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 .     

Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications.     

Human embryonic stem cell lines: socio-legal concerns and therapeutic promise

Stem cell lines would be very valuable for the repair of diseased or damaged organs. Stem cells derived from adult tissues raise few ethical problems, and would not be rejected if derived from the patient. They show considerable plasticity and might be appropriate for some clinical conditions, but they tend not to grow well in culture. Stem cells derived from the early human embryo proliferate indefinitely in culture and can give rise to many different tissues, but their derivation requires destruction of the embryo, which is not ethically acceptable in some countries. Other countries allow strictly regulated destructive research on human embryos, usually those that have been produced for infertile couples in infertility clinics. Embryos that are no longer required for the couple's own reproductive project could be donated for research rather than just discarded. Different approaches are being developed to avoid immunological rejection of embryonic stem cells used for therapy. Derivation of embryonic stem cell lines by somatic cell nuclear transfer ('cloning') from the patients themselves might be one possible approach, but is unlikely to be used in routine clinical practice if more cost-effective methods are available.     

Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction

Therapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for "therapeutic cloning," though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or "fertilization failures" might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming.     

克隆技术引发的伦理之争

自从克隆羊多莉诞生以来,有关克隆人的伦理学争论就一直喋喋不休。世界上的各种政治组织和各国政府都明确反对生殖性克隆,而科学家们则对克隆技术的不完善心存疑虑。为了克服克隆过程中的伦理学障碍和技术缺陷,科学家们在核移植技术的基础上,又发展了异种核移植技术、诱导多能干细胞技术等。诱导的多能干细胞可以分化成各种组织,甚至能发育成个体,这些方法使克隆技术不再破坏胚胎,避免了伦理学纠纷。尽管科学技术在进步,但是人们对克隆人仍有很多不解和困惑。从自主、不伤害、行善和公正等四大生命伦理学原则着手,在技术层面上提出了尽管克隆人不会搞乱人际关系,不会减少人类基因多样性,也不会克隆出类似希特勒的战争狂人,但是,人类的生殖性克隆却剥夺了克隆人的自主性,对克隆人的生理和心理都有所伤害,违反了公正和行善的原则。因此,是否可以克隆人在伦理上仍然是需要长期讨论的问题。     

Going to the roots of the stem cell controversy

The purpose of this paper is to describe the scientific background to the current ethical and legislative debates about the generation and use of human stem cells, and to give an overview of the ethical issues underlying these debates.
The ethical issues discussed are 1) stem cells and the status of the embryo, 2) women as the sources of ova for stem cell production, 3) the use of ova from other species, 4) slippery slopes towards reproductive cloning, 5) the public presentation of stem cell research and 6) the evaluation of scientific uncertainty and its implications for public policy.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.     

The non-human primate oocyte and embryo as a model for women, or is it vice versa?

The role of the non-human primate (NHP) oocyte and embryo in translational research is considered here including both in vitro activities directly involving oocytes or embryos as well as animal studies that impact reproductive function. Reasons to consider NHPs as animal research models along with their limitations are summarized. A case is made that in limited instances, such as in the development and application of the assisted reproductive technologies or in the study of embryonic stem cells, the human oocyte and embryo have acted as models for the monkey. The development of strategies for the preservation of fertility is used as an example of ongoing research in the non-human primate that cannot be conducted in women for ethical reasons. In animal studies, monitoring reproductive potential, responses to embryonic stem cell transplantation, along with translational research in the field of contraceptive development for women are considered as subjects that benefit from the availability of a NHP model.     

A novel two-step procedure to expand cardiac Sca-1+ cells clonally

Resident cardiac stem cells (CSCs) are characterized by their capacity to self-renew in culture, and are multipotent for forming normal cell types in hearts. CSCs were originally isolated directly from enzymatically digested hearts using stem cell markers. However, long exposure to enzymatic digestion can affect the integrity of stem cell markers on the cell surface, and also compromise stem cell function. Alternatively resident CSCs can migrate from tissue explant and form cardiospheres in culture. However, fibroblast contamination can easily occur during CSC culture. To avoid these problems, we developed a two-step procedure by growing the cells before selecting the Sca-1+ cells and culturing in cardiac fibroblast conditioned medium, they avoid fibroblast overgrowth.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

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.     

Parthenotes as a source of embryonic stem cells

Abstract.  The derivation and study of human embryonic stem cell lines, despite their potential therapeutic usefulness, raise considerable ethical, religious, legal and political concerns because it inevitably leads to the destruction of viable embryos. In an attempt to bridge the division between ethical questions and potential scientific and medical benefits, considerable efforts have been devoted to the search for alternative sources of pluripotent cell lines. In this review we discuss the use of artificial parthenogenesis as a way to create entities, called parthenotes, that may represent an alternative ethical source for pluripotent cell lines. We describe the biological differences between parthenotes and embryos, in order to provide a rationale for the discussion on whether their use can be acceptable as a source of stem cells. We present data derived from animal models on the extent parthenogenetic stem cells are similar to biparental cell lines and discuss these aspects in the context of their extension to the human species. Finally, we present experiments recently carried out in our laboratory that allowed us to generate human parthenotes through artificial activation of human oocytes and to use them as a source for the derivation of parthenogenetic pluripotent cell lines.     

Stem cells and cell therapy. Contribution to the ethical debate

Divergent and sometimes conflicting positions with respect to human stem cells and cell therapy do not merely reflect disagreement among scientists and conflicts of interest. They attest the ethical tension resulting from recent progress in understanding the earliest stages of development of the human being that can be observed in vitro. Can the extremely potent notion of the human person starting with conception apply to the very first stage of artificial in vitro fertilisation and disregard the fact that to be a real substitute for natural conception, implantation in the uterus that enables the oocyte to nest and a new human being to develop must also be included? Several arguments are presented that plead in favour of making a clear distinction between the status of in vitro cells obtained by artificial fertilisation and that of the embryo, which becomes a developing human being from the moment it implants in the endometrium of the uterus. This subject could have remained in the sphere of the individual conscience, but it has now become a theme for social debate! The revision of the French 1994 so-called Bioethics Laws, which was recently approved on first reading on 22 January 2002, authorises research on spare embryos from in vitro fertilisation under certain conditions. However, for the sole reason that there is a risk of opening the door wide to reproductive cloning, which is unanimously rejected and condemned, all research on stem cells deriving from the nuclear transfer of a somatic cell is prohibited, irrespective of the distinction between cloning for therapeutic purposes and reproductive cloning. It is undeniable that if the efficacy of somatic stem cells could be demonstrated, they would offer a far more preferable solution, for several reasons, than those involving stem cells obtained from spare embryos from IVF or nuclear transfer. Nevertheless, how will a comparison of the two methods be possible if one of them is prohibited a priori? At present, many fear that French researchers will be prevented from doing essential research that, even if it has far to go, is indispensable if we wish to attempt to control the failures of natural procreation and open the way towards the new regenerative medicine that so many look forward to.     

Bioethical aspects of regenerative and reproductive medicine

The birth announced in 1997 of Dolly, the lamb cloned from the somatic mammary cells of an adult ewe, and the discovery of human embryonic stem cells in 1998 have been the most exciting developments in the biological sciences in the past decade. Reproductive somatic cell nuclear transfer (SCNT) in additional species has been inefficient in that relatively few births, harmful side effects and high fetal and neonatal death rates have resulted from many attempts. Ongoing debates about the ethics of reproductive SCNT have revealed that some researchers regard human reproductive SCNT as morally unacceptable in all circumstances, others see merit in reproductive SCNT in certain circumstances and others await more information before making judgment about the ethical status of the procedure. Regenerative medicine and emerging biotechnologies started to revolutionize the practice of medicine. Advances in stem cell biology, including embryonic and postnatal somatic stem cells, have made the prospect of tissue regeneration a potential reality. Mammal cloning experiments have provided new impetus to the prospect of regenerative medicine through stem cell research. The procedure of SCNT could be used to create the raw material to replace defective or senescent tissue as a natural extension of the biology of stem cells. Researchers working in reproductive medicine should consider the potential hope given to many patients against the requisite and ethically contentious creation of human blastocysts for therapeutic intent.     

Restoring stemness

This essay is focused on a specific line of research toward regenerative therapies that is based on the use of embryonic stem cells but tries to avoid cloning techniques that are the heart of current ethical debates.     

Recent progress and problems in animal cloning

It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.     

Neurotrophin signaling in oocyte survival and developmental competence: a paradigm for cellular toti-potency

While not fulfilling the criterion of a "stem cell" in being capable of self-renewal, mature and fertilized oocytes are the original "toti-potent" cells, whose capacity for expansion and differentiation can only be approximated by stem cells of embryonic or adult origin in vitro. As such, the mechanisms by which oocytes acquire and manifest competence to support embryo development is of fundamental interest to efforts to control and re-specify somatic cell fate and toti-potency. This is underscored by the unparalleled capacity of oocyte cytoplasm to successfully re-specify the genetic program of animal development following cell nuclear replacement (i.e., cloning). Thus, the knowledge gained by understanding the acquisition of oocyte developmental competence could ultimately facilitate the creation of adult stem cells in vitro from terminally differentiated cells, ex ovo. In this paper, we review the concept of oocyte developmental competence, and focus on our own research and that of others implicating a role for neurotrophins in this process, and that of oocyte cell survival. Lastly we propose a role for neurotrophin signalling in embryo stem cell survival.     

Mouse cloning with nucleus donor cells of different age and type

We have tested different cell types as sources for nucleus donors to determine differences in cloning efficiency. When donor nuclei were isolated from cumulus cells and injected into recipient oocytes from adult hybrid mice (B6D2F1 and B6C3F1), the success rate of cloning was 1.5-1.9%. When cumulus cell donor nuclei were isolated from adult inbred mice (C57BL/6, C3H/He, DBA/2, 129/SvJ, and 129/SvEvTac), reconstructed oocytes did not develop to full term or resulted in a very low success rate (0-0.3%) with the exception of 129 strains which yielded 0.7-1.4% live young. When fetal (13.5-15.5 dpc), ovarian, and testicular cells were used as nucleus donors, 2.2 and 1.0% of reconstructed oocytes developed into live offspring, respectively. When various types of adult somatic cells (fibroblasts, thymocytes, spleen cells, and macrophages) were used, oocytes receiving thymocyte nuclei never developed beyond implantation, whereas those receiving the nuclei of other cell types did. These results indicate that adult somatic cells are not necessarily inferior to younger cells (fetal and ES cells) in the context of mouse cloning. Although fetal cells are believed to have less genetic damage than adult somatic cells, the success rate of cloning using any cell types were very low. This may largely be due to technical problems and/or problems of genomic reprogramming by oocytes rather than the accumulation of mutational damage in adult somatic cells.