Current status of in vitro differentiation of stem cells into gametes

From an evolutionary point of view, the gametes are the cells in the body that matter the most as they are the ones who transmit their genes to the next generation ensuring continuation of the species. Being able to generate mature oocytes in vitro is of great interest. Oocytes are the key to totipotency and are able to reprogram somatic cells in approximately one day. In addition, in contrast to a clump of pluripotent stem cells, once the developmental program has started, fertilized oocytes develop into a clump of cells with positional information and the possibility to differentiate into both the embryonic and the extraembryonic lineages that form a complete developing and viable organism. How to instruct pluripotent stem cells to become oocytes in vitro is still unclear and even though the first steps to obtain mouse oocytes have recently been successfully demonstrated, inducing meiosis progression and folliculogenesis in vitro are still far from being understood and have not yet been accomplished. In humans, the specific molecular niche that leads to correct oogenesis is less understood. Here, we discuss the current status of in vitro differentiation of human pluripotent stem cells into gametes, in particular to oocytes

Genetic Basis of Dilated Cardiomyopathy in Dogs and Its Potential as a Bidirectional Model

Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment

BEAUVERICIN DISTURBS NUCLEAR AND CYTOPLASMIC MATURATION OF PREBUPERTAL SHEEP OOCYTES

Beauvericin (BEA) is a mycotoxin produced by several Fusarium species and a common contaminant of food and feed. Due to its ionophoric properties it can disturb mitochondrial function, and enhance reactive oxygen species (ROS) production and membrane lipid peroxidation [1]. It has been reported that BEA may affect the quality of oocytes by reducing granulosa cell function in pigs [1]. Here we determined the effect of BEA on meiotic competence and cytoplasmic maturation of oocytes from pre-pubertal sheep. Cumulusoocyte complexes (COCs) recovered at local slaughterhouses from the ovaries of sheep younger than 6 months were underwent in vitro maturation (IVM) [2]. COCs were exposed to BEA concentrations selected on the basis of previous studies (0.5, 1, 3, 5 μM) [3]. COCs cultured in IVM medium with 0.02% DMSO (vehicle) were used as controls. After IVM, cumulus cells were removed and oocytes stained with MitoTracker Orange CMTM Ros, 2′,7′- dichlorodihydrofluorescein diacetate and Hoechst 33258 and fixed in 2% paraformaldehyde in PBS. Oocytes at the metaphase II stage were analyzed by confocal laser scanning microscopy for their cytoplasmic maturation expressed as mitochondria (mt) distribution pattern [2]. Data were analysed by Chi-square test and differences were considered to be significant when P<0.05. A total of 464 oocytes were analyzed in four replicates. BEA, at 5 μM, was found to reduce the maturation rate (45/94, 47.9% vs 59/93, 63.4%, for exposed and controls, respectively; P<0.05) whereas it was not effective at the lower tested concentrations (58/94, 61.7%; 45/93, 48.4%; 47/90, 52.2% for 0.5, 1 and 3 μM, respectively; P>0.05). In addition, BEA at 5 μM affected the bioenergetic status of oocytes, as it increased the rate of oocytes showing abnormal mt pattern (5/45, 11% vs 0/59, 0%, for exposed and controls, respectively; P<0.05) and reduced the rate of oocytes with healthy perinuclear/pericortical mt pattern (20/45, 44% vs 39/59, 66%, for exposed and controls, respectively; P<0.05). No effects were noticed on mt pattern at lower tested concentrations (37/58, 64%; 25/45, 56%; 29/47, 62%, for 0.5, 1 and 3 μM respectively; P>0.05). These data indicate that BEA, in the exposure conditions used in the present study, hinder nuclear and cytoplasmic maturation in prepubertal sheep oocytes

Modelling human embryogenesis : embryo-like structures spark ethical and policy debate

BACKGROUND: Studying the human peri-implantation period remains hindered by the limited accessibility of the in vivo environment and scarcity of research material. As such, continuing efforts have been directed towards developing embryo-like structures (ELS) from pluripotent stem cells (PSCs) that recapitulate aspects of embryogenesis in vitro. While the creation of such models offers immense potential for studying fundamental processes in both pre- and early post-implantation development, it also proves ethically contentious due to wide-ranging views on the moral and legal reverence due to human embryos. Lack of clarity on how to qualify and regulate research with ELS thus presents a challenge in that it may either limit this new field of research without valid grounds or allow it to develop without policies that reflect justified ethical concerns. OBJECTIVE AND RATIONALE: The aim of this article is to provide a comprehensive overview of the existing scientific approaches to generate ELS from mouse and human PSCs, as well as discuss future strategies towards innovation in the context of human development. Concurrently, we aim to set the agenda for the ethical and policy issues surrounding research on human ELS. SEARCH METHODS: The PubMed database was used to search peer-reviewed articles and reviews using the following terms: 'stem cells', 'pluripotency', 'implantation', 'preimplantation', 'post-implantation', 'blastocyst, 'embryoid bodies', 'synthetic embryos', 'embryo models', 'self-assembly', 'human embryo-like structures', 'artificial embryos' in combination with other keywords related to the subject area. The PubMed and Web of Science databases were also used to systematically search publications on the ethics of ELS and human embryo research by using the aforementioned keywords in combination with 'ethics', 'law', 'regulation' and equivalent terms. All relevant publications until December 2019 were critically evaluated and discussed. OUTCOMES: In vitro systems provide a promising way forward for uncovering early human development. Current platforms utilize PSCs in both two- and three-dimensional settings to mimic various early developmental stages, including epiblast, trophoblast and amniotic cavity formation, in addition to axis development and gastrulation. Nevertheless, much hinges on the term 'embryo-like'. Extension of traditional embryo frameworks to research with ELS reveals that (i) current embryo definitions require reconsideration, (ii) cellular convertibility challenges the attribution of moral standing on the basis of 'active potentiality' and (iii) meaningful application of embryo protective directives will require rethinking of the 14-day culture limit and moral weight attributed to (non-)viability. Many conceptual and normative (dis)similarities between ELS and embryos thus remain to be thoroughly elucidated. WIDER IMPLICATIONS: Modelling embryogenesis holds vast potential for both human developmental biology and understanding various etiologies associated with infertility. To date, ELS have been shown to recapitulate several aspects of peri-implantation development, but critically, cannot develop into a fetus. Yet, concurrent to scientific innovation, considering the extent to which the use of ELS may raise moral concerns typical of human embryo research remains paramount. This will be crucial for harnessing the potential of ELS as a valuable research tool, whilst remaining within a robust moral and legal framework of professionally acceptable practices