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.     

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.     

Creating Donors: The 2005 Swiss Law on Donation of ‘Spare’ Embryos to hESC Research

In November 2004, the Swiss population voted to accept a law on research using human embryonic stem cells. In this paper, we use Switzerland as a case study of the shaping of the ostensibly ethical debate on the use of embryos in embryonic stem cell research by legal, political and social constraints. We describe how the national and international context affected the content and wording of the law. We discuss the consequences of the revised law's separation of stem cell research from other forms of embryo research, its definitions of embryo and of spare embryos, and the introduction of donorship into the Swiss ethical debate on IVF. We focus on the exclusion of the potential embryo donors' voices and perspectives from the debate, and consider the effects of this exclusion on ethical discourse and the political process.     

Production of F0 mice from embryonic stem cells injected eight-cell stage embryos which stored at refrigeration temperature

At refrigeration temperature, mouse embryos can retain their developmental ability for a couple of days. Previous research reports have focused on the effect of cool temperature on the development of 2-cell stage embryos, morulae or blastocysts and determined that the embryo still has the ability to produce offspring after about 48 h storage at refrigeration temperature. Here we examined whether refrigeration temperature affects the development of the eight-cell stage and if the stored eight-cell stage embryo can still be used as a host embryo for ES cell injection. Our results show that eight-cell stage embryos can develop into blastocysts and yield pups after cold storage for 24 and 48 h. After ES cell injection, stored eight-cell stage embryos can support ES cells developing to F0 pups. In summary, cool storage can preserve the developmental ability of eight-cell stage embryos for at least 48 h, allowing transportation of the embryos at refrigeration temperature between different labs and their subsequent use as host embryos for ES cell injection.     

NO ETHICAL BYPASS OF MORAL STATUS IN STEM CELL RESEARCH

Recent advances in reprogramming technology do not bypass the ethical challenge of embryo sacrifice. Induced pluripotent stem cell (iPS) research has been and almost certainly will continue to be conducted within the context of embryo sacrifice. If human embryos have moral status as human beings, then participation in iPS research renders one morally complicit in their destruction; if human embryos have moral status as mere precursors of human beings, then advocacy of iPS research policy that is inhibited by embryo sacrifice concerns renders one morally complicit in avoidable harms to persons. Steps may be taken to address these complicity concerns, but in the final analysis there is no alternative to achieving clarity with respect to the moral status of the human embryo.     

Discovering pluripotency: 30 years of mouse embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells isolated from an early embryo and grown as a cell line in tissue culture. Their discovery came from the conjunction of studies in human pathology, mouse genetics, early mouse embryo development, cell surface immunology and tissue culture. ES cells provided a crucial tool for manipulating mouse embryos to study mouse genetics, development and physiology. They have not only revolutionized experimental mammalian genetics but, with the advent of equivalent human ES cells, have now opened new vistas for regenerative medicine.     

Embryos,The Principle of Proportionality,and the Shaky Ground of Moral Respect

The debate concerning the moral permissibility of using human embryos in human embryonic stem cell (hESC) research has long centred on the question of the embryo's supposed right to life. However, in focussing only on this question, many opponents to hESC research have escaped rigorous scrutiny by making vague and unfounded appeals to the concept of moral respect in order to justify their opposition to certain hESC practices. In this paper, I offer a critical analysis of the concept of moral respect, and its use to support the intuitively appealing principle of proportionality in hESC research. I argue that if proponents of this principle are to justify its adoption by appealing to the concept of moral respect, they must explain two things concerning the nature of the moral respect owed to embryos. First, they must explain which particular aspect of the embryo is morally relevant, and why. Second, they must explain why some uses of embryos in research fail to acknowledge what is morally relevant about the embryo, and thereby involve a violation of the moral respect that they are due. I shall show that providing such explanations may be more difficult than it first appears.     

Moral qualms, future persons, and embryo research

Many people have moral qualms about embryo research, feeling that embryos must deserve some kind of protection, if not so much as is afforded to persons. This paper will show that these qualms serve to camouflage motives that are really prudential, at the cost of also obscuring the real ethical issues at play in the debate concerning embryo research and therapeutic cloning. This in turn leads to fallacious use of the Actions/Omissions Distinction and ultimately neglects the duties that we have towards future persons.     

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.     

转录因子Oct4、Sox2和Nanog在早期胚胎发育过程中的表达调控

胚胎发育的分子机理是现今胚胎工程及发育生物学研究迫切需要了解的。由于胚胎发育的早期阶段就是胚胎干细胞阶段,因此两者在研究上有很好的重合性。通过对调节胚胎干细胞基因转录的三个具有代表性的转录因子Oct4、Sox2和Nanog的综述,展现出胚胎发育分子机理研究概况。     

Determinants of pluripotency: From avian,rodents, to primates

Since mouse embryonic stem (ES) cells was first derived in 1981, the ability of this unprecedented cell type to self‐renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore, they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest, due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However, this is no longer a barrier, with the recent discovery of methods that can convert differentiated somatic cells into ES‐like cells or induced pluripotent stem (iPS) cells, by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo‐derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells, as well as the different determinants identified thus far to sustain their pluripotency. J. Cell. Biochem. 109: 16–25, 2010. © 2009 Wiley‐Liss, Inc.     

BROADENING THE SCOPE OF DEBATES AROUND STEM CELL RESEARCH

Over the last decade, stem cell research has generated an enormous amount of public, political and bioethical debate. These debates have overwhelmingly tended to focus on two moral issues: the moral status of human embryos and the duty to care for the sick and vulnerable. This preoccupation, especially on the question of moral status, has not only dichotomized the debate around two fundamentally incommensurable positions, it has come at the cost of other important issues largely being ignored. In highlighting some of the bioethical and regulatory deficiencies of this fixation, we draw on recent developments in the experimental use of autologous adult stem cells to argue for a more inclusive approach to the ethical issues surrounding stem cell research.     

The origin and identity of embryonic stem cells

Embryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.     

From Ethical Exceptionalism to Ethical Exceptions: The Rule and exception Model and the Changing Meaning of Ethics In German Bioregulation

Germany is an interesting case with respect to the governance of reprogenetics. It has a strong profile in the technosciences and high aims regarding the global bioeconomy, yet her regulation of human genetics, reproductive medicine and embryo research has for a long time been rather restrictive. German biopolitical exceptionalism has often been explained by reference to Catholicism and the legacy of the Nazi past. The Germans, so goes the common story, have learnt the lessons of history and translated them into unconditional respect for human dignity, which in turn translates into unconditional protection of human life, including the human embryo, and the firm repudiation of any eugenic distinction between ‘life worth to live’ and ‘life not worth to live’. This, however, is not the whole story. Alongside deontological strictness we find another strand of governing body politics and reprogenetics in Germany, the rule‐and‐exception model, running from the mid‐1970s abortion law via the 2002 Stem Cell Act to the 2011 regulation of pre‐implantation genetic diagnosis. In contrast to the former, that strongly draws on Kant and his concept of human dignity, the latter bears resemblances to Carl Schmitt's concept of state of exception. The article will show that the rule‐and‐exception model builds the exception into the rule and transforms the meaning and mandate of ethics, namely from safeguarding ethical standards to deciding about the exception. Given that the exception has now tended to become the rule, the question is whether the lessons of history will govern German reprogenetics for much longer.     

Mouse and human embryonic stem cell models of hematopoiesis: past, present, and future

Human embryonic stem (ES) cells provide a unique model and an important resource to analyze early hematopoietic development. Other systems to study mammalian hematopoiesis include mouse ES cells, dissection of timed mouse embryos, or use of human postnatal hematopoietic tissue typically isolated from bone marrow or umbilical cord blood. All these models have particular strengths and weaknesses. The extensive studies on murine hematopoiesis provide a basis for work on the human developmental system. Since there are likely some important species differences, use of human ES cells now provides an optimal means to evaluate basic cellular and molecular mechanisms that regulate the beginning stages of human blood development, prior to derivation of hematopoietic stem cells (HSCs). Eventually, research on human ES cells may provide an alternative source of HSCs and other blood products for hematopoietic cell transplantation or other cellular therapies.     

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.     

The somatic integration definition of the beginning of life

The somatic integration definition of life is familiar from the debate on the determination of death, with some bioethicists arguing that it supports brain death while others argue that some brain‐dead bodies exhibit sufficient somatic integration for biological life. I argue that on either interpretation, the somatic integration definition of life implies that neither the preimplantation embryo nor the postimplantation embryo meet the somatic integration threshold condition for organismal human life. The earliest point at which a somatic integration determination of life could be made would be the beginning of the fetal stage, 9 weeks postfertilization. Bioethical implications are considered, specifically with respect to the moral status of the postimplantation embryo in embryo research and abortion.     

Evaluation of developmental competence of vitrified-warmed early cleavage stage embryos and their application for chimeric mouse production.

The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.     

Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo

So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.