Autonomy in the Development of Stem Cell-Derived Embryoids: Sprouting Blastocyst-Like Cysts, and Ethical Implications.

The experimental production of complex structures resembling mammalian embryos (e.g., blastoids, gastruloids) from pluripotent stem cells in vitro has become a booming research field. Since some of these embryoid models appear to reach a degree of complexity that may come close to viability, a broad discussion has set in with the aim to arrive at a consensus on the ethical implications with regard to acceptability of the use of this technology with human cells. The present text focuses on aspects of the gain of organismic wholeness of such stem cell-derived constructs, and of autonomy of self-organization, raised by recent reports on blastocyst-like cysts spontaneously budding in mouse stem cell cultures, and by previous reports on likewise spontaneous formation of gastrulating embryonic disc-like structures in primate models. Mechanisms of pattern (axis) formation in early embryogenesis are discussed in the context of self-organization of stem cell clusters. It is concluded that ethical aspects of development of organismic wholeness in the formation of embryoids need to receive more attention in the present discussions about new legal regulations in this field.

Rise of the rabbit model for implantation research. A commemoration of the scientific work of Bent G. Boving (1920-2019).

Bent G. Boving, a remarkable pioneer of research on mechanistic aspects of embryo implantation and specifically of using the rabbit model for this purpose, passed away peacefully on 15 November 2019, not long before he would have been able to celebrate his 100th birthday on 23 February 2020. His work has been very stimulating for other researchers, in part possibly because it elicited controversial discussions. This article attempts to give an overview of his scientific contributions, which still harbor some treasures, including potentially stimulating ideas for future research on implantation.

Self-Organization of Stem Cell Colonies and of Early Mammalian Embryos: Recent Experiments Shed New Light on the Role of Autonomy vs. External Instructions in Basic Body Plan Development.

"Organoids", i.e., complex structures that can develop when pluripotent or multipotent stem cells are maintained in three-dimensional cultures, have become a new area of interest in stem cell research. Hopes have grown that when focussing experimentally on the mechanisms behind this type of in vitro morphogenesis, research aiming at tissue and organ replacements can be boosted. Processes leading to the formation of organoids in vitro are now often addressed as self-organization, a term referring to the formation of complex tissue architecture in groups of cells without depending on specific instruction provided by other cells or tissues. The present article focuses on recent reports using the term self-organization in the context of studies on embryogenesis, specifically addressing pattern formation processes in human blastocysts attaching in vitro, or in colonies of pluripotent stem cells ("gastruloids"). These morphogenetic processes are of particular interest because, during development in vivo, they lead to basic body plan formation and individuation. Since improved methodologies like those employed by the cited authors became available, early embryonic pattern formation/self-organization appears to evolve now as a research topic of its own. This review discusses concepts concerning the involved mechanisms, focussing on autonomy of basic body plan development vs. dependence on external signals, as possibly provided by implantation in the uterus, and it addresses biological differences between an early mammalian embryo, e.g., a morula, and a cluster of pluripotent stem cells. It is concluded that, apart from being of considerable biological interest, the described type of research needs to be contemplated carefully with regard to ethical implications when performed with human cells.

Stem cell terminology and 'synthetic' embryos: a new debate on totipotency, omnipotency, and pluripotency and how it relates to recent experimental data.

The rapid progress in the stem cell field, in particular in cell reprogramming, combined with certain recent observations from experimental embryology, has ignited a new discussion on stem cell terminology. The current use of terms describing stem cell potentiality is inconsistent and can be confusing, in particular the widely used term pluripotency and its distiction from totipotency. For cells possessing a complete differentiation potential (but lacking an autonomous embryo-structuring capacity) the term omnipotency (or, as recently proposed, plenipotency) has been coined. The present commentary takes up this discussion and confronts it with recent reports on 'engineering' viable fish embryos or gastrulating human germ disc models using 'pluripotent'/omnipotent cells, as well as on symmetry breaking in aggregates of mouse embryonic stem cells. It is concluded that we should start contemplating not only the terminology but also, even more urgently, the ethical implications of the perspective of constructing embryonic anlagen in humans.

Time to reconsider stem cell induction strategies.

Recent developments in stem cell research suggest that it may be time to reconsider the current focus of stem cell induction strategies. During the previous five years, approximately, the induction of pluripotency in somatic cells, i.e., the generation of so-called 'induced pluripotent stem cells' (iPSCs), has become the focus of ongoing research in many stem cell laboratories, because this technology promises to overcome limitations (both technical and ethical) seen in the production and use of embryonic stem cells (ESCs). A rapidly increasing number of publications suggest, however, that it is now possible to choose instead other, alternative ways of generating stem and progenitor cells bypassing pluripotency. These new strategies may offer important advantages with respect to ethics, as well as to safety considerations. The present communication discusses why these strategies may provide possibilities for an escape from the dilemma presented by pluripotent stem cells (self-organization potential, cloning by tetraploid complementation, patenting problems and tumor formation risk).

Epithelial-mesenchymal transition in rhesus monkey embryonic stem cell colonies: the role of culturing conditions.

Colonies of rhesus monkey embryonic stem cells (rhESC; cell line R366.4) have been described before to show a spatially ordered process of epithelial-mesenchymal transition in vitro. In the present investigations, we have studied variables of culturing conditions which influence the reproducibility of the formation of crater-like ingression centers in the colonies. Critical parameters are found to be age and density of mouse embryonic fibroblast (MEF) feeder cell layers, the mode of mitotic inactivation of the MEFs (mitomycin C, or irradiation), and the mode of rhESC isolation during subculturing (enzymatic/mechanical cell cluster isolation; type of enzyme). The described culturing system appears to offer a reproducible in vitro model potentially useful for studies on cellular processes involved in gastrulation in the primate.

Induced pluripotent stem cells: how to deal with the developmental potential.

Recent developments in research on embryonic stem cells and induced pluripotent stem cells suggest that potentiality of cells should be a new focus in stem cell research ethics and policy. Successful reconstitution of viable embryos from induced pluripotent stem cells using tetraploid complementation has been reported and indicates a way for direct cloning of individuals from these cells. This together with recent observations on gastrulation and pattern formation processes in cultures of embryonic stem cells has considerable ethical relevance after the advent and worldwide spread of induced pluripotent stem cell technology. Available knowledge of the molecular basis of mammalian embryology now makes it possible to envisage ways to deal technically with the ethical dilemma of stem cell potentiality.

Totipotency/pluripotency and patentability.

UNLABELLED: In their article entitled " COMMENTARY: Is totipotency of a human cell a sufficient reason to exclude its patentability under the European law" (Stem Cells 2007;25:3026-3028), K.T. Vrtovec and B. Vrtovec conclude that arguments based on differentiation potential should not be an obstacle to patenting human embryonic stem cells (and related cells referred to as totipotent or pluripotent). While concentrating on formal legal aspects, however, these authors fail to consider a major biological and ethical argument already found in the literature, namely that an obstacle to patenting is to be seen in the potential of cells (e.g., of embryonic stem cell lines), if this potential allows (re)constitution of an embryo when tetraploid complementation is performed.

Human embryonic stem cells: the real challenge for research as well as for bioethics is still ahead of us.

Research on human embryonic stem cells (ESCs) has aroused a lot of controversy for years. Stimulated by recent work on mammalian embryology and new developments in stem cell research, an International Symposium entitled 'Stem Cell Research: A Challenge for Embryology, Regenerative Medicine and Bioethics' was held in Bonn (Germany) in 2006, bringing together embryologists, stem cell researchers and ethicists interested in human ESC research and the ensuing ethical debate. Two contributions to this Symposium are being published in Cells Tissues Organs, and the present paper aims to provide an introduction to these as well as personal impressions of the author about the perspectives that surfaced at the meeting, confronting them with relevant reports about stem cell research published recently. This paper highlights discussions about the mechanisms of specification of the main body axes during development, the role of extrinsic or intrinsic signals, and about the remarkable potential of ESCs to develop a basic body plan (individuation capacity) resembling properties of early embryonic cells (as shown by the formation of embryoid bodies and entire embryos if tetraploid complementation is performed). Another topic is 'alternative sources for human ESCs' recently proposed by the US President's Council on Bioethics ('organismically dead embryos', biopsied blastomeres or 'biological artifacts', e.g. created by 'altered nuclear transfer' and reprogramming of somatic cells). The possibility to rescue such (epi)genetically handicapped cells shows that this is not a way leading out of the ethical cul-de-sac. Recent reports about reprogramming somatic cells (fibroblasts) to gain ES-like potential highlight again the importance of focusing on the developmental potentiality as the major challenge for ethical considerations. Such a change of focus may be the only way out of the ethical impasse.

Epithelial-mesenchymal transition in Rhesus monkey embryonic stem cell colonies: a model for processes involved in gastrulation?

A characteristic feature of embryonic stem (ES) cells is their ability to give rise to differentiated cell types that are derived from all three primary germ layers. In the embryo of higher vertebrates, formation of mesoderm and definitive endoderm (gastrulation) occurs at the primitive streak through a spatially highly ordered process of cell ingression, combined with epithelial-mesenchymal transition (EMT). With respect to ES cell differentiation in vitro, however, germ layer derivative formation has not been studied in much detail, and data on any degree of spatial order that may be attained here are lacking. In the investigations to be reviewed here, rhesus monkey ES cells (line R366.4) were grown on mouse embryonic fibroblast feeder layers for up to 10 days during which time they formed multilayered disc-like colonies with an upper epithelial and a lower mesenchymal cell layer. Processes of epithelialization as well as EMT were studied by transmission electron microscopy, immunohistochemistry combined with confocal laser scanning microscopy, and marker mRNA expression (in situ hybridization, RT-PCR). It was found that under the culture conditions used most of the ES cell colonies developed transitorily a central pit where the epithelial upper layer cells underwent an EMT-like process and appeared to ingress to form the lower, mesenchymal layer, accompanied by appropriate changes of morphology and molecular markers. Similarities and differences in comparison with gastrulation/primitive streak formation in vivo are briefly discussed, as are ethical implications with respect to human ES cells. It is concluded that this rhesus ES cell colony system may be an interesting in vitro model for studies on some basic processes involved in early embryogenesis such as EMT/gastrulation and may open new ways to study the regulation of these processes experimentally in vitro in nonhuman primates.

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation.

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells.

BACKGROUND: Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. METHODS: Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. RESULTS: Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. CONCLUSION: Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo.

Early human development: new data raise important embryological and ethical questions relevant for stem cell research.

Recent research has considerably changed our views about the developmental biology of early mammalian embryos compared with the ideas that were predominant throughout the previous 30 years or so. New data obtained recently suggest that the mammalian embryo uses traits of axes determination mechanisms that are not too different from the modes used by other vertebrates. In particular, it appears that asymmetry cues derived from the oocyte cytoplasm and modified/specified during sperm penetration appear to be crucial in normal embryogenesis, rather than the environmental influences exerted, e.g. at embryo implantation in the uterus. On the other hand, recent advances in research on the equivalents of a Spemann-Mangold organizer in species other than amphibia (including mammals) provide a background for new discussions of early embryonic patterning (axis formation) processes in the embryonic disc. In combination, these new views appear to be of considerable interest in the debate on the developmental properties and the ethical status of embryonic stem cells. The present review focuses specifically on the new aspects of axis determination and pattern formation processes in early mammalian embryos and relates this to questions about the developmental potential of embryonic stem cells (totipotency vs pluripotency/omnipotency), i.e. facts that appear to be worth considering in the recent debate about the ontological status of the early human embryo as well as of human embryonic stem cells.