人胚胎干细胞系NS1的建立

目的:探讨利用长期冷冻的废弃胚胎建立人胚胎干(hES)细胞系.方法:将临床体外受精(IVF)及精子卵浆内注射(ICSI)周期中剩余冷冻5年以上的废弃胚胎复苏后继续培养至扩张囊胚,链霉蛋白酶去除透明带后采用全囊胚培养法获取原代类胚胎干细胞,再用机械法传代分离纯化hES细胞,命名为NS1.取不同代次的培养细胞,进行碱性磷酸酶染色、转录因子OCT-4、阶段特异性胚胎抗原SSEA-4、肿瘤排斥抗原TRA-1-60、TAR-1-81、核型及体内分化实验对NS1进行全能性鉴定.结果:12枚废弃胚胎在体外继续培养后,有4枚发育为囊胚,最终得到1个干细胞系,经鉴定,这些细胞都具有hES细胞的生物学特性.结论:长期冷冻的废弃胚胎可用来建立hES细胞系.     

体外延长培养筛选低评分胚胎中具有发育潜能胚胎的可行性探讨

目的探讨了体外延长培养筛选低评分胚胎中具有发育潜能胚胎的可行性。方法将来自IVF/ICSI的第三天(D3)低评分胚胎进行体外延长培养,观察囊胚形成情况。结果307枚低评分胚胎经体外延长培养后形成53枚囊胚,囊胚率为17.26%,这些囊胚来自85例患者中的33例。结论①体外延长培养能有效筛选出D3低评分胚胎中具有发育潜力的胚胎;②D3胚胎形态评分不能完全预测其发育潜力。     

人废弃胚胎体外培养成囊胚的实验室结局

背景：有报道称可以利用废弃胚胎建立人类胚胎干细胞系,对形态发育滞后的胚胎冻融后移植仍有再利用的医学价值。 目的：观察人废弃胚胎在体外继续培养形成囊胚的潜能。 方法：收集接受体外受精-胚胎移植或卵胞浆内单精子注射患者治疗后第3天废弃的胚胎404枚,进行囊胚序贯培养。 结果与结论：①废弃胚胎404枚,形成囊胚 65枚,总囊胚形成率为16.1%。②囊胚形成与患者的病因分类、年龄、受精方式无相关性(P > 0.05)。③胚胎来源于≥三原核(3PN)的(6~8)细胞组的囊胚形成率最高(P < 0.05)。卵裂球数随细胞数的增加,囊胚率也增高,但>8细胞囊胚率反而下降、与<3细胞组的结果相近。④Ⅰ,Ⅱ胚胎形成囊胚速度快,形成率显著高于Ⅲ,Ⅳ胚胎(P < 0.05)。⑤有囊胚形成患者的临床妊娠率显著高于无囊胚形成患者(P < 0.05)。提示囊胚形成与患者病因分类、年龄、受精方式无相关性。优质胚胎与(6~8)细胞的胚胎囊胚形成率高,多原核胚胎形成囊胚的价值应引起关注。废弃胚胎再利用能预测临床结局,为人类胚胎干细胞提供有利资源。 关键词：人废弃胚胎;胚胎培养;囊胚形成;结局分析;胚胎干细胞 doi:10.3969/j.issn.1673-8225.2012.05.038     

复苏囊胚和复苏D3胚胎行囊胚培养后移植的临床结局比较

目的探讨胚胎的形态学指标与囊胚形成的关系,并比较复苏囊胚移植和复苏第3天(D3)卵裂期胚胎行囊胚培养后移植的临床结局。方法回顾性分析囊胚培养的2139枚新鲜D3胚胎及968枚复苏D3胚胎的囊胚形成情况;分析353例囊胚移植周期的妊娠情况,其中复苏囊胚移植周期136例(A组)和复苏D3胚胎行囊胚培养后移植周期217例(B组),比较两组的临床妊娠率、胚胎种植率和早期流产率等妊娠结局。结果新鲜D3胚胎和复苏D3胚胎的I-II级胚胎囊胚形成率、可用囊胚形成率和优质囊胚形成率均极显著高于III、IV级胚胎(P0.01),两组间相同评分级别的囊胚形成率无显著性差异(P0.05)。A组与B组相比,两组的年龄、移植胚胎数、胚胎种植率、临床妊娠率、早期流产率、多胎率和异位妊娠率之间无显著的差异(P0.05),但后者的移植周期取消率远高于前者(P0.01)。结论高评分D3胚胎的囊胚形成率高于低评分胚胎,对一些形态学上认为无冷冻价值的非优质胚胎也可成功培养至囊胚,筛选出具有发育潜能的胚胎;复苏D3胚胎行囊胚培养后移植周期具有更高取消移植周期的风险,因此选择冷冻囊胚解冻移植更具有优势。     

废弃胚胎继续囊胚培养对IVF/ICSI周期的意义

目的探讨体外受精/卵胞浆内单精子注射(IVF/ICSI)周期中第3天(D3)废弃胚胎形成囊胚的潜能及与妊娠结局的关系。方法回顾性分析1512个IVF/ICSI周期中进行囊胚培养的10355个废弃胚胎,比较了不同胚胎评分的废弃胚胎囊胚形成情况,还对这些囊胚移植后的临床结局进行了分析。结果废弃胚胎的囊胚形成率为17.45%,优质囊胚率为9.38%。按不同胚胎评分对囊胚形成率和优质囊胚率进行比较,优质胚胎(Ⅰ-Ⅱ级)最高(P0.05),Ⅲ级胚胎次之,Ⅳ级胚胎最低。Ⅳ级胚胎中,正常受精(2PN)胚胎的囊胚形成率和优质囊胚率均显著高于异常受精的无原核(0PN)和单原核(1PN)(P0.05)。废弃胚胎来源囊胚移植后可获得妊娠结局。结论 IVF/ICSI周期中的废弃胚胎继续培养后仍具有形成囊胚的潜能和获得妊娠结局的可能。     

低氧对人胚胎干细胞多能性维持及印迹基因表达稳定性的影响

目的 研究低氧对人胚胎干细胞(hESC)生物学特性维持及印迹基因表达状态的影响.方法 在低氧(5％O2)条件下持续培养FY-hES-7细胞并采用细胞免疫荧光染色、RT-PCR等方法对其胚胎干细胞特性进行鉴定.运用全基因组基因表达谱芯片技术及实时荧光定量PCR方法检测FY-hES-7早期(第32代)和晚期(第52代)细胞的55个印迹基因表达状态.所有检测同时以常氧(21％ O2)培养作为对照.结果 低氧条件下长期培养的FY-hES-7细胞具有和常氧条件下培养相似的生物学特性,阳性表达干细胞表面标志SSEA-3、SSEA-4、TRA- 1-60、TRA-1-81及碱性磷酸酶(AKP),但细胞克隆形态较常氧培养下典型,且未分化状态保持时间较常氧条件下更长.低氧长期培养后FY-hES-7细胞的55个印迹基因中,有15个印迹基因(27.27％)表达发生了变化(上调8个,下调7个),而常氧培养后则有39个印迹基因表达水平有变化(70.91％)(上调21个,下调18个),氧浓度对印迹的影响差异具有统计学意义(P＜0.05).结论 低氧更有利于hESC生物学特性的维持.长期培养能对hESC的印迹表达产生影响,但低氧培养条件下hESC的印迹稳定性要明显好于常氧培养.     

姐妹胚胎单囊胚结局的影响因素分析

目的 探讨姐妹胚胎继续单囊胚培养后影响其囊胚形成的相关因素.方法 回顾性分析行D3优质胚胎移植、 剩余姐妹胚胎单囊胚培养的7662个胚胎的相关信息,根据D3胚胎质量分为优质胚胎组及非优质胚胎组;根据形成囊胚的情况分为无囊胚形成组及囊胚形成组,其中囊胚形成组又按时间分为D5组和D6组,按囊胚形成的质量分为优质囊胚组和非优质囊胚组.结果 D3优质胚胎组的囊胚形成率、 优质囊胚形成率、D5/D6囊胚形成率、D5/D6优质囊胚形成率均显著高于非优质胚胎组;0PN、2PN胚胎的囊胚形成率、 优质囊胚形成率均显著高于1PN胚胎;囊胚形成组与无囊胚形成组间D3优质胚胎、1PN、2PN胚胎比例有显著差异;优质囊胚形成组与非优质囊胚形成组之间0PN、1PN、D3优质胚胎比例有显著差异;D5与D6囊胚形成组组间0PN、1PN、2PN、D3优质胚胎比例均存在显著差异.Logistic回归结果表明,原核数、 胚胎质量均对单囊胚培养结局有显著影响.结论 原核数、D3胚胎质量与姐妹胚胎单囊胚培养的结局密切相关.     

人胚胎生殖细胞在人胚胎成纤维细胞饲养层上的生长

目的探讨以人胚胎成纤维细胞为饲养层分离、培养人胚胎生殖细胞的方法和条件。方法分离、培养3～4月胚胎成纤维细胞,取3～15代细胞经丝裂酶素处理后铺板备用;分离6．11周胚胎原始生殖细胞,将其置于人胚胎成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞;用免疫细胞化学方法检测胚胎生殖细胞表面标志SSEA-1和SSEA-4;钙-钴法检测碱性磷酸酶活性;RT-PCR检测转录因子Oct-4的表达。结果人胚胎成纤维细胞可连续传代25代以上(6月),3～15代细胞可以用作饲养层细胞。分离的胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过8代。集落未分化标志检测显示SSEA—1、SSEA-4呈阳性,碱性磷酸酶活性呈强阳性,Oct-4表达阳性。结论用人胚胎成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。     

人胚胎干细胞无血清无饲养层培养体系的建立

目的 拟建立人胚胎干细胞无血清无饲养层的培养体系.方法 将干细胞分别置于Knockout培养基(第1组)、自制无血清培养基(第2组)和添加bFGF及肝素的改良无血清培养基(第3组)中培养25代后观察比较3组干细胞形态学变化、克隆数目及大小.并借助细胞免疫组化、流式细胞术和体外拟胚体形成等方法检测干细胞的未分化状态和全能性.结果 传代25次后,第2组克隆逐渐分化.第1组和第3组的干细胞均呈典型的人胚胎干细胞形态特征;细胞表达Nanog,Oct-4,Tra-1-60,SSEA-4,但不表达SSEA-1;流式检测也显示SSEA-4高表达,SSEA-1低表达;体外分化能形成拟胚体.通过单位视野下克隆个数及大小的比较,结果显示第2组与第1组和第3组间的差异显著.结论 本研究通过改良本实验室已获得国家专利的无血清培养基,初步建立了人胚胎干细胞无血清无饲养层培养体系,培养效果与公认培养体系无明显差异.     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的 探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案.方法 人胚胎干细胞系SYSU-I、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotide probe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型.应用PCR-STR技术检测两株细胞系的基因遗传标记.结果 获得两株hESC细胞系的HLA高分辨分型和STR基因型.结论 可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案.     

Characterization of six new human embryonic stem cell lines (HSF7, -8, -9, -10, -12, and -13) derived under minimal-animal component conditions

Human embryonic stem cells (hESCs) provide a renewable source of a variety of cell types with the potential for use in both scientific research and clinical cell-based therapy. Several hESC lines have previously been isolated and characterized, however, the majority of these lines were generated in the presence of animal serum and animal-derived feeder cells. Therefore, the exposure of the hESC to animal products may have induced phenotypic and/or genomic changes in the hESC lines not characteristic of normal hESC. Moreover, those hESC lines exposed to animal components may not be used for therapeutic applications due to the risk of graft rejection and pathogenic transmission from animal sources. In this study, we characterized six new hESC lines derived from human blastocysts under minimal-animal component conditions and cultured with human fetal lung fibroblasts. The hESC lines retained the ability to self-renew, are karytopically normal, and express stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, TRA-1-60, and TRA-1-81, but not SSEA-1, markers of pluripotent hESC. In addition, we show that telomerase activity decreased in each of the hESC lines following differentiation into embryoid bodies, albeit to different degrees. Finally, we demonstrate that the hESC lines are capable of differentiating into the three embryonic germ layers in vitro and form complex teratomas in vivo. This suggests that the hESC lines described here are valuable models for both future in vitro and in vivo studies, which may aid in the progression toward clinical-grade cell therapy.     

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.     

Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus)

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.     

Derivation of human embryonic stem cells from developing and arrested embryos

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.     

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.     

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.     

Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines

BACKGROUND: Serum-containing and serum-free media were used to derive human embryonic stem (HES) cells from donated oocytes and embryos. METHODS and RESULTS: Inner cell masses (ICM) were isolated by immunosurgery. The HES cells were found to be easily obtained and expanded in a serum-free medium. The efficacy in establishing human embryonic stem cell lines improved in a serum-free medium compared with that in serum-containing media. Four HES cell lines were derived from 13 isolated ICM on mouse embryonic fibroblast feeder layers. All four cell lines possess the same characteristics and differentiating potency: normal 46, XX or 46, XY karyotype; and expressing a series of surface markers such as APase, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1. They can form embryoid bodies in suspension culture and develop teratomas comprising derivatives of three embryonic germ layers when injected into severe combined immunodeficient mice. CONCLUSION: These preliminary results suggest that serum-free cultivation may be superior to serum-containing cultivation for deriving human embryonic stem cells.