Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.

Cell transplantation therapy using pluripotent stem cells

The 2012 Nobel Prize in Physiology or Medicine was awarded jointly to Sir John B Gurdon and Shinya Ya-manaka “for the discovery that mature cells can be re-programmed to become pluripotent”. Professor John B Gordon who pioneered the feld of somatic cell nuclear transfer was the frst to show that a nucleus of a ma-ture cell can be transplanted into an enucleated egg and give rise to a living organism. His pioneering “clon-ing” technique paved the way for genome reprogram-ming and has led to subsequent cloning of differentani-mal species. Professor Shinya Yamanaka revolutionized the fled of stem cell production by showing that the introduction of four selected genes into cells transform them into induced pluripotent stem cells that resemble embryonic stem cells and serve as promising cells for future regenerative medicine.

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi-or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.

A brief review of recent advances in stem cell biology

Stem cells have the remarkable potential to develop into many different cell types, essentially with- out limit to replenish other cells as long as the person or animal is still alive, offering immense hope of curing Alzheimer＇s disease, repairing damaged spinal cords, treating kidney, liver and lung diseases and making damaged hearts whole. Until recently, scientists primarily worked with two kinds of stem cells from animals and humans： embryonic stem cells and non-embryonic ＂somatic＂ or ＂adult＂ stem cells. Recent breakthrough make it possible to convert or ＂reprogram＂ specialized adult cells to assume a stem stem-like cells with different technologies. The review will briefly dis- cuss the recent progresses in this area.

Potential for a pluripotent adult stem cell treatment for acute radiation sickness

Accidental radiation exposure and the threat of deliberate radiation exposure have been in the news and are a public health concern. Experience with acute radiation sickness has been gathered from atomic blast survivors of Hiroshima and Nagasaki and from civilian nuclear accidents as well as experience gained during the development of radiation therapy for cancer. This paper reviews the medical treatment reports relevant to acute radiation sickness among the survivors of atomic weapons at Hiroshima and Nagasaki, among the victims of Chernobyl, and the two cases described so far from the Fukushima Dai-Ichi disaster. The data supporting the use of hematopoietic stem cell transplantation and the new efforts to expand stem cell populations ex vivo for infusion to treat bone marrow failure are reviewed. Hematopoietic stem cells derived from bone marrow or blood have a broad ability to repair and replace radiation induced damaged blood and immune cell production and may promote blood vessel formation and tissue repair. Additionally, a constituent of bone marrow-derived, adult pluripotent stem cells, very small embryonic like stem cells, are highly resistant to ioniz-ing radiation and appear capable of regenerating radiation damaged tissue including skin, gut and lung.

干细胞在牛羊遗传改良中的应用研究进展

干细胞技术是当前动物生物技术的热门研究之一,不仅在再生医学和人类疾病治疗方面优势突出,还在动物遗传资源保存及品种改良等方面有着巨大的应用潜力。干细胞作为一种新型实验材料,可以与基因编辑技术、体细胞核移植技术、体外受精技术及高通量测序等技术相结合,在家畜遗传改良中有着极大的科研潜力和应用价值。本文总结了干细胞分离培养技术,以及近年来干细胞技术在牛、羊遗传育种中的研究进展,并对其应用及发展前景进行了展望。

Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

The nucleus of a somatic cell could be dedif-ferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellu-cida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic

哺乳类动物胚胎干细胞多能性和全能性研究进展

将ＥＳ细胞分离与克隆技术和分子生物学技术相结合就可将新的遗传物质导入家畜的生殖腺细胞。通过囊胚注入胚胎干细胞产生嵌合体小鼠表明对胚胎干细胞进行遗传操作是对动物生殖细胞进行遗传改造的有效方法之一。用于遗传操作的胚胎干细胞的多能性或全能性的检测技术，是改良家畜的有效方法。胚胎干细胞在动物克隆、基因工程及发育生物学等领域的应用前景广阔。

核移植胚胎干细胞的研究及其应用前景

随着核移植技术和干细胞技术的逐渐成熟,目前已获得牛、小鼠核移植胚胎干细胞,以及人-兔异种间核移植胚胎干细胞,这些细胞在体外可分化成多种细胞形态. 已经进行的实验性动物克隆性治疗,显示了诱人的潜力,但人核移植胚胎干细胞研究还面临着许多问题,如建系效率低、卵母细胞来源有限以及伦理学和安全性问题等. 长远地看,随着克隆效率的提高,在道德与法律之间达成共识,核移植胚胎干细胞必将造福人类.

异种动物细胞核移植研究现状及相关问题的探讨

随着体细胞核移植相继在多种动物中取得成功 ,异种细胞核移植技术在拯救濒危野生动物、人类干细胞克隆和细胞核质互作研究等方面具有特殊意义 ,受到广泛重视并取得了很大进展。文章对异种细胞核移植技术研究现状进行了综述 ,并对其应用前景和存在的问题进行了探讨。

昆明小鼠胚胎成纤维细胞分离培养及应用

目的探讨昆明小鼠胚胎成纤维细胞培养及应用。方法取昆明小鼠胚胎分离成纤维细胞,用丝裂霉素C处理成纤维细胞为胚胎干细胞制备饲养层,消化贴壁成纤维细胞为核移植提供供体细胞。结果13.5d、14.5d、15.5d孕鼠均可分离出成纤维细胞,14.5d孕鼠制作的成纤维细胞形态规则,3-5代成纤维细胞经处理可作饲养层,可作核移植供体细胞。结论昆明小鼠可分离成纤维细胞,用于饲养层制作及核移植供体细胞。

胚胎干细胞的代谢组学方法在毒理学中的应用进展

胚胎干细胞(ESC)是高度未分化的细胞,具备体外无限增殖和诱导分化成三个胚层细胞的特性,所以胚胎干细胞可以作为一种毒性评价的工具。代谢组学是近年来发展起来的对某一生物或细胞内源性的所有低分子量代谢产物进行定量和定性分析的一门新学科,它以生物体液,细胞提取物,细胞培养液和组织等为研究对象,研究手段主要是核磁共振和质谱。该文综述了胚胎干细胞和代谢组学相关的实验技术及其在毒理学中的应用现状,同时对基于胚胎干细胞的代谢组学在毒理学研究中的应用和发展趋势进行了探讨。

人胚胎干细胞的研究

来自着床前的囊胚和早期人胚胎的人胚胎干细胞是未分化的多能干细胞 ,具有无限增殖和分化的潜力 ,这种特性使之在基础研究和移植治疗中具有广泛的应用。尤其是胚胎干细胞可以产生任何类型的可供临床使用的细胞、组织和器官的潜力 ,将会带来一场医学革命。

囊胚注射转基因ES细胞制作嵌合体的研究

在小鼠胚胎成纤维细胞制作的饲养层上培养并成功的维持了携带LacZ基因的胚胎干细胞系（S8），在此基础上，以S8为供体细胞，以远交系昆明白小鼠3．5d胚胎为受体，通过显微注射法将供体细胞转移到受体的囊胚腔内，经过恢复培养，移植到代孕鼠昆明白雌鼠的子宫中；后代在嵌合体出生一周后进行判定。本试验用8～13代的S8细胞共注射胚胎597枚，经1～3h恢复培养，有585枚胚胎重新具有膨大的囊胚腔，细胞轮廓分明，滋养层细胞间连接也清晰可见，胚胎成活率为97％；胚胎移植后，代孕母鼠共移植胚胎228枚，经17～19d的妊娠期后，产仔37只（2只死胎），产仔率为16％；有35只仔鼠（雄鼠18只，雌鼠17只）存活到可以判断毛色，共获得8只S8细胞毛色嵌合体小鼠，嵌合体的产生率为21．6％。结果表明用S8细胞经囊胚注射后能够获得嵌合体，并且嵌合体明显发生了性偏离现象。本试验为国内利用囊胚注射携带LacZ基因的胚胎干细胞获得嵌合体小鼠的首例报道。

核移植与治疗性克隆

核移植与治疗性克隆在畜牧业生产以及生物医学上具有广阔和诱人的应用前景。文章分析指出卵母细胞质量与供核细胞重新编程是影响体细胞核移植效率及克隆动物异常的主要因素,阐述了治疗性克隆所面临的一些基本问题及出路:治疗性克隆以核移植技术为基础,核移植所面临的一些问题也直接影响着治疗性克隆的临床应用;核移植胚胎干细胞分离培养效率的高低以及向重要功能细胞定向分化是治疗性克隆的前提;成体干细胞可用于一些重大疾病的治疗,但不能完全替代克隆性治疗;伦理问题也阻碍治疗性克隆的发展。核移植及治疗性克隆技术要想尽快更好地应用于临床和造福于人类,需要不断完善各技术环节和加强一些基础理论的研究。

不同分化状态的供体细胞对小鼠克隆胚胎发育的影响

目的:探讨不同分化程度的供体细胞对克隆胚胎发育潜能的影响。方法:采用"一步法"核移植技术将供体细胞核直接注入小鼠卵母细胞,成功构建克隆胚胎。颗粒细胞克隆胚胎经含有10mmol/L的SrCl2和5ug/mLCB的培养液中激活处理5～6h,R1胚胎干细胞克隆胚胎经含有10mmol/L的SrCl2的培养液中激活处理3h,然后移入CZB培养液中继续培养。结果:颗粒细胞克隆胚胎体外的激活率、卵裂率和囊胚率与胚胎干细胞克隆胚相比具有显著差异,克隆胚胎体内移植后胚胎干细胞克隆胎儿出生率稍高(2.3%vs.1.2%),但没有显著差异。结论:供体细胞分化程度越低,克隆胚胎体内外发育潜能越高。

干细胞核移植效率及核移植胚胎干细胞

干细胞为一类具有无限的或者永生的自我更新能力的细胞,包括胚胎性干细胞和成体干细胞。胚胎性干细胞有胚胎干细胞、畸胎瘤细胞和原始生殖细胞。成体干细胞主要有骨髓间充质干细胞、造血干细胞、神经干细胞、表皮干细胞、脂肪干细胞等。随着体细胞核移植技术与干细胞培养技术的成熟,两者相结合便产生了核移植来源胚胎干细胞(embryonic stem cells via nuclear transfer,ntES细胞),其不仅用于基础的研究,而且也用于临床医学的组织修复和移植的研究。现就干细胞作为核供体时的核移植效率,ntES细胞系的建立、其性质及诱导分化等的研究进展进行综述。

组织工程研究中的胚胎干细胞问题

胚胎干细胞研究是组织工程基本科学问题中一个重要的组成部分。本文回顾了近年来胚胎干细胞领域的研究进程 ,尤其是人体胚胎干细胞研究的最新成果 ;提出了组织工程中“治疗性克隆”的战略目标。并且确立了未来人体胚胎干细胞研究解决组织工程种子细胞问题的具体步骤 ;指出了每项具体研究中所要解决的关键问题。

猪骨髓间充质干细胞的分离培养及核移植后重构胚胎发育能力的研究

不同类型的细胞核移植效率不同,原因之一可能是不同类型细胞核移植后进行重编程的潜力不同。本实验对猪骨髓间充质干细胞(porcine bone marrow mesenchymal stem cells,pMSCs)体外分离培养的方法进行了优化,对猪骨髓间充质干细胞的增殖及生长特性进行了观察分析,并以其作为供体细胞进行核移植,对此类型细胞进行重编程的潜力进行了评估。结果表明用密度梯度离心法分离猪骨髓间充质干细胞优于全骨髓贴壁法;猪骨髓间充质干细胞数目在培养第6天达到峰值,传代培养10h时,贴壁率达到78.50%;传代培养后第4天分裂指数最高,为24.00‰;以猪骨髓间充质干细胞(pMSCs)和猪胎儿成纤维细胞(PF)分别作为供核细胞构建核移植胚胎,其体外囊胚发育率分别为14.63%与15.07%(P>0.05),孤雌对照组囊胚发育率为30.91%(P<0.05);而三组囊胚细胞数分别为30.67±17.7、24.1±6.5和25.8±11.4(P>0.05)。实验表明,体外培养的猪骨髓间充质干细胞生长增殖旺盛,生物学性状稳定,并适合作为核移植供体细胞。