Establishment of a novel non-integrated human pluripotent stem cell-based gastruloid model

Abstract

Embryo loss and pregnancy disorders are prevalent worldwide, with both conditions critically associated with dysfunctioning gastrulation processes. Gastrulation and post-gastrulation organogenesis are crucial stages of embryonic development that establish the blueprint for body part formation. These processes involve the sequential generation of three germ layer cells and primordial germ cells, as well as the assembly of the precursor tissues for body parts. However, due to ethical limitations associated with studying human embryogenesis, a more detailed understanding of gastrulation and post-gastrulation organogenesis remains elusive. To ensure that the knowledge obtained from gastruloids is biologically meaningful and clinically relevant, it is critical to create high-fidelity human embryo models that closely mimic embryogenesis in vivo. Here, we developed a two-stage derivation gastruloids in vitro based on human pluripotent stem cells. Morphological tracking mimicks the developmental processes of models from Carnegie Stage 4 (CS4) to early CS7. Our gastruloids exhibit key structures characteristic of human embryos, including amniotic cavity, embryonic disc, primitive streak, primary yolk sac, secondary yolk sac, and blood islets. Comparison of our cell lineage development maps showed that gastruloids closely resembled human natural CS7 gastrula. Our gastruloids exhibited transcriptional characteristics that mimicked the molecular pathways observed in natural embryos development. Importantly, we found that in our model, extraembryonic mesoderm originates from the yolk sac and primordial germ cells originate from the posterior epiblast of the embryonic disc. Moreover, we found that thalidomide affects the differentiation of three germ layer cells, resulting in the arrest of human gastruloid development. In conclusion, by establishing a human gastruloid, we were able to gain valuable insights into the mechanisms responsible for human gastrulation and shed light on the causes of early embryo loss and pregnancy disorders.

A comprehensive review: synergizing stem cell and embryonic development knowledge in mouse and human integrated stem cell-based embryo models

“…The key commonality across all six studies is the induced formation of the hypoblast layer, facilitating the development of a bi-laminar disc structure. The models are known as heX embryoid, human extra-embryoid (hEE), peri-gastruloid, human gastruloid, E-assembloid and bilaminoid models ( Ai et al, 2023 ; Hislop et al, 2023 ; Liu et al, 2023 ; Okubo et al, 2023 ; Pedroza et al, 2023 ; Yuan et al, 2023 ) ( Figure 4 ). In heX embryoids ( Hislop et al, 2023 ), hypoblast development relied on the continuous induction of GATA6 transcription in primed hES cells.…”

“…By day 11, the peri-gastruloids had developed bilaminar discs, amniotic and yolk sac cavities, initiated gastrulation, formed PGCs, and even exhibited features of early organogenesis ( Liu et al, 2023 ). In the fifth paper, primed hES cells were used to form post-implantation-like aggregates known as human gastruloids ( Yuan et al, 2023 ). Upon exposure to culture media supporting nEnd formation (first stage medium), hES cells underwent differentiation into hypoblast-like cells, which subsequently organized into a hypoblast-like cell layer and formed a primary yolk sac.…”

“…Subsequent exposure to BMP4 and bFGF in the second stage medium facilitated the formation of the amnion. Remarkably, by day 7, the human gastruloids exhibited anterior-posterior polarity, accompanied by the emergence of AVE-like cells, mesoderm, and PGCs ( Yuan et al, 2023 ). Finally, embryo-like assembloids (E-assembloids) ( Ai et al, 2023 ) were derived from naïve hES cells and partially differentiated hES cells, termed Signaling Nest Cells (SNLs), which transiently emerged during trophoblast induction and secrete WNT and BMP ligands.…”

Programming human cell fate: overcoming challenges and unlocking potential through technological breakthroughs

“…Complex organoid systems also rely on the co-culture of a target cell type with one or more auxiliary cell types to promote target cell fate acquisition by mechanical constraints, cell-cell signalling and growth factor secretion, which combine to create a supportive cell niche. This is exemplified by recent advances in stem cell-based models of human embryos, which precisely co-culture several defined cell types to generate higher-order complexity in vitro ( Weatherbee et al, 2023 ; Ai et al, 2023 ; Yuan et al, 2023 preprint ; Hislop et al, 2023 preprint ; Pedroza et al, 2023 ; Oldak et al, 2023 ). Microfluidic approaches can also precisely control spatiotemporal signalling cues.…”

Deciphering the Code of Pattern Formation: Integrating In Silico and Wet Lab Approaches in Synthetic Biology

“…These intricate models traverse key phases, starting with pre-gastrulation events where extra-embryonic compartments play a pivotal role as signaling hubs, guiding the precise specification of cell fate. They seamlessly progress through the establishment of polarity, the initiation of gastrulation, and ultimately reach stages reminiscent of the early phases of organogenesis [40][41][42][43][44][45][46] (depicted in Table 1).…”