3D biomimetic niche modulates embryo development in vitro

Domain: chemistry
Published: 2026-01-02
Editorial outcome: major_revision

Abstract
Embryo development undergoes critical morphological transformations post-implantation, largely driven by the complex and dynamic microenvironment of the uterus. Despite advances, current 3D culture models inadequately recapitulate the uterine environment necessary for studying embryo-uterus interactions. In this work, we engineer a hydrogel inspired by the properties of the decidua, incorporating Matrigel to support blastocyst implantation and embryo development in vitro. Our findings reveal that embryos cultured within this hydrogel system successfully progress to an early organogenesis-like stage, including the development of first and second heart fields, mimicking natural embryogenesis. Moreover, we identify that the mechanical properties, particularly stress relaxation, play a crucial role in facilitating focal adhesion (FA) formation between the trophoblast and the hydrogel. Additionally, the degradation of the hydrogel by embryo-secreted metalloproteinases (MMP2 and MMP9) creates a favorable environment for continued embryonic growth and development. These insights contribute to a deeper understanding of how the external environment regulates embryo development and offer an enhanced approach for in vitro embryo culture.

Introduction
Embryonic development is a complex and highly regulated process1–4. In vivo, embryonic development is closely orchestrated by the dynamic physical conditions within the uterus, coupled with chemical signals. Embryo implantation triggers rapid and extensive changes in the endometrium and the ECM6, while the trophoblast cells interact closely with the uterine ECM7. Throughout development, the environment surrounding the embryo transitions from a 2D to a 3D state, and from an ECM-free to an ECM-encapsulated state. Trophoblastic cells engage in highly integrated physicochemical interactions with the uterine ECM and endometrial cells7. This indicates significant correlations between the developmental stage of the blastocyst and the remodeling of its physical microenvironment, as well as interactions between blastocysts and the uterus that involve factors such as viscoelasticity (e.g., stress relaxation, creep), modulus (e.g., Young’s modulus), the ECM fibrous network (e.g., pore size, fiber diameter), cell adhesion (e.g., focal adhesions, integrins), and cellular tension. However, detailed studies exploring the specific effects of the viscoelastic microenvironment and ECM fibrous network on embryonic development and embryo-uterine interactions remain insufficient.

Mechanical forces are critical during the various stages of embryo morphogenesis and organ formation8–11, including embryo hatching from the zona pellucida12, gastrulation13, and organ size determination10. Despite ample evidence demonstrating the profound influence of mechanical forces on cell behavior, the precise mechanisms by which these forces impact embryonic tissue and organ development remain elusive. Further, a deep understanding of the mechanics between the early uterine decidua and embryonic structure is also lacking.

The challenges associated with replicating the intricate processes of implantation and post-implantation development have long been recognized, dating back to the early proposals of embryonic in vitro culture techniques in 196314,15. Despite the progress made in this field16–20, current in vitro embryo culture systems continue to face significant hurdles, including abnormal trophectoderm development and developmental blocks during implantation. These limitations underscore the need for a model that can better replicate the complex interactions between the embryo and its physical environment within the uterus. Our findings, along with recent studies, have reinforced the idea that both biochemical and physical factors within the microenvironment play pivotal roles in embryonic development21,22. Specifically, factors such as tissue stiffness, viscoelasticity, and cellular tension have emerged as crucial determinants of cell fate, embryonic patterning, and tissue morphogenesis23–27. However, the specific role of these physical microenvironmental factors in modulating embryo-uterus interactions has not been fully explored.

The impact of the viscoelastic microenvironment on embryonic development and its underlying mechanisms remains largely unexplored in current studies of blastocyst in vitro culture. Therefore, developing an in vitro embryo culture method conducive to blastocyst implantation and subsequent development is crucial. To address this, we establish a three-dimensional (3D) embryo culture system (Fig. 1A) inspired by the characteristics of the decidual ECM. Within this model, embryos progress to the early organogenesis stage in vitro. Using this innovative culture system, we successfully conduct real-time monitoring and observation of embryo development. We then use the model to examine how stress relaxation affects embryo development and how ECM spatial structure influences embryo growth. Consequently, this 3D system introduces a approach for examining in vitro embryo-ECM interactions, with potential applications in diverse realms, including reproductive biology and regenerative medicine.

Results
Constructing a decidual-ECM-mimicking 3D system for in vitro culture of mouse embryos
Mouse embryo implantation occurs in vivo at E4.5, triggering decidualization of the uterus and rapid proliferation of the decidua tissue, which forms a 3D growth environment encapsulating the embryo. Consequently, an in vitro culture environment that mimics this 3D metamorphosis may be more conducive to optimal embryo development. Therefore, there is an urgent need to establish a 3D environment that serves as a substitute for in vivo uterine tissue. To better simulate the decidua-embryo interaction compared to existing methods, we constructed a 3D environment designed to match the characteristics of the decidua. We selected Matrigel as it is widely employed in embryo culture and, importantly, as its mechanical properties are comparable to those of the decidua, making it a suitable biomaterial for mimicking the in vivo environment28–30. We prepared Matrigel at concentrations of 20% to 50% (termed 20-MG, 30-MG, 40-MG, and 50-MG, respectively) and measured their moduli. The moduli of 20 to 50-MG (83.4 Pa, 85.5 Pa, 89.1 Pa, 104.6 Pa, respectively) were comparable to those of E5.5 to E8.5 decidua (121.6 Pa, 133.2 Pa, 138.7 Pa, 138 Pa, respectively) (Fig. S1A and B). Furthermore, the pore area of 40-MG was most similar to that of the E5.5 decidua (Fig. 1B, C, and Supplementary Movies 1–5). Thus, 40-MG was selected as the material for constructing the 3D environment.

The 3D culture system production process is described in Fig. S1C. The developmental rate of five E3.5 embryos cultured in a 5 μL volume was higher than that of other groups (Fig. S2A). Additionally, embryos developed more readily in 40-MG than in 20-MG, 30-MG, and 50-MG, consistent with our modulus data (Fig. S2B). Finally, we found that the developmental efficiency of embryos with two cavities and an egg cylinder was greater in the 3D environment than in the 2D environment (Fig. S2C). These results suggest that the 3D environment is more suitable for in vitro embryo culture and better supports the study of ECM-embryo interactions.

To evaluate the impact of the 3D environment on embryo development, we quantified various developmental stages. On IVC day 1, embryos maintained a blastocyst-like structure with a developmental rate of 92.9 ± 0.8% (Fig. S3A and C). By IVC day 2, the embryo cells had aggregated, resulting in a developmental rate of 92.3 ± 0.7% (Fig. S3B and C). A cavity, either epiblast (EPI) or extra-embryonic ectoderm (ExE), appeared on IVC day 3, with developmental rate of 69.7 ± 17.7% (Figs. 1D, E, S3D and E). The EPI and ExE cavities then fused to form an egg cylinder by IVC day 4, with a developmental rate of 59.8 ± 18.9% (Fig. 1D and E). The yolk sac began to grow on IVC days 5–6, with a developmental rate of 28.2 ± 2.3% (Fig. 1D and E). Early neural structures of the embryo appeared on IVC day 7. By IVC day 8, the head and tail of the embryo were clearly observed, and the early heart-like structures were visible, with a developmental rate of 23.6 ± 0.6% (Fig. 1D, E, and Supplementary Movie 6).

To characterize the molecular properties of 3D IVC cultured embryos, we analyzed the expression of critical developmental genes in embryos from IVC day 2 to day 9, as well as natural E4.5–E8.5 embryos. On IVC day 1, we found that the embryos expressed OCT4 and CDX2 (Fig. S3A) and formed a rosette-like structure in the ectoderm region by IVC day 2, similar to natural in vivo embryos (Figs. S3B, S4A and B). By IVC day 3, the EOMES+ segment of the ExE cavity and the SOX2+/OCT4+ segment of the EPI cavity were observed, aligning with natural embryos (Fig. S4C, D, and E). On IVC day 4, the amniotic cavity was formed by the fusion of the ExE and EPI cavities, again reflecting the development seen in natural embryos (Fig. S4F and G). Additionally, T+ cells migrated anterolaterally during development, indicating the onset of gastrulation (Fig. S4H, K and L).

On IVC day 5, a few FOXA2+ cells began to appear in the embryos, showing a developmental stage comparable to that of natural embryos (Fig. S4I). By IVC day 7, the number of FOXA2+ cells had increased and their expression patterns matched those seen in E7.5 embryos (Fig. S4J). Early organogenesis initiated on IVC day 8, with the expression of the early brain and nerve development markers, OTX2 and PAX6, corresponding to the neural structures in E8.5 embryos (Fig. S5A). Notably, OTX2+ and FOXA2+ cells, which are markers for early gut, brain, and notochord development, were observed in both natural and in vitro embryos (Figs. 1F, S5B, and C). To further confirm this, we detected the expression of MHC, NKX2.5, MLC, and GATA4 in the early heart-like structures. Additionally, the formation and morphology of the early heart field were similar to those of E8.5 embryos (Fig. 1G and H). These findings validate the successful establishment of a 3D culture system with decidual ECM characteristics.

Application of the decidual-ECM-mimicking 3D system
To validate the effectiveness of our embryo culture platform, we first assessed whether the heart-like structures of IVC day 8 embryos exhibited early functional heart activity. Tissue staining with a calcium (Ca2+)-binding fluorescent dye revealed transient Ca2+ contractions in the heart-like structure of IVC day 9 embryos (Figs. 2A, B, C, S6A, and Supplementary Movie 7). Additionally, administration of isoprenaline increased the beat rate of these heart-like structures (Figs. 2A, B, C, S6B, and Supplementary Movie 8), similar to the response observed in natural E8.5 embryos upon cardiac β-adrenaline stimulation. For live imaging, embryos were cultured and photographed at two-hour intervals over a period of nine days. On IVC day 0.5, the embryo size exhibited fluctuations, initially decreasing before expanding. By IVC day 1, the embryos began penetrating the zona pellucida. By IVC day 1.5, they displayed mobility within the hydrogel. On IVC day 3, distinct cavities corresponding to EPI and EXE formed. On IVC day 4, the egg cylinder was observed. On IVC day 5, the yolk sac started to expand. On IVC day 6, the embryonic body structure became visible. This developmental progression culminated on IVC day 9 with the first detection of the early heartbeat (Fig. 2D and Supplementary Movie 9). These results suggest that the culture system with the decidua ECM properties is capable of supporting in vitro embryo development and can serve as a platform for early embryonic developmental tracing.

Transcriptomic comparison of IVC and natural embryos
We performed single-cell RNA sequencing (scRNA-seq) of three IVC day 9 embryos, three natural E6.5 embryos and three natural E8.5 embryos to investigate how the 3D culture system, designed with decidua ECM properties, affects gene expression during embryo development. After stringent quality control, 14,315 E6.5 cells, 14,761 E8.5 cells, and 10,756 cells from IVC day 9 embryos were analyzed (Fig. S7A and B). UMAP analyses revealed a similar contribution of cells to the developing lineages in both the E8.5 and IVC day 9 embryos (Figs. 3A and S7C).

Using Seurat and published datasets, we identified 19 distinct cell types based on gene expression patterns, which were clearly represented in both E8.5 and IVC day 9 embryo datasets (Fig. 3B).

A Pearson correlation matrix indicated a high level of congruence in gene expression between the cell-type clusters of the E8.5 and IVC day 9 embryos (Fig. 3C). The comparison of cell-type proportions between E8.5 embryos and IVC day 9 embryos showed close similarities between individual clusters, with some variability (Fig. 3D). In IVC day 9 embryos, GPX3, GSTM1, and CD63 were upregulated across all 19 gene clusters compared to E8.5 embryos, while HSPH1, PPA1, and TCOF1 were downregulated (Figs. 3E and S8). PPA1, GSTM1, GPX3, and HSPH1 play key roles in intracellular metabolism and in mitigating external stress, thereby maintaining intracellular stability. This suggests that embryos cultured in vitro primarily rely on nutrients supplied by the culture medium, and metabolism may become dysregulated in IVC day 9–10. The reduced expression of TCOF1 points to potential developmental abnormalities in the head and facial structures during the IVC day 9–10 stage31. Extracellular vesicles (EVs) serve as significant microenvironmental regulators, with CD63 being recognized as an EV marker. Higher levels of released EVs may reflect poorer embryo quality assessment32.

On IVC day 9, the embryos displayed expression profiles similar to E8.5 embryos, including the expression of hematopoietic-related genes and the presence of blood cells in the yolk sac (Fig. S9). Additionally, genes related to neural and early gut development were observed (Fig. S9). We also identified the expression of key genes associated with cardiac development, indicating the emergence of the first heart field (FHF) and second heart field (SHF) progenitor cells (Fig. 3F–J). Additionally, scRNA-seq analysis confirmed that endothelial cells exhibit similar expression profiles both in vivo and in vitro (Fig. S9).

Stress relaxation regulates embryonic development in vitro
The stiffness and viscoelasticity of the ECM significantly influence cellular behaviors such as spreading, growth, proliferation, migration, differentiation, and organoid formation33,34. We observed that the stress relaxation properties of 40-MG were similar to that of E5.5 decidua (Fig. 4A). To investigate whether viscoelasticity regulates embryonic development, we modified our 3D culture system by incorporating two cross-linking agents commonly used in bioengineering: glutaminase and PEG-NHS, to further covalently cross-link the Matrigel35–39. After optimizing the concentrations of glutaminase and PEG-NHS in ES and TS cells and drawing on previous reports38,40,41, we determined that 50 U/g glutaminase and 1 mg/mL PEG-NHS provided the optimal environment for cross-linking Matrigel (Fig. S10A–D). We measured the hydrogel modulus and found no significant differences in the resulting glutaminase cross-linked (40-MGT) and the PEG-NHS cross-linked (40-MGN) hydrogels (Figs. 4B, C, S10E and F). The pore sizes on the surface of 40-MG, 40-MGT, and 40-MGN remained largely unchanged (Fig. S10G, H, Supplementary Movies 10 and 11). However, stress relaxation rates decreased (Figs. 4D, S10I, and J).

We found that embryo development was markedly inhibited in the 40-MGT, with a developmental rate of 31.5 ± 13.0% on IVC day 3 and 27.7 ± 13.0% on IVC day 4. In the 40-MGN, the rates were slightly higher at 37.4 ± 14.1% and 34.7 ± 13.3% (Fig. 4E and F). Additionally, embryos cultured in 40-MGT and 40-MGN did not form rosette-like structures with OCT4+ cells by IVC day 2, and the number of OCT4+ cells remained low (Figs. 4G, S11A and B). By IVC day 3, only a limited number of OCT4+ cells formed a cavity (Fig. 4H), and by IVC day 4, CDX2+ cells were predominantly observed (Fig. 4I). Bright field images also revealed abnormal embryo morphologies (Fig. S11C). Atomic force microscopy (AFM) measurements showed that the modulus difference of the E5.5–E7.5 embryos decidua was small (Fig. S1A). Additionally, we observed that the stress relaxation of the E5.5–E8.5 embryo decidua gradually slowed down (Fig. S11D and E). Our investigation revealed a gradual deceleration in stress relaxation within the 3D culture system over time (Fig. S11F); notably, 40-MG itself underwent these changes more rapidly (Fig. S11F). These findings suggested that stress relaxation played a key role in embryo development during in vitro culture.

To further validate the impact of stress relaxation on embryo development, we utilized the widely used alginate plus Matrigel system (Figs. 4J and S12A). We combined both high and low molecular weight alginates with 40% Matrigel, resulting in comparable moduli (Fig. S12B and C). Notably, the low molecular weight alginate mixed with 40% Matrigel exhibited faster stress relaxation (Fig. S12D and E) and superior developmental efficiency (Figs. 4K, L and S12F). Furthermore, the addition of glutaminase to 20-MG maintained an unaltered modulus while showing slower stress relaxation (Fig. S12G and H), which corresponded with an increased embryo development rate (Fig. 4M and N). These findings further confirm that stress relaxation plays a critical role in embryo development.

Enhancement of integrin and FA aggregation in TE cells facilitates embryo-hydrogel interactions
Integrins are key adhesion molecules present at the embryo-uterine interface during implantation, playing a critical role in the adhesion and migration of trophoblast cells42,43. The interaction between the ECM and integrins is crucial for mechanosensing during early embryo development19,44. Stress relaxation significantly affects the cell morphology of the implanted embryos. Specifically, under 40-MG conditions, we observed more pseudopods compared to 40-MGT or 40-MGN conditions (Fig. 5A), indicating the development of a more invasive trophectoderm (TE)19. Additionally, integrin subunit expression in embryos cultured in 40-MG was higher than in those cultured in 40-MGT or 40-MGN (Fig. S13A), suggesting that elevated integrin expression is a significant factor in promoting embryo implantation in vitro. The aggregation of integrins subsequently led to vinculin recruitment and the formation of stable FAs in embryos cultured in 40-MG (Fig. 5B, C, and D). The 3D environment modulates cell contraction, thereby regulating FA formation24. Consequently, enhanced integrin expression and FA formation under 40-MG conditions promote better embryo implantation and development, highlighting the critical role of stress relaxation in the 3D system.

It is known that hydrogels regulate the formation of adhesive patches by modulating cell contraction25. To investigate how 40-MG promotes FA formation, we analyzed the localization of phosphorylated myosin light chain (pMLC) in TE cells. In embryos cultured in 40-MGT and 40-MGN, pMLC was predominantly localized in the nuclei, whereas in 40-MG, it was found at the periphery of embryos (Fig. 5E and F). To further investigate the interaction between TE cells and the hydrogel, we cultured trophospheres (spheroids derived from trophoblast stem cells, TS cells) in 40-MG, 40-MGT, and 40-MGN. The localization patterns of FAs and pMLC in these trophospheres were consistent with those observed in embryos (Fig. S13B–F). Inhibiting myosin activity with blebbistatin on IVC days 1–2 reduced FA formation in embryos cultured in 40-MG and increased nuclear localization of pMLC in TE cells, confirming the importance of TE cell contraction in this process (Fig. S14A and B). This inhibition also led to decreased embryonic development rates in the 40-MG environment (Fig. S14C–E).

On IVC day 2, we sequenced the hydrogel-contact regions of embryos from 40-MG, 40-MGT, and 40-MGN groups. Analysis of differentially expressed genes revealed that the 40-MG group exhibited elevated levels of cellular processes, including cell migration, cell adhesion, regulation of actin cytoskeleton reorganization, compared to 40-MGT or 40-MGN groups (Fig. 5G). Since stress relaxation influences FA formation, we explored whether altering the RGD content in the material could enhance FA formation and mitigate the adverse effects of slow stress relaxation on embryo development (Fig. 5H). To do this, we created a group with intermediate RGD content by mixing equal proportions of RGD-free and RGD containing high molecular weight sodium alginate, followed by assessment of embryo development. We found that as the RGD content increased, embryo development initially improved and then decreased, though it remained significantly higher than the group without RGD (Figs. 5I, J, and S15).

Overall, our findings indicate that rapid stress relaxation facilitates swift FA aggregation, while slow stress relaxation combined with RGD inclusion enhances embryo development (Fig. 5K). Furthermore, 40-MG promotes TE cell contraction, integrin aggregation, and FA formation, thereby establishing effective mechanotransduction between the embryo and the ECM.

The fibrous network in hydrogel regulates embryonic development in vitro
Another crucial property of ECM is its fibrous network structure. Pore size and density are pivotal in defining the microenvironment for cell growth. Extensive evidence suggests that the porous architecture of tissues can offer guidance for cell behaviors45. To investigate whether the spatial network of the ECM plays a regulatory role in embryo development, we initially assessed alterations in the spatial network of decellularized natural decidual tissues from E5.5 to E7.5 (Fig. 6A, Supplementary Movies 12 and 13). Our observations revealed that the spatial network of these tissues exhibited a compact structure at E5.5, which gradually expanded from E6.5 onwards (Fig. 6A). This expansion was characterized by an increase in pore size (Fig. 6B) and fiber diameter thickness (Fig. S16A). Similarly, SEM images of the interface between the embryo and 40-MG showed that the fibrous network around the embryo became loosened over time (Fig. 6C and D), accompanied by an increase in fiber diameter thickness (Fig. S16B).

To investigate the impact of MMP on this process, we added MMP inhibitor GM600124,46 to IVC embryos from day 0 to day 4, which slowed down the loosening process (Figs. 6E, F and S16C). Moreover, GM6001 also reduced the expression levels of MMP2 and MMP9 in the embryos cultured in 40-MG (Fig. S16D), indicating that MMP secretion by TE cells regulates ECM. The addition of GM6001 decreased the developmental rates of embryos in 40-MG on IVC days 3 and 4 (38.6 ± 9.5% and 34.2 ± 11.0%, respectively) (Fig. 6G and H), suggesting that the proper regulation of ECM spatial network is crucial for optimal embryonic development.

FAK is known to regulate the expression of MMP2 and MMP9, which are crucial for various cellular processes47. Our results showed higher levels of FAK activation in TE cells of embryos in 40-MG compared to those treated with the FAK inhibitor FI 14 (Fig. 6I). The inhibitor significantly reduced the developmental rate of the embryos on IVC days 3 and 4 to 18.3 ± 12.3% and 10.8 ± 11.7%, respectively (Figs. 6J, K and S16E). Following the addition of FI 14, p-FAK was undetectable (Fig. 6I). To further validate the impact of spatial structure on embryonic development in 3D system, we added collagenase IV to 50-MG, which significantly increased developmental rate of embryos on IVC days 3 and 4 to 53.74 ± 12.0% and 50.11 ± 11.3%, respectively, compared to embryos cultured in 50-MG alone (Fig. 6L and M). This suggests that the controlled degradation of ECM components can be beneficial for in vitro embryonic development by providing additional space for growth and by facilitating cellular interactions. In conclusion, our findings indicate that in vitro embryos degrade surrounding materials through the secretion of MMP2 and MMP9, thereby creating additional growth space conducive to in vitro embryonic development (Fig. 6N).

Methods
Animal and ethical approval
ICR 8-week-old females and 8-week-old males mouse, SD 8-week-old females and 8-week-old males rats were purchased from SPF (Beijing) Biotechnology. All animals were maintained under specific-pathogen-free conditions at the same facility, with ad libitum access to food and water, a 12h light/12h dark cycle, and controlled temperature (18–23°C) and humidity (40–60%). At E3.5, pregnant mice were humanely euthanized, and embryos were randomly collected without sex selection. The sex of mouse embryos cannot be determined prior to or during experiments, and there is no evidence that early embryo development is sex-dependent. Therefore, sex-based analysis was not applicable to this study.

Atomic force microscopy (AFM) testing
The Young’s moduli of deciduae at different developmental stages (E5.5 to E8.5) and various concentrations of Matrigel were measured using atomic force microscopy (AFM, Nanowizard4, Bruker, USA). AFM measurements were conducted with silicon nitride cantilevers (NP-O10, Nanoworld, USA) with a nominal spring constant of 0.12 N/m and a 30-μm-diameter silica bead (Thermo Fisher Scientific). Each sample was measured at five distinct points, and five force curves were collected at each point.

The applied mechanics model and Hertz model could be used for data fitting and calculation with the spherical indenters involved in the indentation, and the key factors have also been specified in eqn. Following the Hertz model describing the indentation of elastic bodies, the force-to-penetration equation is:

F = 4/3 R^1/2 E* δ^3/2 (1)

here, F is the applied force, R is the radius of the indenter tip, δ is the indentation depth, and E* is the reduced modulus, which can be defined by the combination of the plane strain moduli of the indenter (E1) and the substrate modulus (E2)48:

1/E* = (1-v1^2)/E1 + (1-v2^2)/E2 (2)

where v1 and v2 are the Poisson’s ratios of the indenter and the substrate, respectively, and the indenter is much stiffer than the substrate (E1 ≫ E2), so the reduced modulus E* is simply:

E* = E2 / (1-v2^2) (3)

For decidua, the Poisson ratio (v2) is assumed to be 0.549 Eq. (1), Eq. (2) and Eq. (3) become:

F ≈ 16/9 E R^1/2 δ^3/2 (4)

The data in the loading section of the load-displacement curve were used to determine Young’s modulus using a fit of all data points from the contact point to the maximum load point.

Decellularization of decidua
Decidua was decellularized by shaking in solutions of SDS (1%, 0.1%, and 0.01%) and Triton X-100 (1%, 0.1%, and 0.01%), each solution for 3h. This was followed by washing with water for 1h. The decellularized decidua was then fixed for further use.

Scanning electron microscopy (SEM) observations
Decellularized decidua, varying concentrations of Matrigel, and embryos embedded into 40-MG were fixed in 2.5% glutaraldehyde at room temperature for 4h. The samples were then dehydrated in a graded ethanol series (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ethanol for 15 min each), followed by 100% ethanol for 15 min. After supercritical drying (CPD300, Leica), the samples were coated with platinum using a sputter coater (JEC-3000FC, JEOL) and observed using scanning electron microscopy (SEM, SU8010, Hitachi, Japan).

Contact angle measurement
Contact angle measurements were performed using a KRÜSS Contact Angle machine with KRÜSS ADVANCE software version 1.11.0.15801. Prior to testing, samples, such as 40% Matrigel (354230, BD Biosciences), were stored at 4°C. For the sessile drop technique, a 2 μL droplet of the sample was deposited on a PDMS surface that had been treated with plasma for varying durations. Measurements were taken within 5 s of droplet deposition. Contact angles were analyzed from recorded videos using ImageJ software.

Culture system
The PDMS prepolymer (Sylgard 184, Dow Corning, United States) was prepared with a mass ratio of 10:1 between the matrix and crosslinker. The prepolymer was mixed thoroughly and poured into a four-well plate (176740, Thermo Fisher Scientific). A total of 0.05g of PDMS prepolymer was added to each well, followed by curing at 80°C for 2h. The PDMS wells were then treated with plasma at 30 W for 5 s. After treatment, 5 μL of 40-MG was added to each well, and 5 embryos were embedded in the 40-MG. The plates were cured at 37°C for 8-10 min, after which culture medium was added. Embryos were cultured at 37°C in a 5% CO2 atmosphere. For 40-MGT, 50 U/g Transglutaminase (G8661-5g, Solarbio) was added to the 40-MG. For 40-MGN, 1 mg/mL PEG-NHS (N164064-1g, Aladdin) was added to the 40-MG.

Embryo recovery and culture
Pregnant mice were humanely euthanized at 3.5 days post coitum by cervical dislocation. Embryos were then flushed from the uterus using a flush medium and seeded onto a designated well a plate. This time point was recorded as IVC day 0. Embryo culture was conducted at 37°C in a 5% CO2 atmosphere.

In vitro culture medium
Flush medium: CMRL 1066 (11530037, Invitrogen) + 5 × penicillin-streptomycin (60162ES76, YEASEN) + 10% fetal bovine serum (FBS, SE200-ES, Vistech). Medium for IVC days 0-1: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement (35050061, Thermo Fisher Scientific) + 1 × MEM Non-Essential Amino Acids Solution (11140050, Thermo Fisher Scientific) + 0.5 × N-2 Supplement (17502048, Gibco) + 0.5 × B-27™ Supplement (17504044, Gibco) + 10% FBS. Medium for IVC day 2: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement + 1 × MEM Non-Essential Amino Acids Solution + 0.5 × N-2 Supplement + 0.5 × B-27™ Supplement + 20% FBS. Medium for IVC day 3: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement + 1 × MEM Non-Essential Amino Acids Solution + 30% KnockOut™ Serum Replacement (10828028, Gibco). Medium for IVC day 4: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement + 1 × MEM Non-Essential Amino Acids Solution + 30% rat serum (RS) + 0.5 mg/mL glucose (Sigma, D9434). Medium for IVC day 5: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement + 1 × MEM Non-Essential Amino Acids Solution + 40% RS + 1 mg/mL glucose. Medium for IVC day 6–7: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement + 1 × MEM Non-Essential Amino Acids Solution + 50% RS + 1 mg/mL glucose. Medium for IVC days 8-10: CMRL 1066 + 1 × penicillin-streptomycin + 1 × GlutaMAX™ Supplement + 1 × MEM Non-Essential Amino Acids Solution + 50% RS + 2 mg/mL glucose.

Inhibitor treatment
A concentration of 10 µM of each inhibitor, including Fak-specific inhibitor (FI 14, SML0837-10MG, Sigma), MMP inhibitor (GM6001, HAT08408, TargetMol), and blebbistatin inhibitor (S7099, Selleck), was added per well. Vehicle control (DMSO, D2650-100ML, Sigma) was also added per well. 0.02 mg/mL Collagenase IV (07909, Stemcell) was also added per well.

Immunofluorescence staining
(1) Characterization of embryos from IVC day 1 to day 5 and natural embryos (E4.5-E6.5):
Embryos were fixed with 4% paraformaldehyde (PFA, 60536ES76, Yeasen) at 4°C for 6h, then permeabilized with 1% Triton X-100 (9002-93-1, Sigma) in phosphate buffered saline (PBS, PM5090-50, Coolaber) at 4°C for 6h. Blocking was performed with 0.1% Tween-20 (P1379-25, Sigma), 0.01% Triton X-100, and 1% bovine serum albumin (BSA, B09354-100g, Innochem) in PBS at 4°C for 6h. Embryos were incubated overnight at 4°C with primary antibodies diluted in 0.1% Tween-20, 0.01% Triton X-100, in PBS. The primary antibodies used were CDX2 (1:100, 3977S, Cell Signaling Technology); OCT4 (1:100, sc-5279, Santa); SOX2 (1:100, ab97959, Abcam); EOMES (1:100, ab23345, Abcam); FOXA2 (1:100, 8186S, Cell Signaling Technology); T (1:100, ab209665, Abcam); Phalloidin (1:500, 40737ES75, Yeasen); VINCULIN (1:100, 26520-1-AP, Proteintech); p-MLC (1:100, 3674S, Cell Signaling Technology); p-FAK (1:100, PA5-17084, Thermo Fisher Scientific). The embryos were then incubated with the following secondary antibodies at 4°C for 2h: Alexa Fluor 647 goat anti-mouse (1:500, A21235, Invitrogen), Alexa 488 goat anti-rabbit (1:500, A11034, Invitrogen) and Cyanine 3 goat anti-rabbit (1:500, A10520, Invitrogen) at room temperature for 10 minutes. Imaging was conducted using a Zeiss LSM 880 confocal microscope.

(2) Characterization of embryos from IVC day 6 to day 10 and natural embryos (E7.5-E8.5).
The embryos were fixed with 4% PFA at 4°C for 12h, then permeabilized with 2% Triton X-100 in PBS at 4°C for 12h. Blocking was performed with 0.1% Tween-20, 0.01% Triton X-100 and 1% BSA in PBS at 4°C for 12h. Embryos were incubated overnight at 4°C with primary antibodies diluted in 0.1% Tween-20, 0.01% Triton X-100 in PBS. The primary antibodies used were FOXA2 (1:100, 8186S, Cell Signaling Technology); T (1:100, ab209665, Abcam); SOX2 (1:100, ab97959, Abcam); PAX6 (1:100, AF8150, R&D Systems); OTX2 (1:100, AF1979, R&D Systems); Myosin light chain (1:100, ab25310, Abcam); GATA4 (1:100, sc-25310, Santa); Myosin heavy chain (1:100, 53-6503-82, eBioscience). The embryos were then incubated overnight at 4°C with the following secondary antibodies: Alexa Fluor 647 goat anti-mouse (1:500, A21235, Invitrogen); Cyanine 3 donkey anti-goat (1:500, A21432, Invitrogen); Cyanine 3 goat anti-rabbit (1:500, A10520, Invitrogen) and Alexa Fluor 647 donkey anti-sheep (1:500, A21448, Invitrogen). Nuclear staining was performed 10 μg/mL Hoechst 33342 (H3570, Invitrogen) at room temperature for 10 minutes. Imaging was conducted using a Zeiss LSM 880 confocal microscope.

Live imaging of IVC embryos
Pregnant mice were humanely euthanized at 3.5 days post coitum by cervical dislocation. Embryos were then flushed from the uterus using a flush medium and seeded onto a designated well of a plate. This time point was recorded as IVC day 0. Embryo brightfield images were captured every 2 h over a 9-day period using a real-time dynamic cell monitoring and functional analysis system (JS170405-102, Juli-stage) with a 4× objective at 37°C and 5% CO2.

Ca2+ measurement
The embryos were washed twice with PBS, incubated at 37°C for 30 min with 8 μM of the green fluorescent intracellular Ca2+-binding dye Fluo8-AM (21080, AAT Bioquest) and 0.04% Pluronic F127 (P3000MP, Invitrogen) in HBSS solution (14025092, Gibco). After incubation, the embryos were washed twice again with PBS. They were then mounted in glass-bottom dishes with HBSS solution and observed under a confocal microscope (Zeiss 880). Isoproterenol was added to achieve a final concentration of 1 μM. The images obtained were analyzed with ImageJ software, and the time constant of Ca2+ decay was calculated by a single exponential fit using Origin software (OriginLab).

Rheological test
Rheological experiments are conducted on a rheometer (MCR-302, Anton Paar GmbH, Austria) equipped with a parallel plate (25 mm diameter, 3882). For the time sweep model, 200 μL of hydrogel solution was applied to the measuring stage at 4°C. After trimming the sample to match the geometry, the temperature was elevated to 37°C for 90 min. The shear storage (G’) and loss (G”) moduli were measured at a fixed frequency and strain (1 Hz, 1%) to monitor the hydrogel moduli. For the stress relaxation test model, the hydrogel solution was added at 4°C and maintained at 37°C for 60 min. The stress relaxation test was subsequently initiated at a fixed strain (1%). The stress profiles were normalized to the initial maximum stress and fitted using a generalized Maxwell–Weichert model with three parallel elements (n=3)50:

σ = σ0 + σ1 e^(-t/τ1) + σ2 e^(-t/τ2) (5)

Here, σ represents the normalized stress, while σ0, σ1, and σ2 are the normalized stresses in the spring and dashpot 1 and 2 models, respectively. The constants τ1 and τ2 describe the stress decay in the dashpot 1 and 2 models. All tests are repeated at least three times.

Nanoindentation test
To determine stress relaxation properties, a nanoindenter (Optics11 Piuma, The Netherlands) was used. The generalized Maxwell model, which is mathematically expressed using the Prony series, was selected. The following analytical formulation describes the load decay during the holding phase as a function of time51:

P(t) = P1 + Σ (k=1 to N) Pk e^(-t/τk) (6)

where P(t=∞) = P1 represents the equilibrium load, which is the value reached at the end of the relaxation time. All samples were measured using a probe with a 3 μm radius and a cantilever stiffness of 0.024 N/m. Cantilever bending calibrations were performed before each series of experiments by indenting a rigid surface beneath the probe. Each sample was tested over a 400 × 600 μm grid scan with 200 μm spacing between measurements. The indentation protocol consisted of a 4-second loading phase to a 15,000 nm indentation depth, held for 40 seconds, followed by a 4-second unloading phase. Three experiments were conducted on each tested sample to ensure reliability. All single indentation values were calculated using GraphPad Prism 9 software.

Trophosphere formation
Mouse trophoblast stem (TS) cells were obtained from the Zhou laboratory (Institute of Zoology, Chinese Academy of Sciences, Beijing, China), which were isolated from mouse blastocysts. TS cell lines tested negative for mycoplasma contamination. TS cells were cultured at 37°C and 5% CO2 in RPMI 1640 (61870036, Gibco) supplemented with 20% FBS, 2 mM L-glutamine (TMS-002-C, Sigma), 0.1 mM 2-mercaptoethanol (2-ME, 21985023, Gibco), 1 mM sodium pyruvate (11360070, Gibco), and 1% penicillin-streptomycin, plus FGF4 (25 ng/mL, 100-31, PeproTech), FGF2 (25 ng/mL, 450-33, PeproTech) and Heparin (1 μg/mL, 07980, STEMCELL Technologies) in the presence of inactivated ICR MEFs. Trophosphere were obtained by seeding TS cells (approximately 12 cells per microwell) into a 96-well ultra-low attachment microplate (7007, Corning) using the same medium as for TS cell culture52.

Preparation of sodium alginate hydrogel
Sodium alginate with a high molecular weight (209.97 kDa) was purchased from Sigma-Aldrich (A2033), Low-molecular-weight sodium alginate (135.2 kDa) was prepared as follows: high molecular weight alginate was irradiated with a 3 or 8 Mrad Cobalt source, followed by dialysis and purification. Arginine-Glycine-Aspartate peptides (RGD, SynthBio) were coupled to alginate using carbodiimide chemistry53,54. To prepare the mixed hydrogels, the appropriate 1% (w/v) alginate and 40-MG were added to a Luer lock syringe (Cole-Parmer). In a separate syringe, appropriate volumes of calcium sulfate and 1066 medium were added (high molecular weight alginate required 19.52 mM calcium sulfate; low molecular weight alginate required 122 mM calcium sulfate). The two syringes were connected with a coupler, and the solutions were mixed by passing them back and forth six times. A 10 μL volume of the hydrogel was used to culture five embryos.

Real-time RT-PCR
Total RNA was extracted from 10 embryos collected into a 0.2 mL tube, followed by reverse transcription using the CellAmp™ Whole Transcriptome Amplification Kit (Real Time) Ver. 2 (3734, Takara). Quantitative PCR was then performed using TB Green® Premix Ex Taq™ (Tli RNaseH Plus, RR420, Takara) on a QuantStudio™ 6 Flex system (ABI). INTEGRIN α3, INTEGRIN α4, INTEGRIN α5, INTEGRIN α6, INTEGRIN α7, INTEGRIN αV, INTEGRIN β1, and INTEGRIN β8 primer sequences were obtained from the referenced publications55. Detailed sequence information can be found in Supplementary Table 1.

RNA-seq analysis (SmartSeq2)
RNA was extracted from embryos cultured in 40-MG and 40-MGT on IVC day 2 for sequencing. Embryos were removed from 40-MG and 40-MGT cultures, washed three times with PBS, and then placed under a microscope for lysis. Embryos cultured under 40-MG and 40-MGT conditions on day 2 of in vitro culture (IVC) were sectioned as shown in Fig. 5G. Trophoblast cells from the deposited regions were then isolated and lysed for subsequent sequencing. Trophoblast cells from the deposited regions of embryos from three separate embryos were sequenced for both 40-MG and 40-MGT conditions. The raw sequencing data were processed with Cell Ranger using the default mapping arguments. Gene expression analyses and cell-type identifications were conducted using Seurat V3.1. IntegrateData is a function in the Seurat package used to integrate multiple single-cell datasets into a unified dataset. The data are processed to remove batch effects through the IntegrateData function. In this process, the dim parameter is set to 1:20, and other parameters are set to their default values. The Nonlinear dimensionality reduction was performed using t-SNE. Figures were generated in Seurat using the DimPlot and VlnPlot functions.

Single-cell RNA sequencing (scRNA-seq) library generation
Three E8.5 embryos from three different pregnant mice and three IVC day 9 embryos were selected for sequencing. Embryos were washed three times in PBS containing 0.4% BSA, then dissociated with 3 mL Tryple (12605010, Gibco) with 0.02% DNase (R1600, Solarbio) at 37°C in 5% CO2 for 12 minutes with gentle agitation. Following dissociation, 2 mL of PBS containing 20% FBS was added, then the sample was centrifuged at 12,000 × g for 2 min. Cell pellets were resuspended in PBS containing 0.4% BSA and then filtered through a 40-μm cell strainer. The cell count was determined using a hemocytometer. Single-cell suspensions (1,000,000 cells/mL) were loaded into a 10x Genomics Chromium Chip according to the manufacturer’s instructions (10x Genomics, Chromium Next GEM Single Cell 3’ GEM, Library and Gel Bead Kit v. 3.1). The 10x Genomics scRNA-Seq libraries were generated by Annoroad Gene Tech. (Beijing) Co. Ltd.

Video editing
All videos presented in the manuscript were processed and edited using VideoStudio to ensure clarity, proper sequencing, and high-quality visualization of the experimental results.

Quantification and statistical analysis
Data were statistically analyzed using One-way ANOVA with Welch’s correction for unequal sample sizes, followed by the Bonferroni post hoc test, or two-tailed Student’s t-tests, as appropriate. Analyses were performed using GraphPad Prism 7 for Windows. Data are presented as the mean ± standard deviation (s.d.). The number of independent experiments conducted for each analysis is indicated in the figure legends. All experiments were repeated at least three times unless otherwise stated. The following software were used in this study: Imaris 8.2, ImageJ 1.47, GraphPad Prism 7, Excel 2019, and ZEN Blue Edition (Zeiss).

Ethical statement
All animal procedures were approved by the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (Ethical approval No. IOZ20180058).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Guo et. al., devised a hydrogel mimicking mechanical properties of decidual tissue, which promotes growth of blastocyst through implantation and putatively to early organ differentiation in vitro. The authors sustain that stress relaxation supports adhesion of the trophoblast to the gel, and finally investigate the role of MMPs to create a proper environment for embryo growth. Several similar papers have been published trying to mimic the mechanical properties of the uterus, but particularly, the lead contact of this manuscript has published a recent study reporting very similar results (https://doi.org/10.1002/advs.202202282) using a matrix with some different components to the one presented in this paper. Despite producing a new matrix, the embryos obtained are similar in quality and morphology to the previous study, with only slightly higher efficiency of embryo development in this paper, and the cultured embryos are evidently defective after six days of in vitro culture. The authors then dig into the specific role of these physical microenvironmental factors in modulating embryo-uterus interactions, for example: stress relaxation, integrins, focal adhesions, ECM network and MMPs. These results add value to the manuscript, since these parameters have not been deeply explored during implantation. However, the experiments focusing on all these aspects are superficial, and the authors find interesting correlations but do not mechanistically demonstrate this relationship of these parameters to their statements. Major points: Fig. 1. To stringently compare the morphology of cultured embryos, control in utero developed embryos should be shown next to the bright field images of in vitro implanted embryos. The morphology of cultured embryos at day 6 and after seems to be aberrant, with a poorly developed epiblast (embryo proper). Fig. 1F-H. Only one section of the embryos is shown, which may be misleading to properly appreciate the morphology of the embryo. The authors should show whole-mount immunostainings of the embryos dissected out from the yolk sac. Besides, in Fig 1F Foxa2 and Otx2 seem to co-localize, which does not occur in utero. In Fig. 1G, MHC and Nkx2.5 seem to be mostly expressed in the whole tissue, and not in the embryonic heart as it is well known for these markers, thus lines 154-155 “additionally, the formation and morphology of the early heart field were consistent with those of E8.5 embryos” are not supported by the data showed. Fig 1A. Due to the lack of proper morphology of the E8.5 IVC embryos, the scheme corresponding to day 8-10 in Fig. 1 panel A is misleading, since it shows a properly formed E8.5 embryo, which is not showed in the results section. Fig. S3. The authors should show co-staining of epiblast, visceral endoderm and epiblast in the same embryo, since it is common to see embryos lacking one or more compartments when cultured in vitro, i.e., presence of epiblast markers but lack of extraembryonic ectoderm (ExE) markers in the same embryo, etc. Line 168. For the scRNA-seq analysis, the authors should discuss more in detail the evident differences in proportion of several cell types (clusters) in IVC embryos compared to in utero. This point is only briefly mentioned in the lines 174-175 “The reduced expression of Tcof1 points to potential developmental abnormalities in the head and facial structures during the IVC day 9-10 stage”. Fig. 3D and video S8. The embryos after day 5.5 are severely defective, with only a cyst developing inside the yolk sac. Line 192-199, Fig 3E, and video S8. The microinjected embryo shown in the video and Fig. 3E doesn’t seem to develop even into a proper egg cylinder, it looks like an aggregate of cells without any evident epiblast and ExE. The TFAP2C staining showed confirms that there is not ExE (the signal is only at the periphery and not as the ExE should look in a normal embryo). After 8 days the shape does not change. Line 254 and Fig. 4K-L. The authors mention a superior developmental efficiency of LA and HA+ 40-MG, however the graphs show a decrease in percentage of embryos grown. Similarly, in Fig. 4M-N the authors show an increase in egg cylinders with 2 cavities using MGT, which is opposite to what they showed using MGT in Fig. 4F; could the authors better explain what is the difference between these 2 conditions? It is not clear whether in Fig. M-N they are using LA or HA, why is the result opposite to Fig. 4F? Fig. 5B-D. Could the authors explain in more detail how do they quantify focal adhesions? Lines 273-276 and Fig 5E. Could the authors elaborate on what can be the functional role of finding phosphorylated myosin in the nucleus using 40-MGT and 40-MGN vs the peripheral localization in MG? How does it relate to focal adhesion formation as stated in the text? The increase of embryo development by adding collagenase is interesting, how does the fibrous network look by SEM after Col IV treatment? What could be the potential mechanism of this beyond the speculation of “controlled degradation of ECM components”? Minor points: Lines 119-120 The embryonic day at which the embryos are taken is not clearly stated in the text, this is crucial information and should be mentioned beyond the methods, it’s not clear whether they start cultures at E3.5 or E4.5. In Fig. S5D there are colors (cell clusters) that do not appear in the legend to the right of the graphs. Lines 183-185. “Furthermore, scRNA-seq analysis confirmed the crucial role of endothelial cells in cardiomyocyte maturation, function, and survival during embryo development, as evidenced by the expression of typical endothelial marker genes in Figure S7F”. It is not possible to confirm the role of endothelial cells in cardiomyocyte maturation by only using scRNA-seq analysis as the authors did, I suggest rephrasing this sentence. Line 247-248 and Fig. S10F. The authors claim that the stress relaxation rate of 40-MG was faster compared to 40-MGN and 40-MGT. Please add statistical analysis to this data since the values seem similar in most conditions. Fig. S12B-C. All these images should be zoomed in. Currently the differences in localization patterns of FAs and pMLC described by the authors are not totally evident in the immunostainings. Line 204. Do the authors mean decellularized decidual tissues instead of metaphase tissues? Line 333, 350 and 355. Authors should replace the word “bionic” since their protocol does not include any electric compartment.", "reviewer_2": "Guo and colleagues present a study to progress mouse in vitro embryo development. I found the study to be comprehensive and interesting. The manuscript is well written, images are clear, and the design seems to be well described. I did think that the title describing the model as “decidual-inspired” might be a bit of an overstatement – this made me expect to see an engineered hydrogel to very specifically match the decidual characteristics, when in fact the study is really only examining and optimising Matrigel characteristics and concentration. I have some specific comments, below: Bar charts should be presented with individual datapoints, especially when n<10 (many instances here). Overall, I found the introduction to be a bit wordy – it could be more concise and still convey all the same points. Some sentences seem to repeat the previous. It would be useful to have a scale on the videos S1-S5 and S11-14 if possible – I found it difficult to relate these back to the images presented in Figure 1B. Legend for Video S9 could have more explanatory detail relating to the manipulation and the arrowhead. mT/mG mouse should be explained briefly in the text for non-familiar readers. Line54: should be integrins (plural)? Line 54-55: repetition of invasive/invasion could be avoided – I suggest removing the first. Line 60: “from being ECM free state” – some sense missing maybe a word(s) omitted? Lines 60/61: “encapsulated within ECM” and also the endometrial cells producing it, surely? The interaction is not only with an isolated ECM. Line 64: please state the specific modulus when referring – otherwise it just means “measure” without the context of which specific measure you are describing. Several instances. Line 69: No need for both “initially” and “primarily” Line95: ECM abbreviation has already been used. Line 104: Please start with Mouse Line 107: I don’t think it has been conveyed in the introduction, nor here, why the need is “urgent” to establish such a model? Line 112: which modulus? (of course the figure tells us, but please include in the body text). Line 119: Should it be Figure S1E here (not S2E)? Line 152: should this be Figure 1F (not 2F)? Line 154: I think “similar to” would be better than “consistent with” here, since they are not looking exactly the same to me. Line 178: I think that rather than “cell-type composition” it would be more appropriate to refer to expression profiles as being similar, since in this case the number and localisation of cells is not shown. Line 214: is this always the case that red localise to TE and green to epiblast? Or simply referring to the specific embryo presented in the Figure? If always, then this suggest that something about the Cre injection affect development and cell fate? Please show other examples. Line 265: integrin subunit expression? Line 276 – still looks quite nuclear to me in the 40-MG image? Line 304: wasn’t sure if metaphase was intended here? Line 313: missing “of” (addition GM6001) Line 319: FI 14 is only called F14 in figure – please be consistent. Line 334: repetition of FOXA2 Line 729: spelling “using” (suing) Line 854: something missing here about the low MW alginate (“…and was”) Line 862: why is 10ul per 5 embryos here, when earlier you optimised conditions to 5/5? Line 865-6: RT-PCR used here to mean real-time, since reverse transcription is already completed? Less confusing to use RT (reverse transcription) and q(Quantitative) PCR. Line 869: how? What are the dissociation and capture steps specific to these samples? Are these as described in lines 876-885 or something other? Line 872: using which approach? There are several. Figure 1 & legend: B – refer here to the linked videos C – how many replicates? Please amend the legend and show the datapoints D – it would be useful to apply the labels shown here to the supplementary figure S2 E – what is presented here? The average and error of the four experiments or across the 134 embryos? The former being more appropriate for statistical analysis. This query applies elsewhere on similar designed experiments. H – no scale bar is visible on the left panels. Are the Day 9 and E8.5 images all on the same scale – the bars don’t seem quite equivalent to me. Figure 3 & legend: D and E – spelling “culture” Figure 5G Where are the methods for SmartSeq2 sample preparation and analysis? Figure 6 I – nuclei are unclear in the 40-MG + FI 14 images, but distinct in the control images, why is this? D and F – why not combine these charts for easier comparison? Figure S1E – not sure the schematic is very useful here, it’s a bit small. Figure S2 – as above, the addition of labelling to the embryos would be useful here. For example the two cavities described in the main text are not obvious. It might be helpful to align the schematic and the experimental timelines so reader can compare directly the stages? Figure S2C – check the numbers here – they don’t agree with the legend. Figure S2D – how many are with and how many without Matrigel? Why not separate them? Figure S5D – the key is incomplete here", "reviewer_3": "The manuscript from Guo et al represents an exciting and thoughtful journey in ex vivo embryology, and in some ways takes wild leaps into synthetic reproduction. In terms of big picture, this is an insightful body of work with substantial impact in the field of early mammalian development, and should meet the high bar for publication in Nature Communications. Issues to consider for a revision: 1. The paper does not mention/cite Bedzhov Nat Protocols 2014, Bedzhov Cell 2014, or Ozguldez Methods Mol Biol 2021 -- which seems odd given the present work follows from these recent publications. The manuscript's introduction transitions into the results by describing the ECM and biophysics, without much background on the current biological state of the art in ex vivo embryogenesis. 2. Much of the scRNA analysis seems to be pseudo-bulk across the entire samples (Figs. 2D, S7), which doesn't take advantage of the clustering work that was done (Figs. 2B and S5). Metabolic genes are mentioned but without any broad-scale expression categorization (i.e. GO, Kegg) at either sample level or between clusters. Recommend at least one or two panels to analyze gene ontology and/or cell identities, perhaps in selected clusters, to give the scRNA experiments a little more depth. 3. Suggest FHF/AHF gene expression be put into Figure 2 to accompany 2E and 2F, perhaps by dotplot, as currently the way this is split between main figures and supplement isn't too convincing. 4. To dovetail above suggestions, possibly find a way to move Figure S5D into Figure 2. Consider swapping Figures 2 and 3A-D, since Figure 3A-D is phenotypic characterization and Figure 2 is mechanistic. Figure 3E suggestions below. 5. Lines 208-221 -- the in vitro -> in vivo -> in vitro experiment: 1. To my knowledge, pre-implantation stages E0-E3.5 are generally easy to culture with high viability, so I don't understand the explanation or reference #29 on lines 210-211. 2. Did the authors perform only one experimental trial here? Do all Cre-injected (GFP-expressing) cells become epiblast in all injections? The authors inject one blastomere presumably at random, so it wouldn't make sense if all injections follow the description of lines 213-214. If the authors are describing one such example, they should state clearly that asymmetric fate of 2-cell stage blastomeres was observed in X embryos out of Y total embryos, and here is an example... [description of Figure 3E experiment]. If asymmetric blastomere fate is a side effect of the injection or damage, and not natural biology, consider removing this experiment (Figure 3E, Video S9) from the paper (or putting in supplement) since it isn't really necessary anyway. 6. Figure 6I lower row - is DAPI channel missing? I don't see any blue in \"Merge\" or zoom Minor points: line 42: remove \"the\" and \"of\" line 78: \"property\" -> \"properties\" line 118: remainder of sentence after common redundant with opening of same sentence line 119: \"Figures S2E\" -> \"Figure S1E\" line 120: \"5uL volume\" ... culture volume is not described for the reader to understand what this means, perhaps can be added to S1E diagram? line 203: delete \"as reported in two papers published in 2018,\" line 205-206: delete \"each\", \"blastomere\" -> \"blastomeres\", \"into\" -> \"in\" line 320-321: rewrite sentence \"However...\" since it doesn't say what condition or how this is unexpected given lowered developmental rate with FI 14. Fig 2A - may be helpful to just do a single 2D UMAP with different sample conditions painted in overlap Fig S3C - either the paper or the legend should have a description of how embryos were scored for \"successful development\" and counted Fig 4E-F, 4K, 5C-D, 5F, 5I-J, 6G-H, 6J-K, 6L-M, S10B, S12A, S12C-F, S13C, S14D-E - consult journal policies, but consider putting data points on these plots (i.e. jitter / stripplot / etc) line 744: \"cured\" -> \"cultured\" ??", "reviewer_4": "I am concerned with the authors' interpretation of the composition and structure of the decidua and the extent to which that matches with Matrigel. In particular, the collagen types present following the endometrium to decidua transition, and their corresponding collagen structures. While type IV collagen is present in the basement membrane (BM), and is a major component of Matrigel, the decidua contains largely non-BM collagens including I, III, V. Type IV is network-forming while types I, III, and V are fibrillar. See references below, noting that these are for human decidua (which is a separate point I will address below). O'Connor BB, et al., The role of extracellular matrix in normal and pathological pregnancy: Future applications of microphysiological systems in reproductive medicine. Exp Biol Med (Maywood). 2020 Jul;245(13):1163-1174. doi: 10.1177/1535370220938741. Epub 2020 Jul 8. PMID: 32640894; PMCID: PMC7400725. Oefner CM, et al., Collagen type IV at the fetal-maternal interface. Placenta. 2015 Jan;36(1):59-68. doi: 10.1016/j.placenta.2014.10.012. Epub 2014 Nov 1. PMID: 25465704; PMCID: PMC4302218. NB: The authors' reference 15, line 111, is referring specifically to a manuscript about the basement membrane, not about the entire decidua or the entire decidual ECM. As noted above, the current study is related to mouse models which undergo a completely different form of decidualization compared with humans. Since mice are estrous cycling and not menstrual cycling, the murine decidua does not undergo anticipatory decidualization. This makes a mouse model less than ideal as a subject for investigation of the authors' current research question, and this severely limits the impact of the work. In terms of the technical content, in lines 112-116 the authors describe measuring Matrigel stiffness and comparing it to decidual stiffness but they relegate this important result to the supplemental figures and don't at all discuss if their measurements are in agreement with others who have measured such things (and found the stiffness of even 100% Matrigel to be significantly lower than the stiffness at the implantation site, Abbas Y, et al., Tissue stiffness at the human maternal-fetal interface. Hum Reprod. 2019 Oct 2;34(10):1999-2008. doi: 10.1093/humrep/dez139. PMID: 31579915; PMCID: PMC6809602.) The methods section of the paper lists AFM, nanoindentation, and rheology measurements of the gels but gives insufficient information for an interested party to recreate the experiments and more importantly completely inadequate details about the data analysis. The authors have a focus on the implications of stiffness and viscoelasticity, in the abstract and throughout the paper, but there is absolutely nothing convincing about their treatment of the topic within the manuscript. Their presented results seem to be focused on microscopy and genetics while making broad statements about the influence of gel mechanics that are simply not supported with the data presented. I'm also extremely skeptical that adding crosslinking agents did not increase the stiffness of the gels, since that is such an extremely well-known phenomenon in many different hydrogel systems and thus again the inadequacy of the presentation of the mechanical methods and results herein is extremely problematic."}, "author_response": "Point-by-point responses to the reviewers’ comments We thank the reviewers for their insightful feedback and valuable suggestions. In response to your and the reviewers’ comments, we have made several significant revisions to the manuscript to further improve the clarity and rigor of our findings. Below is a summary of the major changes that have been made: 1. The title has been changed to: 3D biomimetic niche modulates embryo development in vitro. 2. In response to concerns raised by Reviewers 1, 2, and 3 regarding Figure 3E, we initially performed the experiment five times and repeated it three additional times during the revision process. Our results confirmed the reviewers’ observations that Cre-injected (GFP-expressing) cells showed a partial tendency to contribute to the epiblast of all injections, which hindered proper egg cylinder formation. Based on these findings and in accordance with the recommendation of Reviewer 3 (point 3.6), we have decided to remove this experiment from the manuscript (Fig. 3E, Video S9). 3. Data points have been added to the following plots: Fig. 4E–F, 4K, 5C–D, 5F, 5I–J, 6G–H, 6J–K, 6L–M, S10B, S12A, S12C–F, S13C, and S14D–E. Additionally, for transparency, responses to reviewer comments are indicated in blue in the manuscript, and the corresponding changes made to the text and figures are marked in red."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In their updated manuscript, the authors addressed most of my concerns regarding the scientific content of the manuscript. I will only suggest toning down some of the statements to make it clear that the early organogenesis-stage embryo obtained resemble in part natural embryos, but there are still clear morphological differences. This is properly mentioned at the discussion (line 318). Abstract: Since the organogenesis-stage embryos presented in the paper have remarkable differences when compared to in utero embryos, I suggest slightly rephrasing a part of the abstract to: Our findings reveal that embryos cultured within this hydrogel system successfully progressed to early organogenesis-like stage, including the development of first and second heart fields, mimicking natural embryogenesis. Line 64- I suggest rewriting the sentence to the following: Further, a deep understanding the mechanics between the early uterine decidua and embryonic structure is also lacking. Line 70- There is a missing word in the text, I suggest correcting as follows: These limitations underscore the need FOR a model that can better replicate the complex… Line 129-130: Remove the word anterior: On IVC day 4, the anterior amniotic cavity was formed by the fusion of the ExE and EPI cavities. Lines 137-139: Authors write: \"Notably, the colocalization of OTX2+ and FOXA2+ cells, which are markers for early gut, brain, and notochord development, was observed in both natural and in vitro embryos (Figures 1F, S5B and S5C).\" I consider that this sentence can be removed because in the rebuttal letter the authors mention that there is no co-localization of these markers. \"Regarding the reviewer’s comment on the potential co-localization of Foxa2 and Otx2 in Figure 1F, we would like to clarify that these two markers do not co-localize in our samples. Figure 1F, together with Figures S4B and S4C, shows the same embryo captured from different imaging angles. In Figure S4B, it is clearly shown that Foxa2 and Otx2 occupy distinct regions, and no overlapping expression is observed. We have clarified this point further in the figure legend.\"", "reviewer_3": "This manuscript remains an insightful body of work that meets the high bar for publication in Nature Communications. The authors have carefully re-written and restructured the manuscript so that it is intuitive, presents arguments in logical series, and facilitates understanding and replication of their work. I agree with concerns of the other reviewers, but I do think (both quantitatively and qualitatively) the present study integrates techniques and knowledge to take a much larger step forward that any recent publication -- both in the biophysical interactions of the early embryo and uterus, and how those interactions contribute to embryogenesis. Accordingly, I recommend provisional acceptance of the manuscript at this time. That said, I have a few residual concerns about the current draft of the paper that nonetheless do not deter my enthusiasm and interest in its publication in Nature Communications. 1. Figure S10 and lines 181-198 - The GO analysis identifies metabolism as a key component of gene expression in the various clusters -- providing evidence for the author's assertions. However, Figure S10 is confusing. Are the y and x axis titles swapped? Raw gene counts (if that's what we see in Figure S10) isn't the best metric for scoring a GO term/category, prefer significant/expected or Fisher p-values or KS p-values. Typically GO analyses are listed by the X lowest p-value terms. If there aren't many terms with significant p-values in any given cluster per timepoint, then try grouping clusters or timepoints into larger clusters. If that doesn't work, just leave this data out since it isn't essential to the paper's message. If the data is kept in the paper, the long descriptions in lines 181 to 198 could be moved to supplement or paraphrased for the main paper text. 2. I understand that Figure R1 is beyond the scope of the paper and will be published side-by-side in Peer Review File, but the authors' technical success is a great strength that is worth explicitly mentioning on some level in the main paper. Suggest one or two sentences in the first paragraph of Discussion section to highlight the impact of this study, possibly incorporating data presented in Response to Reviewers 1.1. 3. Lines 105-106. The description begins with step 1 of the 3d culture system (PDMS application) then defers remaining explanation to Figure S1E. It sounds like plasma-treating PDMS is the only thing necessary for the 3D culture system. Suggest removing line 105 and 106, or alternatively summarizing Figure S1E more comprehensively. 4. Figures 4G-I, 6I, S3A, S5A-B, S15A-B - These microscopy images would benefit from a little more contrast. 5. Line 199, remove \"a\"", "reviewer_4": "My views have not changed since the previous review. The methodology and analysis of the mechanical data are muddle and suspect, and the authors have not improved things with their response and edits. They are relying on a factor of two difference in the time constant to explain a great deal, and there is just not data that supports that such a small change is relevant. Further, the time constant is not the only thing that changes when the time-dependent response changes, there are also potentially changes in the time-dependent extent. Given the changes the authors have made with crosslinking the hydrogels, this would be expected to be a major factor: gels that are covalently crosslinked are more elastic and relax to a lesser extent than gels that are physically crosslinked. But the fundamental thing that still does not make sense about the data is why covalent crosslinking did not also result in increases in the elastic modulus of the gels, which really reinforces my suspicions about the mechanical results. (There are also methodological details that are inconsistent with the text and figure captions especially, which further make me question the authors' understanding of the mechanical data.). As such, although this is interesting, I cannot recommend it for publication as the authors still have mechanical explanations as the primary outcome of the study, but data that do not support their interpretations."}, "author_response": "Point-by-point responses to the reviewers’ comments We thank the reviewers for their insightful feedback and valuable suggestions. In response to the editor’s and reviewers’ comments, we have undertaken several key revisions to enhance the clarity, rigor, and completeness of the manuscript. Below is a summary of the major changes: 1. Mechanical characterization strengthened: In response to Reviewer #4, we clarified the viscoelastic modeling approach, provided raw and normalized stress relaxation data, and added microstructural SEM and rheological analyses. We also revised figure captions and methods for consistency. 2. Figure 3E removed: As suggested by Reviewers #1–3, and based on additional experimental validation, we have removed Fig. 3E and the associated video due to observed variability in Cre-injected embryo contributions. 3. Data transparency improved: We added data points to key figures (e.g., Fig. 4E–F, 5C–D, 6G–H, S10B, S12A, etc.) to ensure full quantitative reporting. 4. Textual and interpretive clarifications: Minor textual edits were made to improve grammar, tone down biological claims, and clarify experimental design and marker usage. 5. GO analysis removed: As per Reviewer #3’s recommendation, we removed Figure S10 and associated text due to statistical limitations in the GO term enrichment results. Additionally, for transparency, responses to reviewer comments are indicated in blue in the manuscript, and the corresponding changes made to the text and figures are marked in red in the manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_5": "As requested by the editor, I am focusing my review on the response to Reviewer #4. While I share Reviewer #4's surprise about the relatively small change in time constant affecting the results so dramatically, I agree that in principle, biological outcomes could be impacted by very small changes in matrix mechanics and that we should not assume how sensitive the biological system should be to mechanics a priori. The data presented in this work adds to the growing body of evidence supporting this degree of sensitivity. I also found the author's responses regarding the improper use of rubber elasticity theory to construct expectations for hydrogel mechanics to be quite compelling, and the examples provided support the idea that we should not assume rubber-like models for these materials. Given the evidence provided and the description of the methods and results, I believe that the authors have adequately and reasonably responded to the reviewers' (valid and very important) concerns."}, "author_response": ""}]

Decision letter