昆明小鼠3.5 d和4 d囊胚不同分离方法的比较

背景：远交系的昆明小鼠作为中国自然科学研究主要实验动物,其胚胎干细胞建系的成功率一直很低。 目的：探讨昆明小鼠胚胎干细胞体外分离培养的最佳方法和最适合的采胚时间。 方法：采集孕3.5 d和4 d的囊胚分别以免疫外科法和全胚培养法在小鼠胚胎成纤维细胞饲养层上分离和克隆昆明小鼠胚胎干细胞集落。 结果与结论：免疫外科法和全胚法培养3.5 d囊胚的内细胞团贴壁率和原代克隆形成率差异均不显著(P  > 0.05);全胚法培养4 d囊胚的内细胞团贴壁率显著高于免疫外科法(P < 0.05),但原代克隆形成率显著低于免疫外科法(P  < 0.05);全胚法培养4 d囊胚的原代克隆形成率显著高于3.5 d囊胚(P  < 0.05);免疫外科法分离4 d囊胚内细胞团的贴壁率和原代克隆形成率显著高于以同样方法分离培养的3.5 d囊胚(P  < 0.05)。结果显示用免疫外科法分离4 d囊胚更适合于昆明小鼠胚胎干细胞的体外分离培养。     

小鼠囊胚内细胞团的生长行为

胚胎干细胞(ES细胞)是由着床前内细胞团(ICM)或原始生殖细胞经体外分化抑制培养而建立的克隆细胞系。能够广泛的分化成各种组织细胞．具有潜在的科研及应用价值。来自ICM的E细胞分离中．ICM集落生长状态和最佳传代时间是一个关键环节。本研究通过观察鼠囊胚内细胞团的体外生长行为,为ES建系积累经验。     

人胚胎干细胞不同传代方法的比较

目的探讨不同传代方法对人胚胎干细胞生长和分化情况的影响。方法用1mg/mlⅣ型胶原酶和机械切割两种不同的方法传代人胚胎干细胞,比较这两种方法传代的人胚胎干细胞克隆生长和分化情况。结果 1.机械切割法传代的人胚胎干细胞克隆较传统酶消化法生长快速且不易分化。2.传代后的人胚胎干细胞仍然维持胚胎干细胞的特有形态：表达胚胎干细胞表面标记,具有正常核型和多潜能分化能力（在体外形成拟胚体在体内形成畸胎瘤）。结论采用机械切割法传代人胚胎干细胞更利于细胞的培养扩增,且传代后细胞在继续培养中仍可保持干细胞的所有特性。     

体外分离培养昆明小鼠胚胎干细胞最佳胚龄的筛选:孕2.5d与3.5d及4.5d比较

背景:昆明小鼠基因型与人类近似,建立昆明小鼠胚胎干细胞系有利于其转基因动物的获得,但目前对于何时收集胚胎尚无明确报道。目的:探讨体外分离培养昆明小鼠胚胎干细胞的最佳胚龄。方法:分别从孕2.5d,3.5d和4.5d的昆明系母鼠子宫中分离收集胚胎,于显微镜下观察胚胎的形态、贴壁情况、内细胞团形成情况、胚胎干细胞克隆率、胚胎干细胞亚克隆率等,并进行碱性磷酸酶染色。结果与结论:孕2.5d采集的胚胎多为16细胞期胚胎,孕3.5d采集的胚胎多为桑椹胚,孕4.5d采集的胚胎多为囊胚。孕2.5d胚胎和孕3.5d胚胎的贴壁率、内细胞团形成率、胚胎干细胞克隆率、胚胎干细胞亚克隆率均无明显差异(P0.05);孕4.5d胚胎上述指标均显著高于前两者,更适合作为胚胎干细胞培养、克隆、分离、传代的材料。     

两种传代方法影响人胚胎干细胞转染效率的比较

背景:现有方法将外源分子如DNA导入到人胚胎干细胞用于科学研究的效率普遍较低,如何优化现有条件,提高转染效率显得尤为重要。目的:比较两种不同的传代方法对人胚胎干细胞系H9转染效率的影响,优化胚胎干细胞转染条件。方法:人胚胎干细胞系H9分别采用小克隆传代法和单细胞传代法进行传代,传代后继续培养细胞48 h,用Lipofectamine 3000转染pAdTrack-AKT1荧光质粒2 d后,荧光显微镜下观察荧光质粒的表达,流式细胞仪检测人胚胎干细胞的转染效率;RT-qPCR和Western blot分别检测转染后AKT1在mRNA和蛋白质水平的表达。结果与结论:①荧光显微镜下观察发现单细胞传代组表达荧光质粒的细胞数量更多,流式细胞仪检测单细胞传代法的转染效率[(47.18±2.00)%]高于小克隆传代法的转染效率[(19.52±0.86)%],差异有显著性意义(P<0.01);②单细胞传代组转染后AKT1 mRNA和蛋白的表达均高于小克隆传代组,差异有显著性意义(P<0.01);③结果表明,采用单细胞传代法,增加细胞与转染试剂脂质体的接触面积可提高人胚胎干细胞的转染效率。     

用低形态学评分D3胚胎建立人胚胎干细胞系

目的 寻找人胚胎干细胞(hESC)建系材料来源.方法 选用IVF低形态学评分的D3胚胎行序贯囊胚培养,用免疫外科的方法去除滋养细胞,将得到的内细胞团(ICM)接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞(MEFs)上培养5～8 d,每4～7 d传代1次,分别取不同代的hESC进行碱性磷酸酶(AKP)染色、转录因子OCT-4、阶段特异性胚胎抗原(SSEA)SSEA-4、SSEA-1、肿瘤排斥抗原(TRA)TRA-1-60、TAR-1-81、核型及体内外分化全能性鉴定.结果 130枚废弃的D3低形态学评分(评分＜16)的胚胎培养出囊胚19枚,获得原代克隆5个,成功培养出两株hESC系,它们具有hESC的共同的生物学特性.结论 部分低形态学评分的D3废弃胚胎可发育成囊胚.囊胚形成率与形态学评分相关,这些胚胎可作为建立hESC系的材料来源之一.     

小鼠胚胎干细胞构建嵌合体的方法

目的 利用本实验室新近分离培养的远交系KM小鼠胚胎干细胞进行嵌合体构建实验。方法 从KM小鼠囊胚的内细胞团中分离培养胚胎干细胞，进行近交系C57BL/6J小鼠受体囊胚腔内注射，进行嵌合体构建实验。结果 从内细胞团中分离的胚胎干细胞具有胚胎干细胞的典型辑征，克隆状生长，碱性磷酸酶染色呈强阳性，核型正常，悬浮培养能形成类胚体，已成功构建1只嵌合鼠。结论 用我们分离的KM小鼠胚胎干细胞可成功地构建嵌合体。     

人胚胎干细胞建系培养及体外诱导分化的研究进展

人胚胎干细胞具有发育全能性,在特定条件下能分化成多种类型的细胞.人胚胎干细胞的研究对人胚胎发育机制、人基因功能研究和治疗性克隆有着重大的意义.本文从人胚胎干细胞建系、培养及体外诱导分化等方面作一综述.     

大鼠胚胎干细胞的研究进展

胚胎干细胞是来源于早期胚胎内细胞团或着床后胚原始生殖细胞的一类未分化的全能性（多能性）干细胞，具有无限增殖和全向分化的能力。ES细胞能够自我更新和多向分化的生物学特性具有重要的医学价值，在基因治疗、组织工程学、药学研究等许多领域都具有广泛的应用前景。至今为止人类已建立了数百个小鼠的ES细胞系。但相对大鼠而言．由于其ES细胞在体外培养时较易发生分化，故建系较难。Iannaccone等从大鼠PVG近交系大鼠的囊胚分离克隆了大鼠ES细胞系-RESC-01。本文主要综述了目前分离、培养大鼠胚胎干细胞的技术进展，我实验小组在预实验中得到的结果及面临的问题，讨论其在临床工作中的应用前景。     

人胚胎干细胞体外培养、传代及冻存、复苏研究

目的建立人胚胎干细胞体外培养模式，并对其进行冻存及复苏。方法将人胚胎干细胞置于小鼠胚胎成纤维细胞饲养层上培养，细胞接近融合状态时进行传代，程序降温法冻存，速溶方法复苏，确定解冻后细胞复苏率、克隆生长与分化情况，并对其生物学特性进行鉴定。结果人胚胎干细胞稳定增殖了5个月，传至20代；细胞在小鼠胚胎成纤维细胞饲养层上呈集落生长，核大，核仁明显。程序降温法冻存后复苏，细胞复苏率达到78．3％；解冻后第12代细胞仍具有稳定的46，XX核型；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89．38％。结论成功建立了人胚胎干细胞体外培养模式，为研究人胚胎干细胞及相关疾病的防治提供细胞模型和细胞来源。     

The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores

BACKGROUND: Immune rejection can lead to the failure of human embryonic stem cell (hES cell) transplantation. One approach to address the problem is to establish hES cell line banks. Due to the limited source of human embryos and to ethical reasons, the hES cell lines are not readily available. This study was undertaken to determine whether discarded day 3 embryos with low morphological scores could develop into blastocysts and produce hES cell lines. METHODS: A total of 130 day 3 embryos with low morphological scores were cultured to blastocyst stage, and inner cell masses (ICM) were isolated by immunosurgery. Colonies derived from the ICM were passaged every 4-7 days and evaluated for cell surface markers, differentiation potentials and karyotypes. RESULTS: A total of 19 blastocysts were obtained from 130 embryos (quality score <16), which resulted in the formation of 10 ICM, and two cell lines. Both cell lines satisfied the criteria that characterize pluripotent hES cells. CONCLUSION: Our results suggest that a subset with poor quality day 3 embryos judged on the basis of morphological assessment can form blastocysts and give rise to hES cell lines.     

Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation

Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.     

人胚胎干细胞饲养层培养体系的研究进展

人胚胎干细胞(hES细胞)来源于着床前人囊胚内细胞团(ICM),由于具有体外无限增殖和分化成3个胚层来源的各种细胞的潜能,使其成为当今生命科学的研究热点.建立一个理想的hES细胞培养体系是利用它的前提.目前,最常用的hES细胞的体外培养方式是将其培养在饲养层细胞上.迄今为止,已经有多种细胞用于hES细胞的体外培养.饲养...     

Expression of cell-surface antigens and embryonic stem cell pluripotency genes in equine blastocysts

Embryonic stem-like (ES-like) cells have now been derived from the inner cell mass (ICM) of horse embryos at the blastocyst stage. Because they have been shown to express cell-surface antigens found in both human and mouse ES cells, the present study investigated gene expression patterns in day-7 horse blastocysts from which the horse ES-like cells had been derived originally. The genes studied included Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, SSEA-4, tumor rejection antigen-1-60 (TRA-1-60), TRA-1-81, and alkaline phosphatase activity, and whereas all three of the SSEA antigens were expressed in both the ICM and the trophoblast on day 7, Oct-4, TRA-1-60, TRA-1-81, and alkaline phosphatase activity were localized mostly in the ICM. Upon in vitro differentiation of the horse ES-like cells, their expression of the stem cell markers was abolished. Therefore, the species-specific expression pattern of stem cell markers in horse ES-like cells reflects gene expression in the blastocysts from which they are derived.     

Ultrastructure of the embryonic stem cells of the 8‐day pig blastocyst before and after in vitro manipulation: Development of junctional apparatus and the lethal effects of PBS mediated cell–cell dissociation

Ultrastructural examination of 8‐day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for ～36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the ～36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca⁺⁺‐Mg⁺⁺‐free phosphate buffered saline (PBS) for 5–10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts (～400 μm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell–cell dissociation methods. Anat Rec 264:101–113, 2001. © 2001 Wiley‐Liss, Inc.     

Nonviable human pre-implantation embryos as a source of stem cells for research and potential therapy

Human embryonic stem cells are derived from the inner cell mass of the human blastocyst. Presumably normal (frozen/thawed) human preimplantation embryos that remain unused following assisted reproduction procedures have provided the main source of blastocysts for stem cell derivation. Alternatively, embryos have been generated from gametes donated for the unique purpose of in vitro fertilization, blastocyst culture, and stem cell isolation. This article describes two previously published methods—and the background to those methods—that allow the use of nonviable embryos excluded from transfer and cryopreservation as a source of stem cells. The first method is based on the observation that some blastomeres from embryos with abnormal division during the first 3–5 d in culture can continue very limited development in isolation. When aggregated in a chimaeric form, some of these blastomeres can contribute to the formation of normally organized blastocysts. Blastocysts so obtained provide a route to embryonic stem cells from otherwise nonviable embryos. Thus the inner cell masses of blastocysts obtained from trisomic embryos were placed on feeder cells and cultured for seven additional days, following which the resulting cell colonies were examined for chromosome content. The second method concerns embryos diagnosed with specific chromosome abnormalities many of which are incompatible with life. Some of these aneuploidies do not preclude development to the blastocyst stage in culture. A proportion of these cells were found to be disomic and the cultures were shown to express OCT-4, a molecular marker for pluripotent cells. This apparent correction of the trisomic state in some cells within the colonies suggests that embryos with cromosomal abnormalities incompatible with life may be another source of human embryonic stem cells.     

兔抗小鼠胚胎干细胞免疫血清对8-细胞胚胎发育的影响

目的 制备兔抗小鼠胚胎干细胞免疫血清,观察其对小鼠胚胎发育的影响。方法 用小鼠胚胎培养基稀释兔抗血清,分别稀释至50倍、100倍、200倍,用其对小鼠8-细胞胚胎进行培养,通过胚胎致密化、囊胚率、胚胎体外贴壁生长、细胞计数、胚胎移植和免疫荧光染色观察胚胎发育状况。 结果 形态学上50倍稀释的抗血清能够抑制8 细胞胚胎的发育甚至使其死亡;100倍稀释的抗血清能够延迟8 细胞胚胎的致密化,并使得胚胎在囊胚化过程中出现空泡化的现象;200倍稀释的抗血清作用不明显。未作免疫处理的兔血清与空白对照组差异不显著。100倍稀释的抗血清能够显著抑制囊胚内细胞团（ICM）的发育、囊胚细胞分布紊乱、胚胎细胞数目减少以及囊胚畸形率增加。虽然囊胚的碱性磷酸酶和Oct4均呈阳性,但经胚胎移植后都不能正常发育。 结论 兔抗小鼠胚胎干细胞免疫血清能够抑制小鼠胚胎致密化过程和囊胚内细胞团的发育,使囊胚出现空泡化,细胞分布紊乱,抑制胚胎着床。