High resolution dynamic ultrasound atlas of embryonic and fetal development of the common marmoset.

PURPOSE: The common marmoset (Callithrix jacchus) provides an ideal model to study early development of primates, and an in vivo platform to validate conclusions from in vitro studies of human embryos and embryo models. Currently, however, no established staging atlas of marmoset embryonic development exists. Using high-resolution, longitudinal ultrasound scans on live pregnant marmosets, we present the first dynamic in vivo imaging of entire primate gestation beginning with attachment until the last day before birth. METHODS: Our study unveils the first dynamic images of an in vivo attached mammalian embryo developing in utero, and the intricacies of the delayed development period unique to the common marmoset amongst primates, revealing a window for somatic interventions. RESULTS: Established obstetric and embryologic measurements for each scan were used comparatively with the standardized Carnegie staging of human development to highlight similarities and differences. Our study also allows for tracking the development of major organs. We focus on the ontogeny of the primate heart and brain. Finally, input ultrasound images were used to train deep neural networks to accurately determine the gestational age. All our ultrasounds and staging data recording are posted online so that the atlas can be used as a community resource toward monitoring and managing marmoset breeding colonies. CONCLUSION: The temporal and spatial resolution of ultrasound achieved in this study demonstrates the promise of noninvasive imaging in the marmoset for the in vivo study of primate-specific aspects of embryonic and fetal development.

The emergence of human gastrulation upon in vitro attachment.

While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our understanding of this process. In vitro attachment of natural blastocysts shed light on aspects of the second week of human development in the absence of the morphological manifestation of gastrulation. Stem cell-derived blastocyst models, blastoids, provide the opportunity to reconstitute pre- to post-implantation development in vitro. Here we show that upon in vitro attachment, human blastoids self-organize a BRA+ population and undergo gastrulation. Single-cell RNA sequencing of these models replicates the transcriptomic signature of the human gastrula. Analysis of developmental timing reveals that in both blastoid models and natural human embryos, the onset of gastrulation as defined by molecular markers, can be traced to timescales equivalent to 12 days post fertilization. In all, natural human embryos and blastoid models self-organize primitive streak and mesoderm derivatives upon in vitro attachment.

Huntingtin CAG-expansion mutation results in a dominant negative effect.

Introduction: Huntington's disease (HD) remains an incurable and fatal neurodegenerative disease long after CAG-expansion mutation in the huntingtin gene (HTT) was identified as the cause. The underlying pathological mechanism, whether HTT loss of function or gain of toxicity results from mutation, remains a matter of debate. Methods: In this study, we genetically modulated wild-type or mutant HTT expression levels in isogenic human embryonic stem cells to systematically investigate their contribution to HD-specific phenotypes. Results: Using highly reproducible and quantifiable in vitro micropattern-based assays, we observed comparable phenotypes with HD mutation and HTT depletion. However, halving endogenous wild-type HTT levels did not strongly recapitulate the HD phenotypes, arguing against a classical loss of function mechanism. Remarkably, expression of CAG-expanded HTT in non-HD cells induced HD like phenotypes akin to HTT depletion. Discussion: By corollary, these results indicate a dominant negative effect of mutated HTT on its wild-type counterpart. Complementation with additional copies of wild-type HTT ameliorated the HD-associated phenotypes, strongly supporting a classical dominant negative mechanism. Understanding the molecular basis of this dominant negative effect will guide the development of efficient clinical strategies to counteract the deleterious impact of mutant HTT on the wild-type HTT function.

Deep-learning analysis of micropattern-based organoids enables high-throughput drug screening of Huntington's disease models.

Organoids are carrying the promise of modeling complex disease phenotypes and serving as a powerful basis for unbiased drug screens, potentially offering a more efficient drug-discovery route. However, unsolved technical bottlenecks of reproducibility and scalability have prevented the use of current organoids for high-throughput screening. Here, we present a method that overcomes these limitations by using deep-learning-driven analysis for phenotypic drug screens based on highly standardized micropattern-based neural organoids. This allows us to distinguish between disease and wild-type phenotypes in complex tissues with extremely high accuracy as well as quantify two predictors of drug success: efficacy and adverse effects. We applied our approach to Huntington's disease (HD) and discovered that bromodomain inhibitors revert complex phenotypes induced by the HD mutation. This work demonstrates the power of combining machine learning with phenotypic drug screening and its successful application to reveal a potentially new druggable target for HD.

Mechanisms underlying WNT-mediated priming of human embryonic stem cells.

Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context-dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs). Our previous work demonstrated that during early stages of hESC differentiation, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening but, instead, acts by inducing the expression of the SMAD2 co-factor EOMES. Expression of EOMES is sufficient to replace WNT upstream of activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

In vitro attachment and symmetry breaking of a human embryo model assembled from primed embryonic stem cells.

Our knowledge of the molecular mechanisms surrounding human embryo implantation and gastrulation is lacking, largely due to technical and ethical limitations of experimenting with human embryos. Alternatives to human embryos have been reported, in which 3D clusters of embryonic stem cells are differentiated in a stepwise manner to model aspects of human embryogenesis. Yet it remains challenging to model the events past attachment. We propose a strategy of modeling the post-attachment human embryo by assembling a pre-formed polarized epithelial epiblast and extraembryonic cells, allowing them to self-organize into a structure that mimics the dish-attached human embryo. The model attaches in vitro and, in the absence of exogenous morphogens, breaks anteroposterior symmetry, giving rise to early gastrulation cell types. Our assembloid approach enables in a modular way to upgrade or exchange extraembryonic tissues to access more advanced stages of post-attachment development while complying with ethical policies.

Role of YAP in early ectodermal specification and a Huntington's Disease model of human neurulation.

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP's potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington's Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP's transcriptional activity can partially rescue the HD phenotype.

Mechanical regulation of early vertebrate embryogenesis.

Embryonic cells grow in environments that provide a plethora of physical cues, including mechanical forces that shape the development of the entire embryo. Despite their prevalence, the role of these forces in embryonic development and their integration with chemical signals have been mostly neglected, and scrutiny in modern molecular embryology tilted, instead, towards the dissection of molecular pathways involved in cell fate determination and patterning. It is now possible to investigate how mechanical signals induce downstream genetic regulatory networks to regulate key developmental processes in the embryo. Here, we review the insights into mechanical control of early vertebrate development, including the role of forces in tissue patterning and embryonic axis formation. We also highlight recent in vitro approaches using individual embryonic stem cells and self-organizing multicellular models of human embryos, which have been instrumental in expanding our understanding of how mechanics tune cell fate and cellular rearrangements during human embryonic development.

Self-organization of human dorsal-ventral forebrain structures by light induced SHH.

Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body's axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on human embryonic stem cells (hESCs). We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers.

Huntingtin CAG expansion impairs germ layer patterning in synthetic human 2D gastruloids through polarity defects.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFß signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFß receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids.

The treasure inside human naive pluripotency, generation of trophectoderm and blastoids.

Recent advances in human naive pluripotent stem cell culture have demonstrated their ability to generate trophectoderm and descendant trophoblast cell types. Moreover, the same cells when cultured in three-dimensional configurations self-organize to generate blastocyst-like structures called blastoids. These discoveries represent a major step forward in modeling early human embryonic development.

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Human embryo research, stem cell-derived embryo models and in vitro gametogenesis: Considerations leading to the revised ISSCR guidelines.

The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.

ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

The ethics of human-embryoids model: a call for consistency.

In this article, we discuss the ethics of human embryoids, i.e., embryo-like structures made from pluripotent stem cells for modeling natural embryos. We argue that defining our social priorities is critical to design a consistent ethical guideline for research on those new entities. The absence of clear regulations on these emerging technologies stems from an unresolved debate surrounding natural human embryo research and one common opinion that one needs to solve the question of the moral status of the human embryo before regulating their surrogate. The recent NIH funding restrictions for research on human embryoids have made scientists even more unlikely to raise their voices. As a result, the scientific community has maintained a low profile while longing for a more favorable socio-political climate for their research. This article is a call for consistency among biomedical research on human materials, trying to position human embryoids within a spectrum of existing practice from stem cell research or IVF to research involving human subjects. We specifically note that the current practices in infertility clinics of freezing human embryos or disposing of them without any consideration for their potential benefits contradicts the assumption of special consideration for human material. Conversely, creating human embryoids for research purposes could ensure that no human material be used in vain, always serving humankind. We argue here that it is time to reconsider the full ban on embryo research (human embryos and embryoids) beyond the 14-day rule and that research on those entities should obey a sliding scale combining the completeness of the model (e.g., complete vs. partial) and the developmental stage: with more advanced completeness and developmental stage of the considered entity, being associated with more rigorous evaluation of societal benefits, statements of intention, and necessity of such research.

Depletion of aneuploid cells in human embryos and gastruloids.

Chromosomal instability leading to aneuploidy is pervasive in early human embryos1-3 and is considered as a major cause of infertility and pregnancy wastage4,5. Here we provide several lines of evidence that blastocysts containing aneuploid cells are worthy of in vitro fertilization transfer. First, we show clinically that aneuploid embryos can lead to healthy births, suggesting the presence of an in vivo mechanism to eliminate aneuploidy. Second, early development and cell specification modelled in micropatterned human 'gastruloids' grown in confined geometry show that aneuploid cells are depleted from embryonic germ layers, but not from extraembryonic tissue, by apoptosis in a bone morphogenetic protein 4 (BMP4)-dependent manner. Third, a small percentage of euploid cells rescues embryonic tissue in mosaic gastruloids when mixed with aneuploid cells. Finally, single-cell RNA-sequencing analysis of early human embryos revealed a decline of aneuploidy beginning on day 3. Our findings challenge two current dogmas: that a single trophectoderm biopsy at blastocyst stage to perform prenatal genetic testing can accurately determine the chromosomal make-up of a human embryo, and that aneuploid embryos should be withheld from embryo transfer in association with in vitro fertilization.

Synthetic by design: Exploiting tissue self-organization to explore early human embryology.

Recent advances in synthetic human embryology has provided a previously inexistent molecular portrait of human development. Models of synthetic human embryonic tissues capitalize on the self-organizing capabilities of human embryonic stem cells when they are cultured on biomimetic conditions that simulate in vivo human development. In this Review, we discuss these models and how they have shed light on the early stages of human development including amniotic sac development, gastrulation and neurulation. We discuss the mechanisms underlying the molecular logic of embryonic tissue self-organization that have been dissected using synthetic models of human embryology and explore future challenges in the field. Geared with technological advances in bioengineering, high resolution gene expression and imaging tools, these models are set to transform our understanding of the mechanistic basis of embryonic tissue self-organization during human development and how they may go awry in disease.