Establishment and characterization of two new human embryonic stem cell lines, SYSU-1 and SYSU-2

Background Human embryonic stem cells can propagate indefinitely in vitro and are able to differentiate into derivatives of all three embryonic germ layers. The excitement surrounding human embryonic stem cells lies largely in their potential to produce specialized cells that can be used for transplant therapies. However, further investigation requires additional cell lines with varying genetic background. Therefore, efforts to derive and establish more human embryonic stern cell lines are highly warranted. Methods Surplus embryos （blastocysts） from donors were used to isolate the inner cell mass by immunosurgery. All cells were cultured continuously on irradiated murine embryonic fibroblasts feed layer and likely human embryonic stem cell colonies were subsequently characterized by cell surface marker staining, karyotyping and teratoma formation. Results Two human embryonic stern cell lines （SYSU-1 and SYSU-2） were established from surplus embryos. The two lines express several pluripotency markers including alkaline phosphatase, SSEA- 4, Tra-1-60, Oct-4, Nanog and Rex-1. They remain in undifferentiated state with normal karyotype after prolonged passages and can form embryoid bodies in vitro and teratoma in vivo. Conclusion Two new human embryonic stem cell lines have been established from surplus embryos. They can be used to understand selfrenewal and differentiating mechanisms and provide more choices for regenerative medicine.     

Establishment of human embryonic stem cell line from gamete donors

Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed. Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line ( criES-1 ) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types. Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widesoread impact on biomedical research.     

4种全能性基因转入人胚胎成纤维细胞诱导 多能性干细胞系的建立及其鉴定

目的：利用人类胚胎成纤维细胞(human embryonic fibroblast cells, hEFs)获得诱导性多能干细胞(induced pluripotent stem cells, iPSCs)系并对其全能性进行鉴定。方法：本研究采用慢病毒感染的方法将含有Oct4,Sox2,Nanog和Lin28全能性基因的慢病毒颗粒转导hEFs,获得iPSCs,并对其进行碱性磷酸酶(alkaline phosphatase,AKP)染色,以及检测表面标记,端粒酶活性,EB分化和畸胎瘤形成等,同时进行核型分析以及短串联重复序列(short tandem repeat,STR)分析。结果：所获得的iPSCs表达多能性细胞的表面标记,具有高的端粒酶活性,可在体内体外向内中外三胚层分化,与hESCs相似。经STR分析证实,iPSCs确实来源于hEFs而非胚胎干细胞的污染。结论：4种全能性基因转入hEFs可诱导获得与胚胎干细胞相似的iPSCs,有助于进一步的表观遗传学重编程以及干细胞多能性维持的研究。     

4种全能性基因转入人胚胎成纤维细胞诱导多能性干细胞系的建立及其鉴定

目的：利用人类胚胎成纤维细胞（human embryonic fibroblast cells,hEFs）获得诱导性多能干细胞（induced pluripotent stem cells,iPSCs）系并对其全能性进行鉴定。方法：本研究采用慢病毒感染的方法将含有Oct4,Sox2,Nanog和Lin28全能性基因的慢病毒颗粒转导hEFs,获得iPSCs,并对其进行碱性磷酸酶（alkaline phosphatase,AKP）染色,以及检测表面标记,端粒酶活性,EB分化和畸胎瘤形成等,同时进行核型分析以及短串联重复序列（short tandem repeat,STR）分析。结果：所获得的iPSCs表达多能性细胞的表面标记,具有高的端粒酶活性,可在体内体外向内中外三胚层分化,与hESCs相似。经STR分析证实,iPSCs确实来源于hEFs而非胚胎干细胞的污染。结论：4种全能性基因转入hEFs可诱导获得与胚胎干细胞相似的iPSCs,有助于进一步的表观遗传学重编程以及干细胞多能性维持的研究。     

Cytotoxic effects of mono-（2-ethylhexyl） phthalate on human embryonic stem cells

Background  Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test (EST) based on two human embryonic stem cell (hESCs) lines.

Methods  CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide (DMSO) and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies (EBs) formed after 8 days’ MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR.

Results  As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 µmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines.

Conclusion  MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicity in human embryos.

     

利用细胞因子和丁酸钠将小鼠诱导多能干细胞高效诱导为有功能的肝细胞

Background  Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Unfortunately, the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies. Therefore, it is urgent to find new ways to provide ample hepatocytes. Induced pluripotent stem (iPS) cells, a breakthrough in stem cell research, may terminate these hinders for cell transplantation. For the promise of iPS cells to be realized in liver diseases, it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.      

Methods  In this study, we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches: conditions via embryonic body (EB) formation plus cytokines, conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined, serum free monolayer conditions. Among these three induction conditions, more homogenous populations can be promoted under chemically defined, serum free conditions. The cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, indocynine green (ICG) uptake and release as well as urea secretion. Although efficient hepatocytes differentiation from mouse iPS cells were observed, mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.

Results  Mouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro, which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.

Conclusion We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

     

猕猴胚胎干细胞的分离培养和鉴定

目的从体外培养成熟囊胚中分离并鉴定猕猴胚胎干细胞(embryonic stem cell,ES cell).方法猕猴卵母细胞经体外成熟培养、体外受精和早期胚胎体外成熟培养后,获得猕猴囊胚.当囊胚由透明带自然孵出后,用细玻璃针剥离囊胚中的内细胞团(inner cell mass,ICM)并与饲养细胞进行共培养.由ICM分离,培养并鉴定胚胎干细胞集落.结果由4只FSH超排猕猴中共取得92个处于GV期的猕猴卵母细胞,选取其中的22个用HECM-10培养基培养后,获得6个高质量的囊胚,由此6个囊胚中分离得到3个内细胞团,并由此最终获得1株猕猴ES细胞,即RS5细胞.RS5细胞具高比例核/质比,核仁多,其细胞集落边缘平整,其内各单个细胞清晰.经约5个月的连续传代后,仍保持了正常二倍体的核型,其染色体数目为42条.碱性磷酸酶细胞组织化学染色为阳性,说明RS5细胞为未分化态的胚胎干细胞.经高密度和长时间培养后,RS5细胞可进一步分化为多种类型细胞.结论 RS5细胞株具有自我更新能力和多分化潜能,属于胚胎干细胞.     

昆明种系小鼠胚胎干细胞的分离培养及特性鉴定

目的 提高昆明小鼠胚胎干细胞（ES细胞）建株的成功率。方法 收集小鼠3．5d的囊胚培养,用小鼠胚胎成纤维细胞作为饲养层,形成的ES细胞样集落,经两次亚克隆分离培养ES细胞;进行相差显微镜观察、碱性磷酸酶染色和体内外分化能力鉴定。结果 获得两个稳定ES细胞集落,传至第11代。ES细胞呈集落样生长,碱性磷酸酶染色强阳性,体外分化的细胞沿拟胚体外向性生长,形态多样,在体分化可形成来源于3个胚层的组织。结论 两次亚克地获得的细胞具有ES细胞主要的生物学性状,有助于提高昆明小鼠ES细胞建株的能力。     

建立植入前遗传学检测人胚胎来源的无动物源性干细胞系

【目的】在无动物源性的干细胞培养体系中建立植入前遗传学检测（PGT）胚胎来源的人胚胎干细胞系,为医学研究提供疾病模型。【方法】在我们的研究中,对无动物源性的人类包皮成纤维细胞饲养层（XF-HFF）与成分确定的培养基（CDM）构建的无动物源性培养体系进行了评估。在该培养体系中,利用染色体平衡易位患者PGT来源的废弃胚胎建立一个新的人胚干细胞（hESC）细胞系。【结果】新建立的人胚胎干细胞系在无动物源性培养系统中可长期培养传代（>45个代次）。核型分析显示,在传代45代次后,新建立的人胚胎干细胞仍保持一致的核型。XF-HFF/CDM中的hESC仍保持多能性。通过荧光免疫染色检测到SSEA-3、SSEA-4、SSEA-1、TRA-1-60、TRA-1-81等多能标志物的表达;RT-PCR分析显示,在XF-HFF/CDM培养体系上生长的hESC中存在干细胞标记。小鼠体内实验也证实了hESC在体内维持其多能性。【结论】本研究建立了PGT来源的人胚胎干细胞系,为进一步建立携带遗传疾病基因的hESC系并为临床研究和治疗提供疾病模型打下了基础。     

胚胎干细胞与再生医学

由于胚胎干细胞具有分化能力,再生性强;同时由于处于低分化状态,它还具有分化成多种细胞、组织和器官的能力。科学家普遍认为胚胎干细胞的研究将为临床医学提供广阔的应用前景,可以解决长期困扰临床的供体不足和免疫排斥的难题,从而实现人类用人工培养的组织和器官更换疾病组织和器官的目标。本文重点介绍胚胎干细胞在心肌细胞、胰腺细胞、神经细胞等方面的再生研究。     

诱导性多潜能干细胞的研究进展与应用

分化成熟的体细胞可在体外被重编程为诱导性多潜能干细胞,后者具有与胚胎干细胞相似的自我更新能力和发育多潜能性,同时避免了免疫排斥和伦理问题.患者特异性的诱导性多潜能干细胞将成为再生医学、药物筛选和毒理测试的理想工具.     

人体胚胎干细胞蛋白质组学的研究进展

人体胚胎干细胞是一类具有自我更新能力的多潜能细胞,在一定条件下,可分化成超过200种人体细胞类型,它在发育生物学和再生医学中具有重要的研究价值。其多潜能性使得一系列疾病,包括癌症、阿尔茨海默病和帕金森病的治疗看到了希望。而胚胎干细胞蛋白质组学的研究对揭示胚胎干细胞增殖和分化的机制以及其多潜能的维持具有重大意义。在此,总结在过去几年中已报道的部分关于人体胚胎干细胞蛋白质组学研究取得的进步及其对人体胚胎干细胞研究的促进作用。     

多能干细胞与肾脏再生的研究进展

虽然肾脏移植术为肾功能不全晚期患者提供了一种有效的治疗策略,但尚存在供体器官有限、免疫排斥反应和终身服用免疫抑制剂等缺陷。多能干细胞具有无限自我增殖能力及在体内分化成为多种类型细胞的特点,尤其是胚胎干细胞和诱导性多能干细胞可分化为人体内的任何一种细胞,在组织、器官再生方面显示出良好的应用前景。该文主要对多能干细胞在肾脏再生方面的研究进展进行综述。     

玻璃化冷冻法保存人类胚胎干细胞

[目的]探讨玻璃化冷冻方法保存人类胚胎干细胞的效率.[方法]冷冻保存人类胚胎干细胞,按冷冻方法的不同分为玻璃化冷冻组和慢速冷冻组,比较两组解冻后人类胚胎干细胞的细胞复苏率、克隆生长与分化情况,并比较分析解冻后的与未经冷冻的胚胎干细胞的生长、分化情况,鉴定解冻后人类胚胎干细胞的全能性.[结果]玻璃化冷冻组与慢速冷冻组解冻后的细胞复苏率分别为81.9%和22.8%(P＜0.001).慢速冷冻组解冻后克隆生长较玻璃化冷冻组缓慢且更易分化.两组未分化的胚胎干细胞继续培养的生长与分化情况一致.玻璃化冷冻组解冻后第6代人类胚胎干细胞染色体核型正常,SSEA-4、SSEA-3阳性,表达OCT-4基因.人类胚胎干细胞来源的畸胎瘤包含了来源于3个胚层的组织细胞.[结论]玻璃化冷冻是保存人类胚胎干细胞的有效方法,解冻后的干细胞在继续培养过程中仍可保持其正常核型和全能性.     

胚胎干细胞诱导分化机制及方法

胚胎干细胞是来源于附植前胚胎的内细胞团或附植后胎儿的原始生殖细胞具有发育全能性的细胞。近年来在胚胎干细胞分化机制研究和定向诱导分化方面都有长足进展，但其分化机制和定向分化的方法一直是人们需要解决的难点问题，本文主要就胚胎干细胞的定向分化机制、方法及目前存在的问题等方面进行概括性论述。     

人胚胎干细胞神经分化的方法探讨

【目的】 利用新建系的人胚胎干细胞株SYSU-7,运用胚体形成的方法,探讨不同的分化条件对胚胎干细胞神经分化的影响。【方法】 首先检测人胚胎干细胞株SYSU-7的Oct4、SSEA4(stage-specific embryonic antigen-4)、TRA-1-60和AKP(Alkaline phosphatase)等胚胎干细胞特异性标志物的表达及体内外三胚层分化的能力;之后,应用含20% Knockout Serum Replacement (KSR)的培养液和神经干细胞培养液(neural progenitor medium, NPM)分别诱导细胞形成悬浮的拟胚体(悬浮时间为4 d或7 d),然后将拟胚体贴壁于多聚鸟氨酸(Polyornithine, PO)和纤维粘连蛋白(Fibronectin, FN)包被的培养皿中继续培养4 ~ 10 d。在分化的不同时间点利用免疫荧光染色的方法检测胚胎干细胞和神经细胞相关的标志性抗原。【结果】 SYSU-7胚胎干细胞表达未分化细胞特异性标志Oct4、SSEA4,TRA-1-60,AKP染色呈强阳性,在体内外具有向三胚层分化的潜能;在体外诱导其向神经分化,结果发现KSR培养液比神经干细胞培养液更有利于胚胎干细胞的神经分化,悬浮生长7 d的拟胚体贴壁后出现特征性的玫瑰花环样结构(rosette structure)的时间更早。数量更多。【结论】 SYSU-7细胞系具有自我更新及多向分化潜能等胚胎干细胞特性。本实验为研究人胚胎干细胞的神经分化提供了新的细胞模型和研究思路。     

人胚胎多能干细胞研究中的几个问题

目前在人胚胎多能干细胞的研究中存在材料来源不足、培养体系复杂和自发性分化的问题.这3个基本问题限制了人胚胎多能干细胞的基础和临床应用研究.要建立新的胚胎多能干细胞系应利用细胞核移植和细胞质重新编程功能建立新的途径,并探讨其中的机制和可能的关键因子;干细胞培养时主要进行简化培养体系和抑制细胞分化的研究;对干细胞分化进行临床应用研究时,主要将多种方法联合应用来诱导和筛选目的细胞并对其形态和功能进行评价.     

Human embryonic stem cells: multilineage differentiation and mechanisms of self-renewal

Human ES (hES) cells are pluripotent stem cells isolated from the inner cell mass (ICM) of blastocysts, with the theoretical capacity to differentiate in vitro to produce all somatic and germ cell types. The diverse differentiation repertoire of hES cells makes them ideal candidates for the generation of tissues for transplantation therapies and drug discovery. However, to realize the full potential of hES cells it will be necessary to characterize the mechanisms that control self-renewal and differentiation into specific cell types. We review here the recent developments to differentiate human ES cell into lineages including neural and cardiac. Further, by reference to the self-renewal system established in murine ES we will discuss the possible mechanisms of self-renewal in hES cells.     

成人胰腺干细胞分离、培养及鉴定

目的：从成人胰腺组织中分离并鉴定胰腺干细胞。方法：利用含bFGF的有血清培养基分离、培养出具有高度增殖能力的细胞群，采用RT－PCR技术检测其Nestin mRNA及Insulin mRNA表达情况。同时诱导定向分化，观察其分化后的状态。结果：从成人胰腺组织中分离出的条索状细胞具有高度增殖能力，其Nestin mRNA表达阳性，而胰岛素Insulin mRNA表达阴性，分化后的细胞形成胰岛细胞团样结构。结论：本研究得到的细胞具有高度增殖能力和分化成胰岛细胞的潜能，是来源于胰腺的胰腺干细胞。     

体外诱导人胚胎干细胞定向分化为神经干细胞

目的:探讨体外定向诱导人胚胎干细胞(human embryonic stem cells,hESCs)生成高纯度神经干细胞(neural stem cells,NSCs)的方法.方法:模拟体内神经细胞分化发育的不同阶段及微环境,分三阶段诱导hESCs定向生成NSCs.形态学观察结合免疫荧光细胞化学、流式细胞术和RT-PCR检测胚胎干细胞标志和神经干细胞标志;NSCs分化实验对所诱导的NSCs的分化潜能进行检测.结果:体外培养的hESCs在胚胎成纤维细胞饲养层上连续传代培养50代,仍保持SSEA-4,TRA-1-81阳性,表达Nanog基因,流式细胞术检测SSEA-4阳性表达率为83.44%;经三步法最终可诱导形成纯度高达90%以上的nestin阳性细胞,表达nestin基因,流式细胞术检测nestin阳性表达率为89.38%;诱导生成的细胞反复传代,仍表现为nestin阳性,并可进一步分化为神经元、星形神经胶质细胞和少突胶质细胞.结论:模拟体内神经分化过程的三步诱导法,可诱导hESCs生成较高纯度的NSCs,并能较好维持其干细胞特性和具有进一步分化的能力.