A transgene-free human peri-gastrulation embryo model with trilaminar embryonic disc-, amnion- and yolk sac-like structures

Introduction: Despite its importance in human development, gastrulation remains difficult to study, due to both technical and ethical challenges. Stem cell-based embryo models, including those recapitulating different aspects of pre- and peri-gastrulation human development, are emerging as promising experimental tools to study human embryogenesis. However, it remains unclear whether existing three-dimensional human embryo models are capable of modeling the development of the trilaminar embryonic disc structure, a hallmark of human gastrulation. In this study, we report a transgene-free human embryo model derived solely from primed human pluripotent stem cells (hPSCs) that recapitulates different aspects of peri-gastrulation human development, including formation of trilaminar embryonic layers situated between dorsal amnion and ventral secondary yolk sac. This embryo model is thus termed the peri-gastrulation trilaminar embryonic disc (PTED) embryoid. Cell lineage identities in PTED embryoids are validated based on both immunostaining for lineage markers and benchmarking against in vivo human and monkey gastrula at the transcriptomic level. Primary hematopoiesis and blood cell generation in PTED embryoids are examined. Our data generated through lineage tracing studies further support that in PTED embryoids, embryonic and extraembryonic mesoderm cells, as well as embryonic and extraembryonic endoderm cells, share common progenitors that emerge during peri-gastrulation development. Together, the PTED embryoid provides a promising and ethically less challenging experimental tool to study self-organizing properties of peri-gastrulation human development, shedding light on complex cellular events involved in early human embryogenesis.

Materials and

Methods: hPSCs are first seeded onto circular Geltrex adhesive islands with a diameter of 800 μm printed onto glass coverslips using microcontact printing. Such geometric boundary confinement provides an effective morphogenetic field to promote cellular organization during embryoid development. Under a cell seeding density of 5,000 cells mm-2, about 1,800 hPSCs become attached to each adhesive island. Cells are cultured in mTeSR1 medium for the first two days before other soluble factors are supplemented into mTeSR1 for the following two days to initiate differentiation of hESCs. Notably, soluble factor stimulations drive hESC colonies to transform from a two-dimensional (2D) monolayer structure to a three-dimensional (3D) multi-layered structure. Over a period of about 8 days, the 3D hPSC colonies develop into the PTED, with clearly delineated trilaminar embryonic disc-, amnion- and yolk sac-like structures.

Results, Conclusions, and Discussions: In this study, we report a new human embryoid system derived solely from transgene-free, primed hPSCs that recapitulates different aspects of the peri-gastrulation human embryonic development, including formations of trilaminar embryonic layers flanked by dorsal amnion and ventral secondary yolk sac. Development of PTED embryoids does not involve the formation of some canonical cellular structures in natural primate development, including the primitive streak, rendering them an ethically less challenging model for studying different cellular events involved in early human embryogenesis. Cell lineage identities in PTED embryoids are validated based on both immunostaining for lineage markers and benchmarking against in vivo human and monkey gastrula at the transcriptomic level. Given that in PTED embryoids a yolk sac-like structure emerges, containing both extraembryonic endoderm and mesoderm cells, we conducted lineage tracing studies to examine the origins of these yolk sac-related cell lineages. Our data support the differentiation of extraembryonic endodermal and mesodermal cells from gastrulating epiblast cells, suggesting alternative lineage development pathways for these two extraembryonic cell types. Notably, blood cell generation and primary hematopoiesis, including the transition from hematopoietic endothelial progenitors to blood progenitors, are observed during PTED development. Detailed characterization of blood cell maturation, particularly erythroblast and megakaryoblast differentiation, is also performed in this work. Together, the PTED embryoid provides a promising tool for studying self-organizing properties and lineage development of peri-gastrulation human development, shedding light on complex cellular events involved in early human embryogenesis.

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