Induced pluripotent stem cell lines derived from human somatic cells

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.     

提高兔核移植胚胎体外发育率的研究

 本研究首先比较了不同培养液维持兔胚体外发育的作用，以及多种卵母细胞去核显微操作的去核效率，建立了高效的体外培养体系及去核方法。然后对16-细胞期卵裂球的细胞周期及核移植（Nuclear transplantation,NT）胚的核质比对发育的影响进行了试验，结果表明：G#-1期NT胚的发育率优于G#-2期NT胚（P<0.01）。当G#-1期NT胚的核质比等于卵母细胞时，78.5%(73/93)可在兔眼球玻璃体液(Rabbit vitreous humor,RVH)中发育至囊胚期。这是迄今为止，在动物胚细胞核移植试验中所获得的最高发育率。     

牛乳腺干细胞核移植

近些年来,体细胞核移植虽然取得了很大的进步,但核移植效率仍然很低,而供核细胞的选择对于核移植效率起着关键的作用.本研究针对牛乳腺干细胞(mammary stem cells,MSCs)和牛乳腺上皮细胞(mammary epithelial cells,MECs)作为核移植的供核细胞,比较了重构胚发育率和核移植胚胎干细胞分离率的差异,发现来源于MSCs的重构胚的卵裂率为69%(331/479),囊胚发育率为27%(130/479);来源于MECs的卵裂率为71%,囊胚发育率为17%(84/495).囊胚发育率差异显著(p＜0.05).试验从176个核移植重构囊胚分离培养胚胎干细胞(embryonic stem cells,ES cells),发现来源于MSCs的重构胚的NTES贴壁率为44%(39/88),来源于MECs的NTES贴壁率为28%(25/88),NTEs的贴壁率差异显著(p＜0.05).以上数据表明,来源于MSCs的重构胚具有较高的发育潜能.     

Histocompatible embryonic stem cells by parthenogenesis

Genetically matched pluripotent embryonic stem (ES) cells generated via nuclear transfer or parthenogenesis (pES cells) are a potential source of histocompatible cells and tissues for transplantation. After parthenogenetic activation of murine oocytes and interruption of meiosis I or II, we isolated and genotyped pES cells and characterized those that carried the full complement of major histocompatibility complex (MHC) antigens of the oocyte donor. Differentiated tissues from these pES cells engrafted in immunocompetent MHC-matched mouse recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.     

转敲低MSTN基因绵羊重构胚的移植及鉴定

为培育肌肉丰满绵羊品系,以前期试验中获得的干涉MSTN基因的转基因细胞株为核供体,进行核移植,将获得的重构胚进行体外培养和胚胎移植。结果表明:所获得的重构胚与绵羊成纤维细胞为核供体时的重构胚分裂比率(分别为44.6%和24.1%)及其发育至桑椹胚比率(分别为15.6%和33.0%)相比,差异无统计学意义;将重构胚移植23只受体后,获得1只流产胎儿,对其进行PCR鉴定,结果显示MSTN基因干涉序列已整合入流产胎儿的基因组中。     

Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.     

Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.     

猪体细胞克隆技术研究进展

克隆不仅可以保护濒危动物,而且与干细胞工程技术结合应用于克隆性治疗,具有重大的科学意义和应用价值。体细胞克隆猪因其在人类器官移植方面具有巨大的应用潜力,已成为当今研究的热点,但是体细胞核移植技术仍存在许多问题,其中最主要的是体细胞核移植的环节多,克隆效率低。对体细胞克隆猪的关键环节,即供核细胞、卵母细胞、显微操作、克隆胚的激活、培养及移植技术进行了简要综述。     

猪胚胎细胞核移植

选用杜洛克２～１６细胞期胚胎卵裂球作核供体，湖北白猪卵母细胞作核受体，通过显微操作和电融合法构成重组胚。体外培养时，以融合前２ｈ，１ｈ激活及融合前不激活的卵母细胞做核受体的重组胚，发育率分别为６４．６％（３１／４８），５５．３％（２６／４７）和３４．５％（１９／５５）。６１枚重组胚移入同步发情的５头受体母猪输卵管，１头于妊娠１１７ｄ产下５头核移植仔猪。结果表明，激活卵母细胞作核受体优于未激活卵母细胞。成熟卵母细胞的胞质对于移入的核具有重排能力     

Modulation of hematopoietic stem cell homing and engraftment by CD26

Hematopoietic stem cell homing and engraftment are crucial to transplantation efficiency, and clinical engraftment is severely compromised when donor-cell numbers are limiting. The peptidase CD26 (DPPIV/dipeptidylpeptidase IV) removes dipeptides from the amino terminus of proteins. We present evidence that endogenous CD26 expression on donor cells negatively regulates homing and engraftment. By inhibition or deletion of CD26, it was possible to increase greatly the efficiency of transplantation. These results suggest that hematopoietic stem cell engraftment is not absolute, as previously suggested, and indicate that improvement of bone marrow transplant efficiency may be possible in the clinic.     

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.     

诱导性多能干细胞的研究及应用

自从小鼠的胚胎成纤维细胞和鼠尾成纤维细胞重编程成为诱导多能干细胞（induced pluripotent stem cells,iPSCs）以来,iPS的研究成了干细胞研究领域的热点。与胚胎干细胞相比,iPS细胞有操作简便和高稳定性等优点可以应用于,如创建人类疾病的遗传模型,培育转基因动物用于器官移植,改善动物生产性状和抗病性,以及生物制药等领域。另外,iPSCs的产生对于解决长期以来干细胞研究领域的伦理问题和免疫排斥问题有巨大的意义,iPS结合基因治疗和细胞移植疗法的成果已经应用到了动物疾病模型上。iPS细胞技术给病人特定细胞治疗和基因针对性药品研制带来了巨大的前景。此外,该技术也提供了iPS细胞重编程机制和人类疾病的病理过程研究的新平台。然而,现阶段多能干细胞的研究只是开辟了一个新的领域,iPS技术要应用于临床还有很多工作要做。本文主要针对iPSCs的研究现状与应用前景进行讨论。     

ICR小鼠胚胎干细胞的分离、培养及鉴定

以小鼠胚胎成纤维细胞为饲养层,收集受孕3.5 d ICR小鼠的囊胚和桑椹胚进行培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定。结果表明,ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物NBT、BCIP作用下,未分化的ES细胞显微镜下为黄褐色,分化的不着色;核型鉴定表明ES细胞具有正常的二倍体核型。     

Out of Eden: stem cells and their niches

Stem cells are currently in the news for two reasons: the successful cultivation of human embryonic stem cell lines and reports that adult stem cells can differentiate into developmentally unrelated cell types, such as nerve cells into blood cells. Both intrinsic and extrinsic signals regulate stem cell fate and some of these signals have now been identified. Certain aspects of the stem cell microenvironment, or niche, are conserved between tissues, and this can be exploited in the application of stem cells to tissue replacement therapy.     

供体细胞来源及预处理对牛体细胞核移植效率的影响

以牛外耳皮肤成纤维细胞为核供体细胞,比较了不同同步化诱导方式(血清饥饿法与接触抑制法)、供体细胞冷冻与否、供体动物性别与年龄等因素对体细胞核移植效率的影响。结果表明,接触抑制法处理的供体细胞核移植胚囊胚率显著高于饥饿法(P<0.05);未冷冻的供体细胞核移植胚囊胚率显著高于冷冻供体细胞(P<0.05);成年母牛供体的囊胚率显著高于成年公牛(P<0.05);不同年龄供体细胞的核移植胚囊胚率差异不显著(P>0.05)。试验表明,以接触抑制法诱导未冷冻成年母牛外耳皮肤成纤维细胞为核供体,有利于核移植胚囊胚的发育。     

cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.     

一种有效分离及在体外扩增成年小鼠精原干细胞的方法

尝试建立一种简便有效的成年小鼠精原干细胞分离培养及扩增的方法。从单只成年小鼠的睾丸中经过两步酶消化法分离精原干细胞,并在去除间质细胞、纯化后的支持细胞饲养层上培养与扩增,获得稳定增殖的精原干细胞系。此方法可显著提高建系成功率,保证细胞系的单一基因型,显著降低体外培养精原干细胞的成本。为成体基因来源的转基因动物的制作和疾病模型的建立提供技术支持。