2PN cell donation in Germany. Or: How the German Embryo Protection (Act) undermines itself

In contrast to embryo donation, the permissibility of 2PN cell donation is highly controversial in Germany. This article is based on there being a legal loophole with respect to 2PN cell donation, which results from an inconsistency within the Embryo Protection Act on the normative status of 2PN cells. Following that thesis, the article argues that, on the basis of the normative criterion totipotency (i.e. the capacity to develop into a born human being), 2PN cells should also be considered human embryos within the meaning of the Act and thereby be protected by that Act in the same way as embryos. However, the normative assumption that 2PN cells should already be endowed with human dignity and the right to life has absurd consequences. Moreover, the consistent continuation of the Embryo Protection Act, as well as of the underlying ethical position or argumentation (i.e. the potentiality argument), leads to the even more absurd consequence of having to place every human somatic cell under the protection of human dignity and the right to life. As totipotency or the developmental potential therefore cannot delimit entities considered worthy of protection (i.e. human embryos) from entities considered not worthy of protection (i.e. 2PN cells, gametes, hESC, hiPSC and human somatic cells), it is not a suitable normative criterion. As a paradigmatic case, 2PN cell donation demonstrates that by retaining this normative criterion the now obsolete German Embryo Protection (Act) ultimately undermines itself.     

DEEPER PROBLEMS FOR NOONAN'S PROBABILITY ARGUMENT AGAINST ABORTION: ON A CHARITABLE READING OF NOONAN'S CONCEPTION CRITERION OF HUMANITY

In ‘An Almost Absolute Value in History’ John T. Noonan criticizes several attempts to provide a criterion for when an entity deserves rights. These criteria, he argues are either arbitrary or lead to absurd consequence. Noonan proposes human conception as the criterion of rights, and justifies it by appeal to the sharp shift in probability, at conception, of becoming a being possessed of human reason. Conception, then, is when abortion becomes immoral. The article has an historical and a philosophical goal. The historical goal is to carefully present the probability argument in a charitable manner. The philosophical goal is to offer a unique criticism of Noonan's probability argument against abortion. I argue that, even on a very charitable reading of Noonan's argument for the conception criterion, this criterion is also susceptible to charges of arbitrariness and absurdity. Noonan's claim that probability shifts have anything to do with the moral rights of fetuses cannot be made coherent. I also show that there are problems with Noonan's assumptions about moral rights and the potential to become a being possessed of human reason.     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

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.     

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。     

Alternate nuclear transfer is no alternative for embryonic stem cell research

Recent developments allow for the creation of human stem cells without the creation of human embryos, a process called alternate nuclear transfer ('ANT'). Pursuing this method of stem cell research makes sense for pro-lifers if arguments for the sanctity of the human embryo do not apply to ANT. However, the technology that makes ANT possible undermines the erstwhile technical barrier between human embryos and somatic cell DNA. These advances bring home the force of hypothetical arguments about the potential of the DNA in somatic cells, showing that there is not a morally relevant difference between the potential of an embryo and the potential of the DNA in a somatic cell. Therefore, the supposed distinction between entities that are potential human life and entities that are human life does not give any support to arguments for the sanctity of the human embryo because those arguments extend value to too many entities.     

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.     

The pros and cons of human therapeutic cloning in the public debate

Few issues linked to genetic research have raised as much controversial debate as the use of somatic cell nuclear transfer technology to create embryos specifically for stem cell research. Whereas European countries unanimously agree that reproductive cloning should be prohibited there is no agreement to be found on whether or not research into therapeutic cloning should be permitted. Since the UK took the lead and voted in favour of regulations allowing therapeutic cloning the public debate has intensified on the Continent. This debate reflects the wide spectrum of diverse religious and secular moralities that are prevalent in modern multicultural European democratic societies. Arguments range from putting forward strictly utilitarian views that weight the moral issues involved against the potential benefits that embryonic stem cell research may harbour to considering the embryo as a human being, endowed with human dignity and human rights from the moment of its creation, concluding that its use for research is unethical and should be strictly prohibited. Given the current state of dissension among the various European states, it is difficult to predict whether 'non-harmonisation' will prevail or whether in the long run 'harmonisation' of legislation that will allow stem cell research will evolve in the EU.     

牛体细胞核移植技术研究进展

对近年来牛体细胞核移植技术研究的进展作一综述。其中包括:供体细胞种类、传代次数和所处细胞周期的选择;对供体细胞的特殊处理;卵母细胞的采集;传统去核方法的优化、去透明带核移植技术的建立与发展;胚胎重构、激活和体外培养条件的比较与改进等内容。     

Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs

Handmade cloning (HMC) is now an established procedure used in several species for somatic cell nuclear transfer, but only applied in two related laboratories for pigs. The aim of this review is to facilitate widespread application by summarizing the process of establishment and explaining the background of the incorporated special approaches. Optimized steps of traditional cloning in pigs (in vitro maturation, activation, embryo culture) were merged with those of the micromanipulation-free HMC that has been modified according to the specific needs of sensitive porcine oocytes (partial zona digestion before enucleation, two-step zona-free fusion with the somatic cell; initiation of activation with the second fusion). The zona-free approach required embryo culture to the blastocyst stage before surgical transfer of embryos to the uterine horns of recipient sows in the proper phase of an unstimulated cycle. Eventually a competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background. Further improvements and involvement of additional techniques to increase the overall efficiency and facilitate practical applications are expected in the foreseeable future.     

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]     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。     

Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells

Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.     

Philosophical arguments for and against human reproductive cloning

Can philosophers come up with persuasive reasons to allow or ban human reproductive cloning? Yes. Can philosophers agree, locally and temporarily, which practices related to cloning should be condoned and which should be rejected? Some of them can. Can philosophers reproduce universally convincing arguments for or against different kinds of human cloning? No. This paper analyses some of the main arguments presented by philosophers in the cloning debate, and some of the most important objections against them. The clashes between the schools of thought suggest that philosophers cannot be trusted to provide the public authorities, or the general public, a unified, universally applicable view of the morality of human reproductive cloning.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

Cytology and Histology in Somatic Embryogenesis of Indica Rice

The somatic embryogenesis was established from mature dehulled seeds. The histological research showed that embryogenic calli were initiated first from absorbed cells of scutellum of mature seed. And then the embryoids derived from the surface of embryogenic callus. Having been the same structure like a zygotic embryo of rice, the embryoids possessed the major parts of scutellum, coleoptile and coleorhiza. In an embryoid, several developmental stages of pro-embryoid, including single embryogenic cells, two, four and multiple cell stage pro-embryeids and some abnormal embryoids were observed. It could be concluded from this experiment that the embryoid from somatic cell culture in Indica rice possessed an original form of a plant in structure like a zygotic did and derived from a single cell.     

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.