Mammalian embryos develop inside the uterine environment, making them inaccessible for direct monitoring and perturbations. Therefore, the full in-depth understanding of the developmental process that occurs post embryo implantation in the uterus remains limited. The lab of Prof. Jacob Hanna recently developed a culture system that enables growing mouse embryos from pre-gastrulation to advanced organogenesis stages, in a manner that faithfully recapitulates the normal in-uterus developmental process. This provides a powerful tool to characterize the effect of different perturbations on early mammalian development that can be combined with genetic modification, chemical screens, tissue manipulation, and microscopy methods. In the future, the system could also be used to study the development of additional mammalian species, and possibly provide a tool for the development of organ cultures for biomedical purposes.
The establishment of the mammalian body plan occurs shortly after the embryo implants into the maternal uterus, making it inaccessible for direct monitoring and perturbations. Therefore, our understanding of post-implantation developmental processes remains limited. While methods for in vitro culture of pre-implantation mouse embryos are routinely utilized, in vitro culturing of post-implantation embryos is very challenging. The currently available platforms remain highly inefficient and can only be used for limited short periods, as the embryos begin to display developmental anomalies as early as 24 hours after culture initiation. Therefore, there is a need for stable and efficient platforms for extended culturing of pre-gastrulating mouse embryos until advanced organogenesis stages.
The group of Prof. Jacob Hanna has developed highly conducive ex utero post-implantation mouse embryo culture platforms that enable appropriate development of embryos before gastrulation (Embryonic day [E]5.5) until the hind limb formation stage (E11).1
The embryos are grown in specific growth conditions that include a unique medium composition and a tightly controlled electronic gas regulation module, which was designed by the team to allow precise control not only of O2 and CO2 levels with high sensitivity but also control of the atmospheric pressure (Figure 1a, b). Extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of novel static and rotating bottle culture platforms (Figure 2). Late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings (Figure 1c-f). To ensure that the ex utero cultured embryos recapitulate in utero development precisely, extensive histological, molecular, and single-cell RNA-seq analyses were performed. In utero and ex utero-grown embryos were equivalent morphologically and in the expression of all lineage markers analyzed (Figure 1, 2). Importantly, this culture system is amenable to introducing a variety of embryonic perturbations and micro-manipulations that can be followed ex utero for up to six days. This was demonstrated by successful whole-embryo electroporation of a fluorescent marker, as well as microinjection of lentivirus harboring an EGFP gene (Figure 3). Finally, chimeric mouse embryos were obtained after microinjection of primed pluripotent stem cells to ex utero embryos. Therefore, this system represents a valuable tool to investigate embryogenesis, eliminating the uterine barrier to mechanistically interrogate post-implantation morphogenesis and tissue specification in mammals, providing an unprecedented view of how mammalian organs and limbs form—a process previously hidden inside a mother’s body.
Figure 1 – Ex utero culture system for growing mouse late-gastrulating embryos until advanced organogenesis. a, Schematic of the E7.5 embryo ex utero culture platform. b, Electronic gas and pressure regulation module connected to the roller culture incubator system. c, Bright-field images of embryos developing in utero from E7.5 to E11.5 and equivalent embryos cultured ex utero. d, Percentage of developmentally normal embryos per culture day. “n”, total number of embryos; “x”, number of experiments. e, Quantification of embryonic length for in utero and cultured embryos. Dots represent individual embryos; f. Sox2 and sox9 whole mount immunofluorescence of embryos grown ex utero from E7.5.
Figure 2 – ex utero culture system for growing mouse pre-gastrulation embryos until advanced organogenesis. a, A schematic representation of the protocol for culturing mouse embryos from pre-gastrulation to organogenesis. b, Comparative scRNA-seq analysis of E6.5 +Day 4 ex utero embryos (green dots) and equivalent E10.5 embryos developing in utero (purple dots). UMAP plot depicting all cells considered in the analysis (n = 39374 ex utero; n = 24107 in utero). c, Cell lineage annotation of clusters based on the expression of marker genes described in the mouse organogenesis cell atlas. Points are colored according to their assigned cell cluster.
- A powerful tool for developmental biology labs.
- Potentially could be developed as a research tool for the emerging field of synthetic human embryos.
- Long-term applications could include an analytical tool in IVF labs to assess embryo quality and development of organ cultures for Tissue engineering.
The platforms for ex utero embryo culture were fully developed and characterized by the team, proving equivalent development of these embryos to normal embryonic development, and demonstrating the potential use of this method to mechanistically interrogate post-implantation morphogenesis and tissue specification in mammals.
Aguilera-Castrejon A, Oldak B, Shani T, et al. Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis. Nature. Published online March 17, 2021. doi:10.1038/s41586-021-03416-3