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。     

哺乳动物体细胞核移植研究进展

体细胞核移植技术已经在基础研究领域与产业化应用领域体现出了重要的价值,因而体细胞核移植技术及其相关研究已经成为了生物领域的持续性研究热点,但是围绕体细胞核移植技术仍然存在许多质疑,其中最主要的就是体细胞核移植的效率较低。尽管如此,体细胞核移植研究仍然在近年来取得了令人瞩目的成就,包括小鼠与恒河猴核移植胚胎干细胞系的建立。该文就体细胞核移植的研究历史与进展进行简要的论述,同时针对体细胞核移植研究中的细胞重编程与治疗性克隆研究中的发展与问题进行剖析,希望能够积极推动治疗性克隆的研究进展,加速核移植与干细胞技术在产业化领域中的应用。     

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.     

细胞核重编程对克隆的影响

细胞核重编程是哺乳动物正常受精胚胎和克隆胚胎发育过程中的一个重要特性,主要是对表观遗传学特征进行重新编写,包括染色质重塑、组蛋白修饰、DNA甲基化、印记基因表达、X染色体失活等表观遗传修饰的改变。通过细胞核重编程,首先,受精卵和克隆胚胎的供体核停止其特有的基因表达程序,恢复为全能状态的基因表达程序;然后,受精胚胎和克隆胚胎的细胞再从全能状态重新进入分化状态,最终形成各种组织和器官。近年来,不少研究表明,克隆胚胎的细胞核重编程存在不同程度的表观遗传修饰异常,可能对克隆及其农业和医学应用有着重要影响。本文就正常和克隆胚胎细胞核重编程的研究进展以及克隆胚胎的细胞核重编程异常对克隆的影响作一综述,并对目前有关治疗性克隆前景的不同看法进行了讨论。     

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 fi0 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 thedonor. 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 ceils maintainthe capability of sustained growth in an undifferen tiated state, and form embryoid bodies, which, on furtherinduction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that expressmarkers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NTto rabbit eggs retain phenotypes similar to those of conventional human ES ceils, including the ability toundergo multilineage cellular differentiation.     

核移植与供体细胞

“多莉”羊的诞生是生物界的一个里程碑,它之所以引起如此大的轰动主要是因为它来源于培养的成年绵羊乳腺上皮细胞,这是人类第一次证明分化的体细胞可以被重编程后恢复全能性并最终分化发育成一个动物个体。这说明哺乳动物分化的体细胞核仍具有全套的遗传物质并能够被卵母细胞逆转恢复全能性。然而,关于多莉的供体细胞来源却一直是克隆领域的一个谜。由于体细胞克隆的效率非常低,而用于核移植的供体细胞悬液中往往含有多种类型的细胞,这使得我们很难确切地知道最终获得的克隆动物是来源于哪一种细胞。这种不确定性给我们研究核移植诱导体细胞重编程的机制带来了很大的困难,因此,对供体细胞的研究也是核移植研究领域的一个重要课题,这包括各种组织来源的体细胞是否均可以用于核移植,终末分化的体细胞是否能够用于核移植,组织干细胞是否更有利于体细胞重编程,供体细胞的分化状态是否与核移植的效率有关,死亡的体细胞是否也可以用于核移植等等。本文综述了核移植中与供体细胞相关的最新研究进展。     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning, which is based on human somatic cell nuclear transfer, is one of our major research objectives. Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos, the effects of type, passage, and preparation method of donor cells on embryo development remain unclear. In our experiment, cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell, skin fibroblast, and cumulus cells. The cumulus cell embryos showed significantly higher development rates than the other two (P < 0.05). The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference. Also, fluorescence in situ hybridization (FISH) was conducted to detect nuclear derivation of the embryos. The result showed that the nuclei of the inter-species cloned embryo cells came from human. We conclude that (1) cloned embryos can be constructed through human-rabbit interspecies nuclear transfer; (2) different kinds of somatic cells result in different efficiency of nuclear transfer, while in vitro passage of the donor does not influence embryo development; (3) refrigeration is a convenient and efficient donor cell preparation method. Finally, it is feasible to detect DNA genotype through FISH. Translated from Zoological Research, 2005, 26(4): 416–421 [译自: 动物学研究]     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

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。Abstract: Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.     

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

Engineered heart tissue graft derived from somatic cell nuclear transferred embryonic stem cells improve myocardial performance in infarcted rat heart

The concept of regenerating diseased myocardium by implanting engineered heart tissue (EHT) is intriguing. Yet it was limited by immune rejection and difficulties to be generated at a size with contractile properties. Somatic cell nuclear transfer is proposed as a practical strategy for generating autologous histocompatible stem (nuclear transferred embryonic stem [NT‐ES]) cells to treat diseases. Nevertheless, it is controversial as NT‐ES cells may pose risks in their therapeutic application. EHT from NT‐ES cell‐derived cardiomyocytes was generated through a series of improved techniques in a self‐made mould to keep the EHTs from contraction and provide static stretch simultaneously. After 7 days of static and mechanical stretching, respectively, the EHTs were implanted to the infarcted rat heart. Four weeks after transplantation, the suitability of EHT in heart muscle repair after myocardial infarction was evaluated by histological examination, echocardiography and multielectrode array measurement. The results showed that large (thickness/diameter, 2–4 mm/10 mm) spontaneously contracting EHTs was generated successfully. The EHTs, which were derived from NT‐ES cells, inte grated and electrically coupled to host myocardium and exerted beneficial effects on the left ventricular function of infarcted rat heart. No teratoma formation was observed in the rat heart implanted with EHTs for 4 weeks. NT‐ES cells can be used as a source of seeding cells for cardiac tissue engineering. Large contractile EHT grafts can be constructed in vitro with the ability to survive after implantation and improve myocardial performance of infarcted rat hearts.     

Cloned mice and embryonic stem cell establishment from adult somatic cell

Cloning methods are now well described and becoming routine. Yet the frequency at which cloned offspring are produced remains below 2% irrespective of nucleus donor species or cell type. Especially in the mouse, few laboratories can make clones from adult somatic cells, and most mouse strains never succeed to produce cloned mice. On the other hand, nuclear transfer can be used to generate embryonic stem (ntES) cell lines from a patient's own somatic cells. We have shown that ntES cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntES cells can be used in regenerative medicine in order to rescue immune deficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem (ES) cells. In general, ntES cell techniques are expected to be applicable to regenerative medicine, however, these techniques can also be used for the preservation of the genetic resources of mouse strains instead of preserving such resources in embryos, oocytes or spermatozoa. This review seeks to describe the phenotype, application, and possible abnormalities of cloned mice and ntES cell lines.     

人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

体细胞克隆哺乳动物胚胎发育的表观遗传学

如何提高克隆效率和体细胞核移植后表观遗传重编程的潜在机制的研究是当前生命科学的热点之一。将处于分化状态而进行核移植的体细胞转变成具有全能型的早期胚胎的关键是表观遗传的重编程。文章从基因印迹,x染色体失活,端粒长度等方面来探讨哺乳动物克隆胚胎在发育过程中的表观遗传重编程的机制。     

胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

本文通过定向诱导人胚胎干细胞分化为心肌细胞,对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测,旨在探索分化过程中细胞能量代谢表型的转换机制.用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞;细胞免疫荧光检测人胚胎干细胞标志物,流式细胞术检测人心肌祖细胞和心肌细胞标志物;应用细胞外流量分析(extracellular flux analysis)方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.研究发现,人胚胎干细胞干性保持稳定,均表达Nanog、OCT4、SOX2细胞标志物;在向心肌分化过程中,第7 d心肌祖细胞标志物Isl1表达99%以上,分化第14 d心肌细胞标志物cTnT表达83%以上;人胚胎干细胞糖酵解代谢能力最强,心肌细胞线粒体功能最强,心肌祖细胞处于两种代谢方式的过度阶段.因此推断,在人胚胎干细胞向心肌细胞分化的过程中,细胞糖酵解能力逐渐减弱,线粒体氧化磷酸化能力逐渐增强,细胞的能量代谢类型发生转变.本研究旨在优化人胚胎干细胞定向分化为心肌细胞的方法,揭示...     

核移植与诱导多能干细胞技术在体细胞重编程研究中的应用

体细胞重编程是指分化的体细胞在特定的条件下被逆转后恢复到多能性或全能性状态,或者形成多能干细胞系,或者形成早期胚胎然后发育成一个新的个体的过程。诱导体细胞重编程的方法有许多,如核移植（nuclear transfer,NT）、细胞融合、细胞培养和通过导入特定因子获得诱导多能干（induced pluripotent stem,iPS）细胞的方法等。其中核移植和iPS技术是到目前为止诱导体细胞为多能干细胞最为完全、最具有运用于临床再生医学潜能的方法。然而,它们的效率都很低,机制也不清楚,如何将两个方法结合在一起,提高重编程的效率,揭示重编程的机制,进而促进其在患者特异性治疗中的运用将是下阶段的努力方向。