Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (mTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage mT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Key roles of CCCTC-binding factor in cancer evolution and development

The processes of cancer and embryonic development have a partially overlapping effect. Several transcription factor families, which are highly conserved in the evolutionary history of biology, play a key role in the development of cancer and are often responsible for the pivotal developmental processes such as cell survival, expansion, senescence, and differentiation. As an evolutionary conserved and ubiquitously expression protein, CCCTC-binding factor (CTCF) has diverse regulatory functions, including gene regulation, imprinting, insulation, X chromosome inactivation, and the establishment of three-dimensional (3D) chromatin structure during human embryogenesis. In various cancers, CTCF is considered as a tumor suppressor gene and plays homeostatic roles in maintaining genome function and integrity. However, the mechanisms of CTCF in tumor development have not been fully elucidated. Here, this review will focus on the key roles of CTCF in cancer evolution and development (Cancer Evo-Dev) and embryogenesis.

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

ABSTRACT 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 TGFβ 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 TGFβ 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.

Reconstructing aspects of human embryogenesis with pluripotent stem cells

Understanding human development is of fundamental biological and clinical importance. Despite its significance, mechanisms behind human embryogenesis remain largely unknown. Here, we attempt to model human early embryo development with expanded pluripotent stem cells (EPSCs) in 3-dimensions. We define a protocol that allows us to generate self-organizing cystic structures from human EPSCs that display some hallmarks of human early embryogenesis. These structures mimic polarization and cavitation characteristic of pre-implantation development leading to blastocyst morphology formation and the transition to post-implantation-like organization upon extended culture. Single-cell RNA sequencing of these structures reveals subsets of cells bearing some resemblance to epiblast, hypoblast and trophectoderm lineages. Nevertheless, significant divergences from natural blastocysts persist in some key markers, and signalling pathways point towards ways in which morphology and transcriptional-level cell identities may diverge in stem cell models of the embryo. Thus, this stem cell platform provides insights into the design of stem cell models of embryogenesis.

Generation of human blastocyst-like structures from pluripotent stem cells

Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

Reprogramming of DNA methylation is linked to successful human preimplantation development

Human preimplantation development is characterized by low developmental rates that are poorly understood. Early mammalian embryogenesis is characterized by a major phase of epigenetic reprogramming, which involves global DNA methylation changes and activity of TET enzymes; the importance of DNA methylation reprogramming for successful human preimplantation development has not been investigated. Here, we analyzed early human embryos for dynamic changes in 5-methylcytosine and its oxidized derivatives generated by TET enzymes. We observed that 5-methylcytosine and 5-hydroxymethylcytosine show similar, albeit less pronounced, asymmetry between the parental pronuclei of human zygotes relative to mouse zygotes. Notably, we detected low levels of 5-formylcytosine and 5-carboxylcytosine, with no apparent difference in maternal or paternal pronuclei of human zygotes. Analysis of later human preimplantation stages revealed a mosaic pattern of DNA 5C modifications similar to those of the mouse and other mammals. Strikingly, using noninvasive time-lapse imaging and well-defined cell cycle parameters, we analyzed normally and abnormally developing human four-cell embryos for global reprogramming of DNA methylation and detected lower 5-methylcytosine and 5-hydroxymethylcytosine levels in normal embryos compared to abnormal embryos. In conclusion, our results suggest that DNA methylation reprogramming is conserved in humans, with human-specific dynamics and extent. Furthermore, abnormalities in the four-cell-specific DNA methylome in early human embryogenesis are associated with abnormal development, highlighting an essential role of epigenetic reprogramming for successful human embryogenesis. Further research should identify the underlying genomic regions and cause of abnormal DNA methylation reprogramming in early human embryos.

Unraveling the Spatiotemporal Human Pluripotency in Embryonic Development

There have been significant advances in understanding human embryogenesis using human pluripotent stem cells (hPSCs) in conventional monolayer and 3D self-organized cultures. Thus, in vitro models have contributed to elucidate the molecular mechanisms for specification and differentiation during development. However, the molecular and functional spectrum of human pluripotency (i.e., intermediate states, pluripotency subtypes and regionalization) is still not fully understood. This review describes the mechanisms that establish and maintain pluripotency in human embryos and their differences with mouse embryos. Further, it describes a new pluripotent state representing a transition between naïve and primed pluripotency. This review also presents the data that divide pluripotency into substates expressing epiblast regionalization and amnion specification as well as primordial germ cells in primates. Finally, this work analyzes the amnion’s relevance as an “signaling center” for regionalization before the onset of gastrulation.

Generation of human blastocyst-like structures from pluripotent stem cells

Human blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast, and primitive endoderm, all of which are essential for early development and organ formation1,2. However, due to ethical concerns and restricted access to human blastocysts, we lack a comprehensive understanding of early human embryogenesis. To bridge this knowledge gap, we need a reliable model system that recapitulates early stages of human embryogenesis. Here we report a ∼three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids also exhibited the developmental potential to undergo post-implantation morphogenesis in vitro to form structures with a cellular composition and transcriptome signature similar to human embryos that had been cultured in vitro for 8 or 10 days. In conclusion, human EPS-blastoids provide a new experimental platform for studying early developmental stages of the human embryo.HighlightsA method for generating human blastoids from EPS cells.Human blastoids resemble blastocysts in terms of morphology and cell lineage composition.Single-cell transcriptomic analyses reveal EPI, PE, and TE cell lineages in human blastoids.Human blastoids mimic in vitro the morphogenetic events of pre- and early post-implantation stages.