Human embryogenesis, the process of human development from fertilization to the formation of a complex organism, remains largely mysterious. The study of human embryos is ethically and legally restricted, and the use of surplus embryos donated for research is limited. While in vitro culture methods have advanced, studying the critical developmental steps leading to the blastocyst stage and beyond is still challenging. This research explores the use of human pluripotent stem cells (hPSCs) as a model to study early human development. Specifically, the study investigates whether hPSCs cultured under extended pluripotency (EP) conditions (hEPSCs) can self-organize into 3D structures that mimic aspects of early embryogenesis. The researchers hypothesized that by manipulating the culture conditions with appropriate growth factors and inhibitors, hEPSCs could recapitulate some features of early lineage development, allowing for insights into the regulatory processes involved without the ethical limitations of working with natural human embryos. Understanding the self-organization and differentiation of these cells in 3D culture could offer a valuable model for studying the complex processes of human development and potentially discovering new avenues for regenerative medicine and disease modeling.

Previous studies have demonstrated the ability of PSCs to be reprogrammed to an expanded pluripotency state, exhibiting developmental potency for both embryonic and extraembryonic lineages. Several stem cell-derived models have been developed to recapitulate stages of mouse and human embryo development in vitro. These models have provided significant insights but often lack the complexity of natural development. This study builds upon these existing models by focusing on the use of hEPSCs to generate 3D structures that mimic the key events of human pre-implantation and peri-implantation development. Specific studies on mouse models and the use of hPSCs in 3D culture have paved the way for this research, highlighting the potential of stem cell-based models to understand and dissect intricate developmental processes.

The researchers first converted hPSCs to hEPSCs and then used a multi-inverted-pyramidal microwell-based 3D culture system to allow for cell aggregation and self-organization. They screened various growth factors, cytokines, and small molecules to optimize the culture conditions and promote the formation of cavitated cystic structures. The optimized conditions included BMP4, CHIR99021, FGF2, ROCK inhibitor Y-27632, and A83-01 (pulsed). The structures were cultured under low oxygen tension (5% O2). Immunofluorescence analysis with lineage markers (SOX2, GATA3, SOX17, KRT18, OCT4, FOXA2) was performed to assess lineage specification. Analysis of cell polarity markers (E-CADHERIN, F-ACTIN, PARD6) and investigation of PLC-PKC pathways were conducted to understand the early events of cell polarization and lineage segregation. Quantitative RT-PCR (qRT-PCR) was used to examine the expression levels of genes involved in establishing blastocyst lineage identity. To mimic post-implantation development, the structures were cultured in a human embryo in vitro culture (IVC) platform. Single-cell RNA sequencing (scRNA-seq) was performed on hEPSCs in 2D, hEP-structures at day 5 and day 6, and natural human blastocysts at day 5/6. The scRNA-seq data was analyzed to determine lineage composition and identify transcriptional programs. Co-culture experiments were conducted combining hEPSCs and human trophoblast stem cells (hTSCs) to investigate whether the addition of hTSCs would improve the generation of TE-like cells. Statistical analysis included two-sided Student's t-tests, Kruskal-Wallis tests, Mann-Whitney tests, and ANOVA.

The researchers successfully generated self-organizing cystic structures from hEPSCs in 3D culture. These structures displayed some hallmarks of human early embryogenesis, including cavity formation, and showed similarities to natural blastocysts in morphology and cell number. Immunofluorescence analysis revealed the expression of markers characteristic of the three blastocyst lineages (EPI, HYPO, TE), though not always in the expected spatial arrangement. Analysis of cell polarity indicated a role for the PLC-PKC pathway in promoting cell polarization and GATA3 expression. qRT-PCR confirmed the upregulation of key lineage markers in the structures, although the level of GATA3 expression was lower than other TE markers. When transferred to IVC media, the structures underwent a post-implantation-like morphological reorganization, with evidence of radial organization of the inner compartment. scRNA-seq analysis showed some similarities to natural blastocysts in lineage specification, but also revealed a significant proportion of undefined cells and an overrepresentation of hypoblast-like cells compared to the natural blastocyst. A comparison of key lineage marker genes between the hEP-structures and natural blastocysts revealed some discrepancies in gene expression. Co-culture of hEPSCs with hTSCs did not improve the formation of TE-like cells in the structures.

The study demonstrates that hEPSCs can self-organize into structures resembling human blastocysts, offering a valuable in vitro model for studying early human development. However, the findings also highlight the limitations of this model, particularly the incomplete and imperfect lineage specification. The discrepancies observed in the expression of key lineage markers compared to natural blastocysts suggest that hEPSCs may adopt an intermediate transcriptional state, not fully recapitulating the totipotency of early embryonic cells. The results are consistent with recent reports suggesting limitations in the molecular and epigenetic plasticity of hEPSCs. The inability of human TSCs to enhance TE differentiation in these structures, unlike the successful co-culture in mouse models, emphasizes species-specific differences and potential limitations of the current hTSC line used. The differences observed between the hEPSC-derived structures and natural blastocysts highlight the complexity of modeling human development in vitro and the need for further refinement of the experimental conditions and cell lines used. Despite these limitations, the generated structures demonstrate some key morphological and patterning features of natural embryos, providing a valuable platform for future studies.

This study successfully developed a 3D stem cell-based model of early human embryogenesis, albeit with limitations in complete lineage specification. The hEPSC-derived structures exhibit key morphological features and patterning similar to natural early human embryos. While this model doesn't perfectly recapitulate all aspects of natural development, it provides a promising platform for further investigation into human developmental regulation and offers a valuable tool for future studies of human embryogenesis. Future research could focus on optimizing culture conditions, employing different hPSC lines, exploring co-culture strategies with enhanced hTSCs, and incorporating more comprehensive analyses to improve the accuracy and functionality of this model.

The study acknowledges limitations in the complete and accurate specification of all three lineages in the hEPSC-derived structures. The scRNA-seq analysis revealed a significant proportion of undefined cells, indicating incomplete lineage specification and the adoption of an intermediate transcriptional state. The differences in gene expression profiles between the model and natural blastocysts highlight the limitations in fully recapitulating natural developmental processes. The co-culture experiments with hTSCs did not yield the expected improvement in TE differentiation, which suggests limitations in the current hTSC line used. Further refinement of the culture conditions, cell lines, and analysis methods is needed to enhance the accuracy and biological relevance of this model.