胚胎干细胞体外定向分化为Ngn3+胰岛祖细胞的基因表达谱分析

目的 探讨胚胎干细胞体外定向分化为胰岛祖细胞的基因表达谱.方法 采用逐步诱导分化方案体外传代培养胚胎干细胞,应用RT-PCR和免疫细胞化学方法鉴定各分化阶段相关特异基因的表达,应用Illumina Mouse Ref-8 vl.1小鼠基因表达谱芯片测定胚胎干细胞来源的不同阶段的分化细胞(第4、8、15、20、22和25天)与未分化胚胎干细胞的基因组表达谱.结果 本研究得到了86个阶段特异性表达基因,6组具有相同表达趋势的基因簇,6组阶段差异表达基因组.从数量上看,差异表达基因最多的阶段是后前肠阶段(201个基因),接下来是定形内胚层细胞阶段(17个基因).结论 应用基因芯片对胰腺发育过程中具有相同表达趋势的基因和阶段差异表达基因的分析为早期胚胎发育和胰腺发育研究提供了试验依据.     

人类胚胎干细胞定向分化为胰岛素分泌细胞的研究进展

人类胚胎干(hES)细胞具有极其强大的增殖能力,理论上具有分化为机体所有组织细胞的潜能.治疗性克隆技术可用于制备带有患者基因型的hES细胞及其分化而来的胰岛素分泌细胞,但存在激烈的伦理学争论.新近,通过体外转基因处理可诱导人类体细胞重构形成hES样的多潜能干细胞.本文着重介绍hES细胞定向分化为胰岛素分泌细胞的研究进展.     

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

Aims/hypothesis We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. Methods Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice.Results The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols.Conclusions/interpretation These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.Abbreviations Anti-Shh antibody against sonic hedgehog - D3 undifferentiated D3 stem cell line - EB embryoid bodies - ES embryonic stem - FBS fetal bovine serum - LIF leukaemia inhibitory factor - mES mouse embryonic stem - Ngn3 neurogenin 3 - P gelatine-coated plates - Pdx-1 pancreatic duodenum homeobox 1     

The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells

Background

Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs.

Methods and results

Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase.

Conclusion

We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.     

绿色荧光蛋白基因转染小鼠胚胎干细胞及向神经细胞的分化

目的 建立能够稳定表达绿色荧光蛋白(GFP)的小鼠胚胎干细胞(ESC),并诱导其向神经细胞分化,为临床移植治疗神经系统疾病提供方便追踪观察的神经细胞.方法 分别用脂质体、电穿孔转染法转染小鼠胚胎干细胞系R1,检测48 h转染效率;经G418筛选后,得到稳定高效表达GFP的ESC克隆,通过碱性磷酸酶(AKP)染色检测ESC的生物学特性;利用单层分化法诱导转染GFP的胚胎干细胞向神经细胞分化,通过免疫荧光检测神经细胞特异标记Toil.结果 转染48 h的GFP阳性细胞转染率脂质体组为65%,电穿孔组为79%,两组比较差异无统计学意义(X2=3.14,P0.05).转染GFP的胚胎干细胞AKP染色阳性,并可诱导分化为Tuj1阳性的神经细胞.结论 建立稳定表达GFP基因的胚胎干细胞克隆,获得的转染GFP的胚胎干细胞能够分化为神经细胞.     

胚胎干细胞移植到大鼠梗死心脏后的分化研究

目的研究胚胎下细胞在梗死心脏微环境下向心肌细胞、成纤维细胞的分化情况。方法将大鼠分为两组,梗死组为正常大鼠通过结扎左前降支(LAD)制备,对照组为正常大鼠。将4,6-二氨基(DAPI)标记的具有伞能分化能力的鼠胚胎干细胞(mESCs)注射人急性心肌梗死大鼠(18只)或对照绀大鼠(16只)的心脏,观察胚胎干细胞在休内的分化情况。结果 DAPI标记的移植mESCs在对照和梗死心脏均能成活并形成稳定的移植岛,同时在移植区有巨噬细胞浸润。mESCs移植2～4周后,心脏特异性肌钙蛋白T(cTnT)阳性的移植mESCs比例在正常心脏较梗死心脏高(2.67%±0.79%比1.06%±0.52%,P0.01),但4周后cTnT阳性的DAP1标记细胞在正常和梗死心脏的比例差异无统计学意义(1.17%±0.98%比1.07±1,02%,P0.05)。mESCs在埘照组和梗死组心脏都能分化为成纤维细胞。结论移植mESCs不仪能仔活,还可分化进入大鼠梗死心肌细胞。但是,梗死心脏的微环境不能选择性促进mESCs分化进入心肌细胞。     

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

AIM: To investigate the ability of a genetically altered embryonic stem (ES) cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene. METHODS: Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body (EB) culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone (DTZ), a zinc-chelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells. The outgrowths were incubated in DTZ solution (final concentration, 100 μg/mL) for 15 min before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA. RESULTS: DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells (Nkx-ES cells), which was as much as 2 wk earlier, than those in the culture of parental ES cells (wt-ES). The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters. Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated. CONCLUSION: Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.     

胰高血糖素样肽1诱导小鼠胚胎干细胞E14分化为胰岛素表达细胞

将小鼠胚胎干细胞株E14以高血糖素样肽 1(GLP 1)诱导 ,观察细胞形态 ,用原位杂交和RT PCR检测细胞内胰岛素mRNA的变化 ,免疫组化、流式细胞仪、放射免疫法验证细胞是否能分泌胰岛素样物质。发现E14细胞在GLP 1的作用下可以分化成表达胰岛素样物质的细胞     

间充质干细胞分化为肝样细胞的研究进展

肝炎后肝硬化等慢性进展性肝病、急性肝功能衰竭、肝脏代谢性疾病及肝脏恶性肿瘤等终末期肝病的发病率日益上升,美国贝塞斯达回忆宣布肝移植是目前治疗终末期肝病最有效的方法,但由于肝源有限、移植后的免疫排斥反应以及高额费用等限制了这种治疗方法的开展。近年来,随着对干细胞研究的深入,发现间充质干细胞(MSCs)具有向肝样细胞分化的潜能,且安全性、可行性及疗效均显示了较好的应用前景,为终末期肝病的治疗提供了新的途径。本文就MSCs不同组织来源分化特点、体内外分化为肝样细胞研究、分化后的肝样细胞的生物学特性及MSCs分化为肝样细胞的机制、MSCs应用的一些问题作一概述。     

Development of hematopoietic cells from embryonic stem cells

Embryonic stem cells are pluripotent stem cells that can differentiate into all somatic cell lineages and germ lineage cells in vivo. In vitro differentiation capacity of the cells is rather limited compared with the in vivo pluripotency. However, differentiation into hematopoietic lineages is easily obtained, and it is a powerful tool to investigate hematopoietic development and differentiation. In this article, we describe a differentiation induction method that we established, the OP9 system, a unique method using the macrophage colony-stimulating factor-deficient stromal cell line OP9. The utility of the OP9 system includes hematopoietic development, differentiation, B-cell formation, osteoclast formation, and so on. The usefulness and limits of embryonic stem cell-derived hematopoietic cells in cell therapy are also discussed.     

胚胎干细胞向心肌细胞分化中内皮细胞的作用

目的:研究胚胎干细胞向心肌细胞分化过程中,内皮细胞与心肌细胞之间的功能性联系. 方法:通过检测CGR8-GFP小鼠胚胎干细胞系心肌α-肌球蛋白重链(α-MHC)荧光蛋白表达情况变化,观察抑制内源性内皮细胞、添加外源性内皮细胞以及两者并存情况下,胚胎干细胞分化所得心肌细胞数量的变化. 结果:(1)在胚胎干细胞分化过程中,加入外源性内皮细胞后,心肌细胞形成明显增多.(2)特异性抑制内源性内皮细胞,心肌细胞形成明显减少.(3)外源性内皮细胞与胚胎干细胞共培养能够部分挽救由于内源性内皮细胞抑制所导致的心肌细胞形成障碍. 结论:在胚胎干细胞分化过程中,内源性内皮细胞对于促进心肌细胞形成起着至关重要的作用,是形成心肌细胞发育微环境的关键因子.在胚胎干细胞向心肌细胞分化过程中,外源性内皮细胞可以刺激心肌细胞形成,从而得到大量心肌细胞,具有潜在的临床应用价值.     

Notch1蛋白在胚胎干细胞向神经细胞诱导分化过程中的表达及意义

目的探讨胚胎干细胞（ESC）向神经细胞（NC）定向诱导分化过程中跨膜信号分子Notch1蛋白的表达情况及意义。方法体外培养ESC,在培养基内添加5×10^-7mol/L维甲酸（RA）进行诱导分化。分别取ESC及RA诱导分化1、5、9d的细胞,倒置相差显微镜观察细胞形态变化,免疫细胞化学、Western Blot法及流式细胞术检测各相应时间点Notch1蛋白的表达。结果随诱导时间延长,ESC分化出的成熟神经元标志微管相关蛋白（MAP-2）阳性神经细胞逐渐增多,并形成较为单一密集的神经网络结构。ESC阶段Notch1蛋白为高水平表达,诱导分化后Notch1蛋白表达随时间延长逐渐下降。结论在ESC向NC的诱导分化过程中,Notch1信号逐渐关闭;Notch1可能对ESC向NC的特异性分化起重要作用。     

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, thepotential of pax-4-nucleofected cells in medical treatment is promising.     

胚胎干细胞在预测毒理学中的应用前景

建立和完善既短期、经济,又能大通量筛检环境污染物对人类的毒性一直是预测毒理学中主要研究热点。胚胎干细胞具有自我更新和多向分化的特性,有望成为毒性安全性评价的体外替代实验模型。本文主要对预测毒理学现有方法的缺陷、胚胎干细胞的生物学特性和在预测毒理学中的应用研究进展进行综述。     

胚胎干细胞治疗肝病的进展

胚胎干细胞(ESC)在特定的环境下可以分化成各种组织细胞.ESC向肝脏细胞的定向分化使其可能成为肝脏细胞移植的一个重要细胞来源,为肝脏疾病的细胞移植治疗奠定基础,在治疗肝脏疾病的研究领域中有着广阔的应用前景.