Formation of synthetic blastocysts by self-organization of human naïve pluripotent stem cells

Blastocyst formation evolved uniquely in mammals to enable uterine implantation. The blastocyst comprises three founding tissues, the epiblast, trophectoderm and hypoblast (primitive endoderm in mice). Epiblast is the origin of the embryo proper and the source of pluripotent embryonic stem cells. Trophectoderm and hypoblast give rise to extra-embryonic tissues, the placenta and yolk sac,that support embryo development in the uterus. How blastocysts form three distinct and precisely arranged tissues is a fascinating fundamental biology question. Furthermore, understanding blastocyst development in humans will have applications in reproductive medicine.

Research on human blastocysts is limited by availability of good quality IVF embryos and ethical considerations. Therefore our concept of human embryo development draws heavily on mouse studies. However, although mouse and human blastocysts appear similar, they differ in at least one fundamental aspect. We recently discovered that, unlike in mouse, human epiblast cells can regenerate authentic trophectoderm1. This plasticity is maintained in human naïve pluripotent stem cells, which are also able to produce primitive endoderm. Therefore human naïve pluripotent stem cells have a unique potency to reconstitute an intact embryo.

Based on these findings, we aim in this project to establish the first synthetic human blastocyst model. Using chemical and growth factor signals we will direct formation of the three lineages from naïve stem cells and steer self-organization into structures that accurately recapitulate the composition, organization and functional properties of human blastocysts. We will apply bioengineering approaches to optimize robustness and throughput for blastocyst modeling. We establish the potential of the synthetic blastocysts for development up to gastrulation. We will then explore early human embryo developmental dynamics using molecular and chemical genetics in conjunction with high-resolution time-lapse imaging.

The human synthetic blastocyst we develop will transform access to early human development for fundamental research, providing a highly reproducible and authentic model amenable to any type of experimental and analytical dissection. Model blastocysts developed from naïve induced pluripotent stem cells will also have important applications in directing stem cell differentiation for disease modeling and in investigation of the genetic basis of human infertility. Finally, we expect that knowledge gained from development of model blastocysts and evaluation of their developmental potential will enable improvement of IVF embryo quality for assisted reproduction technology

The primary goal of this project is to develop a robust experimental system for production of high quality synthetic human blastocyst embryos. This is based on our recent discovery that human naive stem cells have unique trophectoderm differentiation potential. As a proof of principle we showed in our pilot experiment that human blastocyst like structure could be generated solely with human naïve stem cells. The blastocyst like structure mimics the morphology of human embryos and contains the three founding cell lineages. We have two main objectives to achieve in this proposal. Firstly, we will optimize conditions and procedures to build a robust and scalable protocol. Secondly, we will thoroughly validate the function of the blastocyst model. In the later phase we will extend our primary research goals to study cellular and molecular dynamics of the formation of the synthetic embryos. We have validated a GATA3:mKO2 reporter line to track trophectoderm lineages. This permits fast screens for factors that could influence on the formation and localization of the trophectoderm. We will generate a SOX17/GATA3 dual reporter to screen for conditions for hypoblast formation. We will implement a microfluidic platform to build an automated process for cell handling, medium change, and imaging. To validate the synthetic blastocyst model we will quantify three lineages by localization of key transcription factors expression. We will determine whole transcriptome identity and relative representation of each cell type by single cell RNA sequencing. We will culture synthetic embryos to examine peri- and post-implantation development potential. Finally we will use our lineage specific fluorescence reporters and time-lapse microscopy to track the developmental dynamics. We will further study the development trajectory by single cell RNA sequencing. Pilot gene perturbation studies of candidate regulators will also be explored.