Stem-cell-based embryo models for fundamental research and translation

Abstract

Despite its importance, understanding the early phases of human development has been limited by availability of human samples. The recent emergence of stem-cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This Review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem-cell-derived embryo models for fundamental discovery and translation.

Figure 1.. Overview of mouse and human development from pre-implantation to the onset of gastrulation.

Prior to implantation, both mouse and human embryos undergo cell divisions culminating in the development of a blastocyst, comprising an outer trophectoderm (TE) layer and an inner cell mass (ICM) that further segregates into epiblast (EPI) and primitive endoderm (PE; hypoblast in humans). The timing of blastocyst implantation differs between mice and humans (E5 in mouse and E7 in human). Furthermore, morphogenesis and lineage developments during peri-implantation mouse and human development show distinct features. Mouse development from E5 - E6.5 leads to the formation of a cup-shaped EPI juxtaposed with TE-derived extraembryonic ectoderm (ExE), enclosing the pro-amniotic cavity. Concurrently, the PE forms the visceral endoderm (VE) that envelops both EPI and ExE. In contrast, soon after human blastocyst implantation, while the EPI undergoes lumenogenesis to form the pro-amniotic cavity, EPI cells adjacent to polar TE cells become specified into the amniotic ectoderm (AM), with remaining pluripotent EPI cells forming the embryonic disc. By E6.5 for mice and E14 for humans, gastrulation is initiated in the posterior EPI compartment. Mouse primordial germ cells (PGCs) emerge at the boundary between posterior EPI and ExE at the onset of gastrulation. Data on primate PGC specification remain sparse. Existing data suggest that human PGCs may emerge in the nascent AM prior to the gastrulation. Human PGC specification requires additional studies for clarification. For peri-implantation mouse and human embryos, only their embryonic regions are shown.

Figure 2.. Mouse and human embryonic and extraembryonic stem cells and their corresponding developmental potencies.

Expanded potential stem cells (EPSCs) are established by isolating individual cells (or blastomeres) from eight-cell stage mouse and human embryos (or morula). By isolating cells from mouse blastocysts, mouse embryonic stem cells (mESCs) with naïve pluripotency, trophoblast stem cells (mTSCs), and extraembryonic endoderm (mXEN) cells representing the stem cell population of the primitive endoderm (PE) have been established. Similarly, human trophoblast stem cells (hTSCs) have been derived from human blastocyst. Primed mouse ESCs, known as mouse epiblast stem cells (mEpiSCs), are derived from the late post-implantation, pre-gastrulation mouse epiblast (EPI). Mouse EPI-like cells (EpiLCs) with an intermediate or formative state between naïve and primed pluripotency have been generated from mESCs in vitro, with a transcriptional profile similar to the early post-implantation mouse EPI. Human ESCs (hESCs) with primed pluripotency have also been derived from pre-implantation human blastocysts. Using strategies such as reprogramming, differentiated somatic mouse and human cells can be converted to a pluripotent state to establish induced pluripotent stem cells, or iPSCs. Using chemical cocktails, primed hESCs can be reverted into a naïve-like pluripotent state. Human hypoblast stem cells (hypoSCs) can be generated using these chemically reset naïve hESC lines.

Figure 3.. Existing embryoids that recapitulate different stages of mouse (top) and human development (bottom), from pre-implantation through gastrulation or early neurulation and organogenesis.

3D blastoid: Embryoid to model pre-implantation blastocyst development. 3D ETX embryoid: Embryoid to model post-implantation embryo development up to early gastrulation. 3D gastruloid and trunk-like structure: Embryoid to model post-gastrulation development of the posterior portion of the embryo. 2D gastrulation model: Embryoid to model germ layer patterning during gastrulation. 3D epiblast patterning model: Embryoid to model epiblast morphogenesis and patterning during early post-implantation development. 3D post-implantation amniotic sac embryoid (PASE): Embryoid to model post-implantation human development up to early gastrulation. 2D neurulation model: Embryoid to model the neurulation process, leading to neural tube development and patterning.

Figure 4.. Bioengineering tools to promote multicellular interaction and self-organization in embryoid development.

(a) Micropatterning to generate 2D circular colonies of hPSCs to model germ layer patterning during gastrulation. Immunofluorescence image shows emergence of concentric gene expression regions, mimicking development of the germ layers (SOX2+ ectoderm, blue; TBXT+ mesendoderm, red) as well as a GATA3+ extraembryonic layer (green). Image from A. Yoney and E.D. Siggia. (b) Microwell array to promote cell aggregation and development of mouse blastoids. Top: Microwell arrays composed of agarose hydrogels to promote aggression of mESCs and mTSCs. Bottom: Merged image showing two blastoids, with a layer of mTSCs surrounding a cavity and a cluster of mESCs mimicking the inner cell mass. Immunostaining: NANOG (red) and GATA6 (green). Images from N. Rivron. (c) Microfluidics to control spatiotemporal morphogen signaling and tissue patterning. Bright-field (top) and immunofluorescence (bottom) images of an array of post-implantation amniotic sac embryoids (PASEs), showing molecular asymmetry and tissue patterning, with TFAP2A+ amniotic cells on one pole (green) and TBXT+ gastrulating cells (magenta) on the opposite pole. Images from Y. Zheng. (d) Chemically and physically defined hydrogels for 3D embryoid development. Immunofluorescence image of a 3D human gastrulation embryoid for modeling epiblast morphogenesis and patterning (SOX2, green; TBXT, magenta). Image from M. Simunovic.