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Published in final edited form as: Curr Opin Genet Dev. 2023 Dec 5;84:102134. doi: 10.1016/j.gde.2023.102134

Topical Section: Embryonic models (2023) for Current Opinion in Genetics & Development

Charlotte E Handford 1, Sergi Junyent 1, Victoria Jorgensen 1, Magdalena Zernicka-Goetz 1,2,#
PMCID: PMC11556421  NIHMSID: NIHMS1950979  PMID: 38052116

Abstract

Stem cell-based mammalian embryo models facilitate the discovery of developmental mechanisms because they are more amenable to genetic and epigenetic perturbations than natural embryos. Here, we highlight exciting recent advances that have yielded a plethora of models of embryonic development. Imperfections in these models highlight gaps in our current understanding and outline future research directions, ushering in an exciting new era for embryology.

Keywords: embryo, embryonic model, stem cells, mouse, human, totipotency, pre-implantation, peri-implantation, post-implantation

Introduction

In recent years, modeling embryo development with stem cells has opened up new avenues in developmental biology. By reconstructing embryo like structures using basic components – precise mixtures of embryo cell types, scaffolding with instructive signals and induction with both transgenes and morphogens – these models allow us to untangle the self-organizing principles that coordinate embryo morphogenesis. An understanding which would otherwise be limited by the technical or ethical challenges associated with prolonged culture of human embryos.

Modeling totipotency with human and mouse pluripotent stem cells

Totipotency is the capacity of a single cell to give rise to all embryonic and extraembryonic tissues of the conceptus. Both the zygote and the 2-cell stage mouse embryo contain totipotent cells. However, when totipotency becomes restricted in the human embryo remains unknown. The creation of a totipotent stem cell model would allow for exploration of the mechanisms controlling totipotency.

Pioneering work by the Pfaff group (Macfarlan et al., 2012) demonstrated a rare and transient 2-cell like-cell (2CLC) population within a culture of pluripotent mouse embryonic stem cells (ESCs). Recently, a surge of publications describe new methods for the robust establishment of totipotent cells in vitro (Figure 1): Totipotent blastomere-like cells (TBLCs), established by inhibition of the spliceosome in mESCs (Shen et al., 2021); Totipotent-like stem cells (TLSCs), generated by reprogramming mESCs through the inhibition of DOTL1, KDM5B and G9a (Yang et al., 2022); Totipotent potential stem cells (TPSCs), established from 2-cell stage embryos and extended pluripotent stem cells (EPSCs) using a defined chemical cocktail that includes DOTL1 inhibition (Xu et al., 2022), and chemically induced totipotent stem cells (ciTotSCs), that use alternative small molecule combinations to capture totipotency in vitro (Hu et al., 2022). TBLCs, TLSCs, TPSCs and ciTotSCs exhibit transcriptional and epigenetic profiles that mimic those of the totipotent cells in the embryo and to a certain extent contribute to both embryonic and extraembryonic lineages upon chimaera formation. However, ciTotSCs may reportedly come closest to replicating totipotency-like profiles, including a 2-cell embryo-like metabolic signature (Hu et al., 2022). Future work will help to resolve which of these promising chemical combinations best recapitulates the dynamic nature of embryonic totipotency.

Figure 1. Current models of in vitro totipotency in mouse and human cells.

Figure 1.

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Schematic representation of the strategies followed to capture in vitro totipotency in mouse (A) and human (B) cells. Gray boxes indicate totipotency hallmarks displayed by in vitro totipotent cells.

Meanwhile, three recent publications have detected totipotency in cultures of naïve human ESCs, that resemble the inner cell mass of the preimplantation blastocyst. The Esteban group (Mazid et al., 2022) developed an enhanced hESC naïve culture media (termed e4CL) that promotes the expression of an 8-cell embryo-like signature in hESCs. Human 8-cell like cells (8CLCs) are enriched using a TPRX1-GFP reporter, can produce trophoblast stem cells (TSCs), self-organize into embryo models at the blastocysts stage (so called blastoids), integrate extensively in interspecies chimaeras and form teratomas. Concurrently the Reik group (Taubenschmid-Stowers et al., 2022) used a set of 8-cell stage ZGA markers to also identify totipotent cells in naïve hESC cultures with matching transcriptional and methylation profiles to 8-cell human embryo blatomeres. These cells can be enriched in hESC cultures by treatment with spliceosome inhibitors or DUX4 overexpression (Yoshihara et al., 2022). However, future studies would benefit from further exploration of conditions to improve upon heterogeneities and stability of the totipotent identity in culture. Nevertheless, these reports demonstrate the capture of the human totipotent state in vitro and open the door for the study of the mechanisms leading to the establishment of this unique state in human cells.

None of the totipotent cells described to date recapitulate a key morphological feature of the early embryo: cell size. Future work will have to address how morphological transitions can be incorporated into the modeling of totipotency in vitro.

Modelling pre-implantation development in 3D

2D models are powerful tools for recapitulating certain aspects of development. Yet, they overlook the spatial organization and structure of the embryo and do not recapitulate all lineages, and therefore ignore key interplay between cell types (Figure 2). In recent years numerous 3D models have emerged that not only mimic the embryo in morphology, but also demonstrate correct localization of embryonic and extra-embryonic lineages (Figure 3).

Figure 2. Mouse and human embryo morphogenesis.

Figure 2.

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Schematic representation of the morphogenetic transformations of mouse (top) and human (bottom) embryos before or shortly after implantation. Key differences between the models include the morphology of the embryo at the onset of gastrulation: in the embryonic day (E)6.5, the mouse embryo is organized in an ‘egg cylinder’ morphology, while the E14 human embryo presents as a flat embryonic disk overlayed by the amniotic cavity and the amnion, and underlaid by the hypoblast and the yolk sac.

Figure 3. Three dimensional stem cell-based embryo models.

Figure 3.

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Schematic representation of the current three-dimensional stem cell-based models of mouse and human embryo development.

Work spearheaded by the Rivron group established pre-implantation mouse embryo models called blastoids by combining mouse ESCs and trophoblast stem cells (TSCs) in vitro to mimic E3.5 blastocysts. These structures mimic natural blastocysts in size and morphology and demonstrate initiation of the blastocyst lineages: epiblast, primitive endoderm, and trophoblast (Rivron et al., 2018). Nevertheless, efficiency to form blastoid structures was low, and formation of the primitive endoderm-like cells was inefficient. Subsequent blastoids have been developed to improve on this model by utilizing EPSCs. By aggregating EPSCs alone (Li et al., 2019) or EPSCs with TSCs (Sozen et al., 2019), two new blastoid models showed higher cavitation efficiency and higher instance of primitive endoderm formation. The addition of TSCs to EPSCs proved critical for trophectoderm formation EPSCs alone showed deficiencies in trophectoderm formation (Posfai et al., 2021). A transcriptional profile similar to that of natural embryos was captured in blastoids made from TBLCs, although formation of primitive endoderm formation was limited (Zhang et al., 2023). Boosting primitive endoderm formation from ESCs using a chemically inductive medium was shown to support developmental progression via reciprocal interactions between the epiblast and primitive endoderm in a partial blastoid model in the absence of TSCs (Vrij et al., 2022).

Although mouse blastoids should have the potential to be implanted within the uterus of a mouse and develop in vivo, several transfer experiments have been performed, but with limited results beyond decidualization likely in part due to the limited development of trophectoderm in blastoids (Seong et al., 2022). Thus, while blastoid models offer exciting avenues to explore pre-implantation development, none of the current models demonstrate the ability to fully transition to post-implantation morphology in vitro or in vivo, and blastoid implantation remains a future objective in the field.

Given technical and ethical limitations surrounding the culture of natural human embryos, stem cell-derived models for the human embryo would be a powerful tool to overcome these hurdles. In the last two years, a surge of human pre-implantation models (Fan et al., 2021; Liu et al., 2021; Sozen et al., 2021; Yu et al., 2021), demonstrated the ability to recapitulate human blastocyst morphology using a variety of protocols and starting cell populations. While each of these models offers powerful insights into our own development, scRNA-seq analysis reveals key transcriptional discrepancies between natural blastocysts and the models (Zhao et al., 2021). Moreover, the efficiency of blastoid formation remains low, hampering their potential for translational applications (e.g. high-throughput drug screening or implantation assays).

Amazing efforts in the field are leading to constant improvements to these models. An inhibition of the NODAL, ERK, and ROCK pathways was shown to improve transcriptional fidelity of blastoids and enhance proper lineage segregation (Yanagida et al., 2021). Furthermore, triple inhibition of the Hippo, TGF-β and ERK pathways was shown to lead to over 80% efficiency of human blastoid formation, with high commitment to each of the three lineages (~97%) (Kagawa et al., 2021). Using this model, blastoids can attach to and interact with endometrial tissues upon hormonal activation, an assay for studying mechanisms of implantation.

To improve these models, several key areas of study remain outstanding. While each of the models demonstrates some degree of early post-implantation remodeling, robust transition from pre- to post-implantation development is lacking; improvement here would be useful to understand the full scope of early human development, and to further study implantation mechanisms. When compared to the natural embryo, transcriptional fidelity of the in vitro models is imperfect. Side-by-side comparison of all models, in addition to natural blastocysts as well as embryo models of non-human primates (reviewed by Nakamura et al., 2021) would not only lead to the generation of enhanced models but would also further our understanding of self-organization principles and give insight into the limitations of in vitro culture. Further improvement in these aspects would allow for high-throughput applications and wide adoption of these models to study molecular dynamics of early development.

Peri-implantation models of mouse and human embryonic development

The Zernicka-Goetz group spearheaded development of mouse and human embryo models during the implantation phase to uncover mechanisms of the morphogenesis of the epiblast and how these processes link with the transition from naïve to primed pluripotency. Early attempts to study the morphogenetic transition from mouse blastocyst to egg cylinder were aided by the development of an in vitro culture system using collagen-coated polyacrylamide matrices (Morris et al., 2012). Next, by using a very small number of ESC cells (to recapitulate the small size of the embryonic ICM) and embedding them in the Matrigel they demonstrated how naive pluripotent cells became polarized, formed a 3D rosette-like structure and opened the lumen to form the pro-amniotic cavity in the mouse embryo (Bedzhov and Zernicka-Goetz, 2014). Subsequently they used a similar approach with human stem cells to uncover mechanisms of human embryo morphogenesis (Shahbazi et al, 2016). These peri-implantation mouse and human embryo models revealed that the exit from the naïve state of pluripotency is not essential for the polarization of cells but is essential for lumenogenesis to form the amniotic cavity (Shahbazi et al., 2016). This peri-implantation embryo model was subsequently used to model mosaic human embryos to understand when and how aneuploid cells become eliminated in the epiblast but not in the trophectoderm lineage (Singla et al., 2020).

Post-implantation models of mouse and human embryonic development

Building on approaches to study aspects of mouse post-implantation development with large aggregates of ESC aggregates (termed embryoid bodies (Doetschman et al., 1985; ten Berge et al., 2008)), so-called “gastruloids” have been shown to undergo axial elongation and patterning and activate some of the transcriptional machinery of gastrulation-stage natural embryos (Beccariet al., 2018; ten Berge et al., 2008; Tsakiridis et al., 2014; Turner et al., 2017; Van Den Brink et al., 2014). When embedded in Matrigel and exposed to a combination of a Wnt agonist and a Bmp antagonist, gastruloids develop into trunk-like structures comprised of a neural tube, somites and a gut-like population (van den Brink et al., 2020a; Veenvliet et al., 2020). Neurulation has also been achieved through the addition of extra-embryonic endoderm cells (XEN) to mESCs, in the absence of external signaling sources (Bérenger-Currias et al., 2022). In this approach, neuroepithelial tissue formed as a result of Wnt inhibition and basement membrane production by the XEN cell population. Furthermore, gastruloids exposed to a cocktail of three cardiogenic factors can form a cardiac domain and develop of a vascular-like network as well as a transcriptional signature reminiscent of that of the first and second heart fields (Rossi et al., 2021). Collectively, this work highlights the utility of these models in deciphering the molecular requirements for development of their respective tissues. Importantly, it also showcases the diversity of biological tissues that can be studied using reductionist systems (Figure 3).

Work pioneered by the Zernicka-Goetz group established so called “integrated” post-implantation mouse embryo models, meaning that they contain not only embryonic but also extra-embryonic tissues. The first integrated model established by this group combined ESCs with TSCs, resulting in “ET-embryos” that developed to recapitulate the architecture of the mouse embryo at the egg cylinder stage, forming the embryonic and extra-embryonic compartments, mesoderm and specifying a PGC population (Harrison et al., 2017). This approach was subsequently improved upon through a high-throughput cell aggregation protocol, leading to the generation of “EpiTS embryoids” that displayed temporally advanced hindbrain/midbrain cell populations, but lacked cells of the forebrain (Girgin et al., 2021). A signature indicative of further developmental progression was also captured in another model that employed the use of an ectopic morphogen signaling centre – an “organizer” – aggregated with ESCs (Xu et al., 2021). Structures arising from this protocol developed cardiac populations, a gut tube, a neural tube and a posterior midbrain region. Notably, though these models progress further in development than their predecessors they lack forebrain development and do not recapitulate the morphogenetic transformations characteristic of the post-implantation embryo. To this end, the Zernicka-Goetz group added XEN cells, representing the third constituent tissue of the embryo at this stage – the extra-embryonic endoderm, to ESCs and TSCs and used the culture media they developed for the post-implantation embryos (Bedzhov and Zernicka-Goetz, 2014). Combined, these three cell types self-organized into structures termed “ETX-embryos” that besides capturing the egg cylinder architecture that undergoes anterior-posterior patterning, also contained a layer of visceral endoderm-like cells (Sozen et al., 2018). The ETX-embryo formed an important signaling center, the anterior visceral endoderm population (AVE) and was able to initiate symmetry breaking leading to gastrulation movements and PGC formation. An exploration of the principles of self-organization in the ETX system identified the driving factors to be differential cadherin expression between the three different stem cell types and cortical stiffness (Bao et al., 2022). The quality of this system was further improved upon through the substitution of XEN cells with ESCs transiently expressing the transcription factor Gata4, a proven driver of primitive endoderm formation (Fujikura et al., 2002; Schröter et al., 2015). This yielded a cell population bearing greater similarity to the primitive endoderm of the natural embryo (Amadei et al., 2021). Importantly, this cell combination resulted in self-organizing ETiX-embryos capable of specifying an anterior-posterior axis, as well as further development to stages of early organogenesis (E8.5) (Amadei et al., 2022). The replacement of XEN cells with ESCs transiently expressing the transcription factor Gata6 has been shown to also yield ETX-embryos although their capacity to develop beyond E7.5 to E8.5 remains undetermined (Dupont et al., 2023). The E8.5 stage of development was also reached by structures derived solely from ESCs, through the additional replacement of TSCs by ESCs transiently expressing the transcription factor Cdx2, critical for development of trophectoderm (Lau et al., 2022; Tarazi et al., 2022). Although these stem cell-derived models develop embryonic-like tissues, the development of the extra-embryonic placental tissues is compromised, possible due to the absence of the maternal environment. Altogether, these studies bring to light the incredible capacity of stem cell-based embryo models to undergo morphogenesis and progress through development ex utero setting the stage for future studies to shine light on the elusive post-implantation period in development in the mouse and other species including human.

Human peri- and post-implantation development differs from that of the mouse in embryonic architecture, as it forms a flat bilaminar disk-like structure rather than an egg cylinder structure. Aspects of human post-implantation development have been modeled in 3D through an extension of the mouse gastruloid protocol resulting in elongated structures made from hESCs (Moris et al., 2020). In a model termed “Post-implantation amniotic sac embryoid” hPSC on Geltrex embedded in ECM form 3D asymmetric epithelial cysts that closely resemble the amniotic ectoderm (Shao et al., 2017). Equivalent approaches have been employed to study early neural tube development either using forced aggregation followed by suspension or shaking culture (Libby et al., 2021;Olmsted & Paluh, 2021). In a model of the epiblast-yolk sac crosstalk hPSCs were shown to differentiate towards a yolk-sac like cells (YSLCs) following exposure to media stimulating the WNT, ACTIVIN-A, and JAK/STAT signaling (Mackinlay et al., 2021). These YSLCs share functional attributes with the AVE, as they were capable of influencing hESC fate in 3D, inducing the expression of markers of pluripotency and anterior ectoderm while repressing markers of mesoderm and endoderm. Meanwhile, in an embryonic 3D model of hESCs seeded on polymeric hydrogels supplemented with Matrigel the resulting spherical epithelial structures broke symmetry following the uniform addition of Bmp4 (Simunovic et al., 2019). Building on this, hESCs pre-treated with Bmp4 to express extraembryonic markers combined with the hESC-derived epithelial spheroids and the resulting structures were capable of symmetry breaking in the absence of exogenous factors (Simunovic et al., 2022).

A recent transgene directed approach generated human embryo-like models from hESCs representing the peri-implantation stage of development combined with cells induced with GATA6-SOX17 to drive a hypoblast-like fate, and GATA3-AP2γ for a trophoblast-like fate (Weatherbee et al., 2023). Interestingly, a comparison of this model with an equivalent model lacking the induction of SOX17 revealed an inhibitory role for this transcription factor in the specification of the anterior hypoblast identity, demonstrating the utility of these embryo models in disentangling the functional contributions of driver transcription factors and exploring the mechanisms governing cell identity transitions in human post-implantation development.

In addition, this human embryo model explored the mechanisms behind amnion and PGC specification and their dependence on the BMP signalling. It also showed that not only the epiblast and amnion are derived from hESC in the model but also extraembryonic mesenchyme. Development of a bilaminar disk structure, reminiscent of the natural embryo was achieved in “iDiscoid” structures formed from wildtype hiPSCs combined with cells with inducible GATA6 (Hislop et al., 2023). In this model morphogenesis of the bilaminar disk was accompanied by, among other features, the formation of an amniotic cavity in the hypoblast-like layer and the specification of anterior hypoblast-like cells. iDiscoids lack representation of the trophoblast lineages but remain a valuable model for the study of the epiblast-hypoblast crosstalk at these stages of development.

Transgene-independent systems employed slightly different approaches. A two-step induction protocol starting from hPSCs and involving the addition of BMP4 from days 4–7 was reported to give rise to “human gastruloids” forming an embryonic disc with an amniotic cavity and yolk sac (Yuan et al., 2023). “E-assembloids” were produced by aggregation of naïve hESCs with cells pre-exposed to Bmp4 (Ai et al., 2023). These were allowed to develop under conditions of signal modulation engineered by timed pulses of a WNT agonist and BMP antagonist over the first 4 days which limited excess differentiation towards extraembryonic mesenchyme. The resulting structures capture the bilaminar disk structure, with a yolk-sac like population surrounded by extraembryonic mesenchyme-like cells the origin of which is suggested to be the epiblast, although further work will be required to confirm this. Alternatively, starting with a cell population in an intermediate pluripotency state (“formative-to-primed”) allowed for spontaneous multi-lineage differentiation in the “human extra-embryoid” model (Pedroza et al., 2023). This model consists of epiblast- and hypoblast-like cells that emerge following aggregation of hPSCs alone in a mixture of defined and conditioned media. Human extra-embryoids transiently maintain an AVE-like cell population in the hypoblast that antagonizes BMP signalling transiently counteracting the emergence of the amnion-like population and leading to a lack of early-amnion cell types. However, the lack of hTSC coupled with the relatively high ratio of hypoblast-like cells in the system may account for discrepancies in signalling dynamics and relative proportion of cell populations present. Nevertheless, the human extra-embryoids offer a system to explore alternative patterning strategies and dissect developmental dynamics. Alternatively, a stem cell embryo model was generated from naïve hPSCs cultured in a cocktail of basal media supplemented with GELTREX and small molecules (Oldak et al., 2023). In this study separate cultures of naïve cells were exposed to media promoting differentiation towards hypoblast, extraembryonic mesoderm and trophoblast fates after which they were aggregated with naïve PSC. The resulting structures appear to capture the bilaminar disk structure, a yolk sac-like cavity, and are surrounded by syncytiotrophoblast-like cells although cytotrophoblast cells are missing in this model. Finally, an EPSC-derived model of human post-implantation comprised of epiblast-like and hypoblast-like cells but lacking trophectoderm recapitulated hallmarks of development including gastrulation-like and early neurulation-like transcriptomic signatures (Liu et al., 2023).

Further work will be necessary to determine the extent to which the signaling dynamics and kinetics of developmental trajectories present in the embryo are captured in the various emergent models allowing the interplay between gene expression and morphogenesis during this critical time period to be elucidated. At the same time, it is imperative that as new human models are developed their use is subject to regulation by scientific, legal and ethical committees. All stem cell-based embryo models are still imperfect in their modeling power, at best recapitulating short moments of the developmental trajectory of an embryo. The efficiencies at which these models can be formed, as well as the heterogeneity between structures and the ability of “embryoids” to model some but not all aspects of embryonic development are current limitations shared amongst all the models. Far from being seen as limitations, these imperfections highlight gaps in our current understanding, and outline future research directions for the field. For instance, further advances in capturing totipotency in vitro will likely enable the creation of embryonic models that better recapitulate development. Combinations of stem-cell based models and engineered biomaterials may help uncover the precise mechanisms of implantation. Advances in future post-implantation human models may offer sources of cell progenitors for regenerative medicine. These possibilities along with the myriad scientific questions that remain unexplored preface an exciting time in the study of embryology.

Acknowledgements

We acknowledge receipt of the following grants to M.Z.-G.: NIH Pioneer Award (DP1 HD104575-01), European Research Council (669198), Wellcome Trust (207415/Z/17/Z), Open Philanthropy/Silicon Valley Community Foundation, Weston Havens Foundation, NOMIS award.

Footnotes

Declaration of Competing Interest

Authors are inventors on the following patents: 1. Patent Applicant: Caltech. Inventors: Magdalena Zernicka-Goetz, Berna Sozen, Victoria Jorgensen. Application Number: 17/692,790. Specific aspect of the manuscript covered in patent application: Reconstructing Human Early Embryogenesis In Vitro With Pluripotent Stem Cells. 2. Patent Applicant: Caltech and Cambridge Enterprise Limited. Inventors: Magdalena Zernicka-Goetz, Gianluca Amadei, Charlotte Handford. Application Number: 63/397,630 Specific aspect of the manuscript covered in patent application: Synthetic Embryos. 3. Patent Applicant: Caltech and Cambridge Enterprise Limited; Inventors: Magdalena Zernicka-Goetz, Bailey Weatherbee, Carlos Gantner. Application Number: 63/403,684 Specific aspect of the manuscript covered in patent application: Stem Cell Derived Model Of The Human Embryo

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