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Human embryonic stem cells (hESCs) have the intrinsic capacity to self-organize and generate patterned tissues. In vitro models that coax hESCs to form embryonic-like structures by modulating physical environments and priming with chemical signals have become a powerful tool for dissecting the regulatory mechanisms underlying early human development. Here we present a 3D suspension culture system of hESCs that can generate post-implantation, pre-gastrulation embryonic-like tissues in an efficient and controllable manner. The efficiency of the development of asymmetric tissues, which mimic the post-implantation, pre-gastrulation amniotic sac, was about 50% in the 3D suspension culture. Quantitative imaging profiling and unsupervised trajectory analysis revealed that hESC aggregates first entered into a transitional stage expressing Brachyury (or T), before their development branched into different paths to develop into asymmetric embryonic-like tissues, amniotic-like tissues, and mesodermal-like tissues, respectively. Moreover, the branching developmental trajectory of embryonic-like structures was affected by the initial cell seeding density or cluster size of hESCs. A higher percentage of amniotic-like tissues was observed under a small initial cell seeding density of hESCs. Conversely, a large initial cell seeding density of hESCs promoted the development of mesodermal-like tissues. Intermediate cell seeding densities of hESCs in the 3D suspension culture promoted the development of asymmetric embryonic-like tissues. Our results suggest that hESCs have the intrinsic capability to sense the initial cell population size, which in turn regulates their differentiation and self-organization into different embryonic-like tissues. Our 3D suspension culture thus provides a promising experimental tool to study the interplay between tissue topology and self-organization and progressive embryonic development using in vitro hESC-based models., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Despite its importance, understanding the early phases of human development has been limited by availability of human samples. The recent emergence of stem-cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This Review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem-cell-derived embryo models for fundamental discovery and translation.

Early post-implantation human embryonic development has been challenging to study due to both technical limitations and ethical restrictions. Proper modeling of the process is important for infertility and toxicology research. Here we provide details of the design and implementation of a microfluidic device that can be used to model human embryo development. The microfluidic human embryo model is established from human pluripotent stem cells (hPSCs), and the resulting structures exhibit molecular and cellular features resembling the progressive development of the early post-implantation human embryo. The compartmentalized configuration of the microfluidic device allows the formation of spherical hPSC clusters in prescribed locations in the device, enabling the two opposite regions of each hPSC cluster to be exposed to two different exogenous chemical environments. Under such asymmetrical chemical conditions, several early post-implantation human embryo developmental landmarks, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and gastrulating cells (or mesendoderm cells), can be robustly recapitulated using the microfluidic device. The microfluidic human embryo model is compatible with high-throughput studies, live imaging, immunofluorescence staining, fluorescent in situ hybridization, and single-cell sequencing. This protocol takes ~5 d to complete, including microfluidic device fabrication (2 d), cell seeding (1 d), and progressive development of the microfluidic model until gastrulation-like events occur (1-2 d).

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of β-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca 2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca 2+ diffusion in hESC colonies. We quantify cell-cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca 2+ waves in hESC colonies induced by sonoporation of single cells.

Early human embryogenesis is a dynamic developmental process, involving continuous and concomitant changes in gene expression, structural reorganization, and cellular mechanics. However, the lack of investigation methods has limited the understanding of how cellular mechanical properties change during early human embryogenesis. In this study, ultrasound actuation of functionalized microbubbles targeted to integrin (acoustic tweezing cytometry, ATC) is employed for in situ measurement of cell stiffness during human embryonic stem cell (hESC) differentiation and morphogenesis. Cell stiffness, which is regulated by cytoskeleton structure, remains unchanged in undifferentiated hESCs, but significantly increases during neural differentiation. Further, using the recently established in vitro 3D embryogenesis models, ATC measurements reveal that cells continue to stiffen while maintaining pluripotency during epiblast cyst formation. In contrast, during amniotic cyst formation, cells first become stiffer during luminal cavity formation, but softens significantly when cells differentiate to form amniotic cysts. These results suggest that cell stiffness changes not only due to 3D spatial organization, but also with cell fate change. ATC therefore provides a versatile platform for in situ measurement of cellular mechanical property, and cell stiffness may be used as a mechanical biomarker for cell lineage diversification and cell fate specification during embryogenesis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.

Dissociation-induced apoptosis of human embryonic stem cells (hESCs) hampers their large-scale culture. Herein we leveraged the mechanosensitivity of hESCs and employed, to our knowledge, a novel technique, acoustic tweezing cytometry (ATC), for subcellular mechanical stimulation of disassociated single hESCs to improve their survival. By acoustically actuating integrin-bound microbubbles (MBs) to live cells, ATC increased the survival rate and cloning efficiency of hESCs by threefold. A positive correlation was observed between the increased hESC survival rate and total accumulative displacement of integrin-anchored MBs during ATC stimulation. ATC may serve as a promising biocompatible tool to improve hESC culture., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In vitro gametogenesis is the process of making germline cells from human pluripotent stem cells. The foundation of this model is the quality of the first progenitors called primordial germ cells (PGCs), which in vivo are specified during the peri-implantation window of human development. Here, we show that human PGC (hPGC) specification begins at day 12 post-fertilization. Using single-cell RNA sequencing of hPGC-like cells (hPGCLCs) differentiated from pluripotent stem cells, we discovered that hPGCLC specification involves resetting pluripotency toward a transitional state with shared characteristics between naive and primed pluripotency, followed by differentiation into lineage-primed TFAP2A+ progenitors. Applying the germline trajectory to TFAP2C mutants reveals that TFAP2C functions in the TFAP2A+ progenitors upstream of PRDM1 to regulate the expression of SOX17. This serves to protect hPGCLCs from crossing the Weismann's barrier to adopt somatic cell fates and, therefore, is an essential mechanism for successfully initiating in vitro gametogenesis.

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development. Human and murine embryonic development has disparities, highlighting the need for primate systems. Here, the authors construct a post-implantation transcriptional atlas from non-human primate embryos and show ISL1 controls a gene regulatory network in the amnion required for mesoderm formation. [ABSTRACT FROM AUTHOR]