞的成熟

获得功能性的肝细胞可用于研究肝细胞再生机理,并可为细胞移植和药物筛选提供生物材料。该研究采用四步法诱导多能干细胞分化成肝细胞,比较传代和不传代分化细胞的形态和肝细胞特异标志的差异表达。结果发现,利用诱导多能干细胞分化可获得白蛋白(albumin,ALB)阳性的成熟肝细胞,不传代法获得的细胞更接近于肝细胞形态,两组均获得甲胎蛋白(alphafetoprotein,AFP)和ALB阳性表达的细胞;GATA6(GATA binding protein 6)和HNF4A(hepatocyte nuclear factor 4 alpha)表达水平没有显著性差异,但不传代法分化细胞的AFP和ALB表达量高于传代法,存在显著性差异。结果提示,细胞因子与细胞间、多细胞间相互作用等因素影响多能干细胞的分化,细胞间相互作用有利于肝细胞定向诱导分化过程中肝细胞的成熟。     

单克隆来源的小鼠胎肝HPP-CFC肝上皮分化潜能的研究

为研究胎肝中造血和肝上皮发育的关系,建立了小鼠胎肝高增殖潜能集落形成细胞（HPP-CFC）培养体系,并进行了单克隆培养以及诱导分化实验.在造血和肝诱导因子的共同作用下,对单克隆来源的HPP集落细胞向造血和肝上皮细胞进行诱导分化,采用透射电镜(TEM)、巢式RT-PCR、细胞免疫荧光检测,从细胞形态、超微结构、上皮细胞分化标志等方面对分化后的细胞进行检测.检测结果显示诱导后的部分细胞具有肝细胞特异性的超微结构并不同程度的表达白蛋白（ALB）、甲胎蛋白（AFP）、细胞角蛋白（CK8,CK18）等肝上皮分化标志,同时还表达间质标志α-SMA和血管内皮细胞标志Flk-1.免疫磁珠分选表明：胎肝来源的HPP-CFC主要来自于CD45⁺细胞,CD45^-细胞不具有形成造血克隆的能力.在肝上皮细胞分化潜能上,流式分选获得的CD49f⁺/Sca-1⁺细胞与未分选细胞无明显差异.该模型的克隆源性通过细胞混合实验进行证明.研究结果表明,改进的胎肝来源的HPP-CFC可能代表了一个新的造血向肝上皮细胞分化的单克隆模型,为研究胎肝中造血和非造血细胞的发育关系提供了一个新的切入点.     

LO-2细胞上清诱导人脐血干细胞向类肝细胞分化

目的：探讨体外培养人脐血单个核细胞向类肝细胞的分化。方法：收集Lo-2细胞培养上清液加入培养基培养分离出的脐血单个核细胞,连续lOd检测甲胎蛋白（AFP）的表达情况,细胞传代后无诱导剂（LO-2细胞培养上清液）情况下检测白蛋白（ALB）表达情况。结果：培养6d后心P出现阳性结果,细胞传代后在无诱导剂的情况下AI卫检测阳性。结论：在L0-2细胞上清的诱导下,脐血单个核细胞能够分化为类肝细胞。     

LCB1基因表达对酵母神经酰胺合成的影响

酿酒酵母(Saccharomyces cerevisiae)LCB1（Long chain base）基因被克隆到酵母诱导表达载体pYES2中,并转入到FY2中,用半乳糖诱导表达。与对照相比,质粒所含LCB1基因的表达,使酵母细胞干重略有下降,而神经酰胺的含量提高为对照的1.9倍。     

小分子化合物体外诱导肝上皮样干细胞-WB细胞表达PDX1

目的：近年来干细胞治疗糖尿病一直是国内外研究人员关注的焦点,而肝细胞向胰岛样细胞转变也是热点之一。本实验应用小分子化合物在体外诱导WB-F344大鼠来源肝上皮样干细胞（简写WB细胞）表达胰腺内分泌前体细胞基因PDX1,建立一种体外诱导WB细胞分化为胰腺内分泌前体细胞的实验方法。方法：选用5-AZA TSA,RA,ITS等小分子化合物,分两步法直接诱导WB细胞分化为表达PDX1的胰腺内分泌前体细胞,用含有不同浓度5-AZA分化培养基诱导WB分化,摸索诱导分化的最佳条件。观察细胞形态变化,RT-PCR及实时定量PCR检测部分基因表达情况,免疫荧光检测PDX1的表达。结果：5AZA 5 uM处理2 d,TSA 1 d,RA联合ITS诱导7天,诱导的WB细胞表达PDX1较1-4 uM 5-AZA诱导强,并表达胰腺内分泌前体细胞的相关基因,NGN3,Neurod,NKX2.2,WB表达的Nestin仍持续表达,Insulin1有少量表达。WB表达的肝干细胞基因如ALB,AFP大量下调,标志分化的基因C/EBP下调。结论：5-AZA,TSA,RA,ITS等小分子化合物能够诱导肝上皮样细胞WB表达PDX1,并且这种诱导分化的细胞具有胰腺内分泌前体细胞特征。本实验进一步证明在体外微环境中,肝干细胞能向胰腺内分泌细胞转化,而肝细胞增极强,为将来干细胞治疗糖尿病提供充足的细胞来源     

肾胚细胞瘤原癌基因novC在tTA调控系统中的表达

nov基因与肾甲细胞瘤密切相关,在肾母细胞瘤细胞中大量表达,而在正常的成年肾中不表达。通过将Bujard′s tTA调控系统引入QT6细胞系中,同时将novC基因克隆到pUHD10-3质粒中,与pUHD-tTA质粒共转染细胞,诱导novC大量表达(其分子量约为48kD),建立了使nov基因大量表达的细胞系统,为研究novC基因对细胞的调控机制建立了体外细胞模型系统。     

H18杂交瘤抗凋亡能力的改造

利用PCR从pGEMTbcl-X_L质粒中获得bcl-X_L基因,构建真核表达载体pEF-bcl-X^{L,脂质体法转染杂交瘤细胞,G418筛选稳定表达株,Western blotting检测目的蛋白表达,流式细胞仪检测Bcl-X_L提高杂交瘤抗正丁酸钠诱导凋亡的功能。 将构建的编码鼠bcl-X_L基因的真核表达载体pEF-bcl-X_L,转染H18细胞后,获得稳定的表达株细胞;稳定表达Bcl-X_L的细胞具有抗正丁酸钠诱导凋亡的功能。鼠bcl-X_L基因在杂交瘤细胞中稳定表达,提高了杂交瘤抗凋亡的能力,对高密度大规模培养杂交瘤细胞具有重要意义。}     

食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定

间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程。胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型。该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCs。结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞。将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性。     

敲除Pofut1不影响胚胎干细胞向神经元分化

蛋白O-连接岩藻糖基转移酶1 (Pofut1)基因缺失可导致Notch分子无法与配体结合并启动信号传递. 为研究Pofut1基因对哺乳动物胚胎干细胞（ESC）向神经分化的影响,利用Pofut1基因敲除的胚胎干细胞与野生型胚胎干细胞,经体外培养诱导拟胚体（EB）分化为神经细胞,计数分化为神经细胞的比例,采用细胞免疫组化染色和real-time PCR等方法,分析神经细胞特异性标志分子的表达. 结果显示,Pofut1基因缺失后,对EBC生长没有明显影响,分化过程中形成的拟胚体数量明显增多,分化的神经样细胞以及神经标志物分子的表达也明显多于对照组;Notch信号缺失对小鼠胚胎干细胞生长无明显影响,但可以促进ES细胞向神经细胞分化.     

Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency

Murine embryonic stem cells (ESC) provide a unique homogeneous cell system for studying early vasculogenic cell differentiation in vitro. In this report, we characterized endothelial development of cultured E14 ESCs and mapped the effects of vascular endothelial growth factor (VEGF) on these cells. After removal of leukemia inhibitory factor undifferentiated state ESCs were precultured for 6 days and then cultured for up to 30 days in differentiation culture medium, with or without supplemental VEGF. ELISA analysis was used to detect endogenous VEGF levels. Early vasculogenic development and expression of selected genes were characterized using flow cytometry for specific antigens and quantitative RT-PCR. ELISA analysis showed no endogenous VEGF after preculture and at day 2 in unsupplemented culture, therafter VEGF levels rise. Directly after preculture a high proportion (36%) of the ESCs showed positivity for endothelial CD31. We describe characteristic endothelial differentiation patterns in embryoid bodies (EB) kept in culture for up to 30 days. VEGF supplementation lead to qualitative changes in the EB vessels, specific activation of vasculogenesis-related genes (CD31, CD144, and ERG) and temporary down-regulation of the VEGF receptor gene flk-1. VEGF supplementation did not produce measurable changes in the endothelial cell fractions as judged by surface antigen presence. We conclude that early ESCs may undergo endothelial differentiation through VEGF-independent pathways, whereas endothelial cell patterns in EBs are cytokine dependent and fully stimulated by endogenous cytokine levels.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.     

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

mRNA-Seq and MicroRNA-Seq Whole-Transcriptome Analyses of Rhesus Monkey Embryonic Stem Cell Neural Differentiation Revealed the Potential Regulators of Rosette Neural Stem Cells

Rosette neural stem cells (R-NSCs) represent early stage of neural development and possess full neural differentiation and regionalization capacities. R-NSCs are considered as stem cells of neural lineage and have important implications in the study of neurogenesis and cell replacement therapy. However, the molecules regulating their functional properties remain largely unknown. Rhesus monkey is an ideal model to study human neural degenerative diseases and plays intermediate translational roles as therapeutic strategies evolved from rodent systems to human clinical applications. In this study, we derived R-NSCs from rhesus monkey embryonic stem cells (ESCs) and systematically investigated the unique expressions of mRNAs, microRNAs (miRNAs), and signalling pathways by genome-wide comparison of the mRNA and miRNA profilings of ESCs, R-NSCs at early (R-NSCP1) and late (R-NSCP6) passages, and neural progenitor cells. Apart from the R-NSCP1-specific protein-coding genes and miRNAs, we identified several pathways including Hedgehog and Wnt highly activated in R-NSCP1. The possible regulatory interactions among the miRNAs, protein-coding genes, and signalling pathways were proposed. Besides, many genes with alternative splicing switch were identified at R-NSCP1. These data provided valuable resource to understand the regulation of early neurogenesis and to better manipulate the R-NSCs for cell replacement therapy.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.