Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages tend to be slower and generate target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors (primed ground state conditions) as a starting point for directed differentiation. As a proof-of-concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. First, we developed a new protocol that can rapidly generate nearly pure definitive endoderm from EpiSCs. Second, we developed a protocol for generating forebrain organoids that model the development of prethalamic and hippocampal neurons. These significantly improved differentiation models present new possibilities for combining mouse genetic tools and resources with in vitro differentiation to characterize the molecular and cellular mechanisms of embryonic development.

YAP creates epiblast responsiveness to inductive signals for germ cell fate

The germ cell lineage in mammals is induced by the stimulation of pluripotent epiblast cells with signaling molecules. Previous studies have suggested that the germ cell differentiation competence or responsiveness of epiblast cells to signaling molecules is established and maintained in epiblast cells of a specific differentiation state. However, the molecular mechanism underlying this process has not been well defined. Here, using the differentiation model of epiblast stem cells (EpiSCs), we have shown that two defined EpiSC lines have robust germ cell differentiation competence. However, another defined EpiSC line has no competence. By evaluating the molecular basis of EpiSCs with distinct germ cell differentiation competence, we identified YAP/YAP1/YAP65, an intracellular mediator of the Hippo signaling pathway, as a critical mediator for establishing germ cell induction. Strikingly, deletion of YAP severely affected responsiveness to inductive stimuli, leading to a defect in WNT target activation and germ cell differentiation. In conclusion, we propose that the Hippo/YAP signaling pathway creates a potential for germ cell fate induction via mesodermal WNT signaling in pluripotent epiblast cells.

SmcHD1 underlies the formation of H3K9me3 blocks on the inactive X chromosome in mice

Stable silencing of the inactive X chromosome (Xi) in female mammals is critical for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher order chromatin structure of the Xi, the underlying mechanism is largely obscure. Here we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells (EpiSCs) and mouse embryonic fibroblasts (MEFs) in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a critical role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.

Derivation of Mouse Parthenogenetic Advanced Stem Cells

Parthenogenetic embryos have been widely studied as an effective tool related to paternal and maternal imprinting genes and reproductive problems for a long time. In this study, we established a parthenogenetic epiblast-like stem cell line through culturing parthenogenetic diploid blastocysts in a chemically defined medium containing activin A and bFGF named paAFSCs. The paAFSCs expressed pluripotent marker genes and germ-layer-related genes, as well as being alkaline-phosphatase-positive, which is similar to epiblast stem cells (EpiSCs). We previously showed that advanced embryonic stem cells (ASCs) represent hypermethylated naive pluripotent embryonic stem cells (ESCs). Here, we converted paAFSCs to ASCs by replacing bFGF with bone morphogenetic protein 4 (BMP4), CHIR99021, and leukemia inhibitory factor (LIF) in a culture medium, and we obtained parthenogenetic advanced stem cells (paASCs). The paASCs showed similar morphology with ESCs and also displayed a stronger developmental potential than paAFSCs in vivo by producing chimaeras. Our study demonstrates that maternal genes could support parthenogenetic EpiSCs derived from blastocysts and also have the potential to convert primed state paAFSCs to naive state paASCs.

Establishment of Mouse Primed Stem Cells by Combination of Activin and LIF Signaling

In mice, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are established from pre- and post-implantation embryos and represent the naive and primed state, respectively. Herein we used mouse leukemia inhibitory factor (LIF), which supports ESCs self-renewal and Activin A (Act A), which is the main factor in maintaining EpiSCs in post-implantation epiblast cultures, to derive a primed stem cell line named ALSCs. Like EpiSCs, ALSCs express key pluripotent genes Oct4, Sox2, and Nanog; one X chromosome was inactivated; and the cells failed to contribute to chimera formation in vivo. Notably, compared to EpiSCs, ALSCs efficiently reversed to ESCs (rESCs) on activation of Wnt signaling. Moreover, we also discovered that culturing EpiSCs in AL medium for several passages favored Wnt signaling-driven naive pluripotency. Our results show that ALSCs is a primed state stem cell and represents a simple model to study the control of pluripotency fate and conversion from the primed to the naive state.

Inhibition of N-myristoyltransferase Promotes Naive Pluripotency in Mouse and Human Pluripotent Stem Cells

Naive and primed states are distinct states of pluripotency during early embryonic development that can be captured and converted to each other in vitro. To elucidate the regulatory mechanism of pluripotency, we performed a recessive genetic screen of homozygous mutant mouse embryonic stem cells (mESCs) and found that suppression of N-myristoyltransferase (Nmt) promotes naive pluripotency. Disruption of Nmt1 in mESCs conferred resistance to differentiation. Suppression of Nmt in mouse epiblast stem cells (mEpiSCs) promoted the conversion from the primed to the naive state. This effect was independent of Src, which is a major substrate of Nmt and is known to promote differentiation of mESCs. Suppression of Nmt in naive-state human induced pluripotent stem cells (hiPSCs) increased the expression of the naive-state marker. These results indicate that Nmt is a novel target for the regulation of naive pluripotency conserved between mice and humans.