哺乳动物孤雌干细胞与孤雌生殖

未受精的孤雌胚胎衍生的孤雌胚胎干细胞(parthenogenetic embryonic stem cells,pESCs),具有与胚胎干细胞(embryonic stem cells,ESCs)相似的多向分化和自我更新能力,且具备来.源广泛、获取高效及低致瘤性等优势,因此成为近年来的研究热点.该文概述了pESCs特殊...     

影响小鼠孤雌胚胎干细胞的建系效率因素初探

目的检测孤雌胚胎干细胞系的建系效率与小鼠品系以及培养体系的关系。方法将小鼠MⅡ期卵子孤雌激活发育至囊胚,然后从囊胚内细胞团分离孤雌胚胎干细胞。结果杂交和近交系小鼠的建系效率没有显著差异,建系的培养体系中加入ERK抑制剂或者采用血清替代品KSR时,建系效率显著提高。结论小鼠孤雌胚胎干细胞的建系效率与小鼠的遗传背景并没有直接关系,而与分离内细胞团的培养体系密切相关。     

孤雌胚胎干细胞基因印迹及X染色体失活状态初步研究

目的:研究孤雌胚胎干细胞(phESC)与受精卵来源胚胎干细胞(hESC)在印迹基因表达、X染色体失活等方面的异同。方法:运用实时荧光相对定量PCR、甲基化特异性PCR和免疫荧光染色等方法检测phESC与hESC在父系印迹基因IGF2R,母系印迹基因SNRPN,IGF2相对表达量及X染色体失活状态。结果:①母系印迹基因SNRPN,IGF2在phESC细胞中不表达,而父系印迹基因IGF2R表达量则相对于hESC有近2倍的上调;②XIST基因在第35代phESC细胞中没有表达,意味着早期的phESC没有进行X染色体失活,而到了第55代,XIST基因开始表达并随着分化时间的延长表达量逐渐上调;③XIST启动子甲基化状态及组蛋白H3赖氨酸27三甲基化免疫荧光染色阳性证实phESC在长期培养后启动了X染色体失活。结论:phESC的X染色体失活状态在培养过程中存在不稳定的情况,建议对phESC进行更深入的表观遗传稳定性研究,以确保这种细胞未来安全、高效的应用。     

小鼠孤雌胚胎干细胞的建立及其向运动神经元分化的初探

文章采用小鼠的孤雌囊胚建立胚胎干细胞系,探究其向运动神经元分化的可能,为临床治疗以及研究基因组印记与神经分化的的关系提供理论基础。结果表明:卵母细胞孤雌激活率达到93.26%,成功建立了8个孤雌胚胎干细胞系,建系率达到23.53%。克隆表达多潜能标记Oct4及细胞表面标记SSEA-1,有高水平的碱性磷酸酶活性,在细胞第10代和第30代时核型分析检测显示为正常的40条染色体。体内、外均分化出三胚层来源的细胞。联合应用全反式维甲酸(RA)、音猬因子(Shh)及细胞外基质,小鼠孤雌胚胎干细胞可被诱导表达运动神经元的标志性标记HB9、Olig2。     

小鼠胚胎干细胞与四倍体胚胎的嵌合

The oviducts of superovulated Kunming white females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1074 late two-cell-stage embryos.The embryos were placed twenty at a time between two platinum electrodes laid 1 mm apart in 0.3M mannitol in the electrode chamber.The blastomeres were fused by a short electric pulse(80V for 50μsec) applied by a pulse generator.Fusion of blastomeres was usually completed in 20-60minutes.After 25 hours of culture,most of the tetraploid embryos developed to the four-cell stage.Zonae pellucidae of 387 four-cell-stage tetraploid embryos were removed by treatment with acid Tyrode‘s buffer.The embryos were plated on an ES cell layer,After 40 hours of coculture,248 embryos aggregated with ES cells were collected and transferred into the uteri of twenty four 2.5-day pseudopregnant recipinets.Ten recipients were pregnant.but no live fetuses were born.Three pregnant recipients were routinely subject to a Caesarean section on day 18 of pregnancy and seven abnormal fetuses were obtained.The results demonstrate that ES cells derived from C57BL/6 mice are pluripotential to a certain extent.     

小鼠孤雌胚胎干细胞系的建立及生物学特性鉴定

目的:探讨建立合适的小鼠孤雌胚胎干细胞建系方法。方法:采用氯化锶联合细胞松弛素B激活B6D2F1杂交小鼠卵母细胞,所获得的囊胚与桑椹胚分别用于孤雌胚胎干细胞的建系,观察两者的建系成功率。结果:共建立了12株小鼠孤雌胚胎干细胞系,这些细胞SSEA-1抗原阳性,SSEA-4,TRA-1-81,TRA-1-60表面抗原阴性,具有AKP活性,保持正常染色体核型,体内外分化分别形成畸胎瘤和拟胚体。结论:采用囊胚和去透明带的桑葚胚建立孤雌胚胎干细胞系获得成功。该方法为人类纯合子的胚胎干细胞建系提供基础,在自体细胞治疗领域中具有潜在的应用价值。     

人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

小鼠孤雌胚胎干细胞集落的建立

ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＳＴＥＭＣＥＬＬＣＯＬＯＮＩＥＳＦＲＯＭＰＡＲＴＨＥＮＯＧＥＮＥＴＩＣＡＬＬＹＤＥＲＩＶＥＤＢＬＡＳＴＯＣＹＳＴＳＯＦＭＯＵＳＥ小鼠孤雌胚胎干细胞集落的建立ＫｅｙｗｏｒｄｓＭｏｕｓｅ,Ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃｅｍ...     

小鼠电激活孤雌胚胎早期体内外发育能力的比较

目的探讨小鼠电激活孤雌胚胎的早期体内、外发育能力。方法 利用不同电脉冲参数和激活液对小鼠卵母细胞进行活化,观察激活后的小鼠孤雌胚体外发育状况和移植后的发育能力。结果非电解质激活液优于电解质液,脉冲强度、脉冲宽度和脉冲次数3个参数各自处于某一范围内时,他们之间存在某种相关性,降低其中1个参数可通过升高另外2个参数得到补偿,经筛选较适宜的电脉冲参数为：1.0 kV/cm、40μs、2 p,或者1.5kV/cm、30/μs、2 p,分别为74.65%和71.19%,体外囊胚发育率分别为43.40%和47.62%。电激活孤雌胚体外发育时序比正常胚胎慢,但囊胚细胞数与对照组差异不显著。它们经胚胎移植后,其中的一部分能够着床,但着床率仅为3.6%,极显著低于对照组（67%,P〈0.01）。结论电刺激能够较好地模拟正常受精过程激活小鼠卵母细胞,但激活后的多数小鼠孤雌胚胎着床能力较低,不能够顺利着床。     

猪早期孤雌激活胚不同发育阶段差异基因表达的研究

收集2细胞、4细胞、8-16细胞时期的猪孤雌激活胚,采用SPEDDRT-PCR方法挑选不同时期的差异表达产物,通过反向northern杂交去除假阳性的条带。将阳性条带克隆入T载体中,经过PCR鉴定后挑选其中的阳性克隆进行测序,筛选了8个代表不同时期表达差异的cDNA片段,编号为DD1-DD8。经过与GenBank中的数据进行同源性分析,发现其中DD1和DD2没有相似的数据, 提交数据库获得GenBank登录号(EU545158, EU545159);其余的DD3-DD8发现了相似性较高的数据,但除DD3外均无基因功能说明需要进行进一步的研究。     

Human parthenogenetic embryonic stem cells: one potential resource for cell therapy

Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem (iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies. However, the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells. Embryonic stem cells (ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy. Recent studies on human parthenogenetic embryonic stem cells (hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics, but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions. To generate various pluripotent stem cells, diverse reprogramming techniques and approaches will be developed and integrated. This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology, and ultimately benefit cell therapy and regenerative medicine. Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA02A101).     

Aggregation of pre-implantation embryos improves establishment of parthenogenetic stem cells and expression of imprinted genes

Parthenogenetic embryonic stem cells (PgES) might advance cell replacement therapies and provide a valuable in vitro model system to study the genomic imprinting. However, the differential potential of PgES cells was limited. It could result from relative low heterology of PgES cells compared with ES cells from fertilization (fES), which produce different expression of most imprinted genes. Here, we described the establishment of PgES cells by aggregating parthenogenetic embryos at the 8-cell stage (aPgES cells), which may increase heterozygy. We found that derivation of aPgES cells in association with an increased number of inner cell mass cells by aggregating was more efficient than that of PgES cells from a single parthenogenetic blastocyst. The aPgES cells have normal karyotype, stain positive for alkaline phosphatase, express high levels of ES cell markers and can differentiate into teratomas composed of the three germ layers. Moreover, compared with PgES cells, the more highly upregulated paternally expressed imprinted genes were observed in aPgES cells, the same change was not shown in aPg blastocysts. This suggested that the aggregation induced effect could modify the expression of paternally expressed imprinted genes. Our studies showed that aPgES cells, the expression of imprinted genes in which more closely resemble fES cells than PgES cells, would contribute to all organs and avoiding immuno-rejection, which may provide invaluable material for regeneration medicine.     

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.     

Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).     

Human parthenogenetic stem cells produce enriched populations of definitive endoderm cells after trichostatin A pretreatment

Human parthenogenetic stem cells (hpSC) hold great promise as a source of pluripotent stem cells for cell-based transplantation therapy due to their ethical method of derivation as well as the enhanced capacity for immunomatching with significant segments of the human population. We report here the directed differentiation of hpSC to produce enriched populations of definitive endoderm. Moreover, we find that treatment of undifferentiated hpSC by trichostatin A (TSA) before applying the directed differentiation protocol significantly increases the proportion of definitive endoderm cells in the final population. TSA-pretreated as well as non-TSA-treated hpSC undergoing differentiation toward definitive endoderm demonstrate a similar temporal sequence of gene expression to that which occurs in the course of definitive endoderm differentiation during vertebrate gastrulation and for differentiation of hESCs to definitive endoderm. Creation of the definitive endoderm lineages from hpSC represents the critical first step toward the development of hpSC-based cellular therapies for diseases of the liver or pancreas.     

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines

Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.     

Haploid embryonic stem cells: an ideal tool for mammalian genetic analyses

Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology. However, the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches. Recently, mouse haploid embryonic stem (haES) cells have been successfully isolated from parthenogenetic and androgenetic embryos, providing an ideal tool for genetic analyses. In these studies, mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination. In particular, haES cells from androgenetic embryos can be employed as novel, renewable form of fertilization agent for yielding live-born mice via injection into oocytes, thus showing the possibility that genetic analysis can be extended from cellular level to organism level.