Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.

Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple sgRNA delivery systems.

Spatial transcriptomic characterization of a Carnegie stage 7 human embryo.

Gastrulation marks a pivotal stage in mammalian embryonic development, establishing the three germ layers and body axis through lineage diversification and morphogenetic movements. However, studying human gastrulating embryos is challenging due to limited access to early tissues. Here we show the use of spatial transcriptomics to analyse a fully intact Carnegie stage 7 human embryo at single-cell resolution, along with immunofluorescence validations in a second embryo.

3D reconstruction of a gastrulating human embryo.

Progress in understanding early human development has been impeded by the scarcity of reference datasets from natural embryos, particularly those with spatial information during crucial stages like gastrulation. We conducted high-resolution spatial transcriptomics profiling on 38,562 spots from 62 transverse sections of an intact Carnegie stage (CS) 8 human embryo. From this spatial transcriptomic dataset, we constructed a 3D model of the CS8 embryo, in which a range of cell subtypes are identified, based on gene expression patterns and positional register, along the anterior-posterior, medial-lateral, and dorsal-ventral axis in the embryo.

Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture.

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-β, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts.

Derivation and Primordial Germ Cell Induction of Intermediate Pluripotent Stem Cells.

Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of the pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. By modulating the FGF, TGF-β, and WNT pathways, we have derived intermediate PSCs (FTW-PSCs) that are permissive for direct primordial germ cell-like cell (PGC-LC) induction in vitro.

Generation of Human Blastoids from Naive Pluripotent Stem Cells.

Under certain culture conditions, naive human pluripotent stem cells can generate human blastocyst-like structures (called human blastoids). Human blastoids serve as an accessible model for human blastocysts and are amenable for large-scale production. Here, we describe a detailed step-by-step protocol for the robust and high-efficient generation of human blastoids from naive human pluripotent stem cells.

An aggregation of human embryonic and trophoblast stem cells reveals the role of trophectoderm on epiblast differentiation.

The interactions between extra-embryonic tissues and embryonic tissues are crucial to ensure proper early embryo development. However, the understanding of the crosstalk between the embryonic tissues and extra-embryonic tissues is lacking, mainly due to ethical restrictions, difficulties in obtaining natural human embryos, and lack of appropriate in vitro models. Here by aggregating human embryonic stem cells (hESCs) with human trophoblast stem cells (hTSCs), we revealed the hESCs robustly self-organized into a unique asymmetric structure which the primitive streak (PS) like cells exclusively distributed at the distal end to the TS-compartment, and morphologically flattened cells, presumed to be the extra-embryonic mesoderm cells (EXMC) like cells, were induced at the proximal end to hTSCs.

Establishment of bovine trophoblast stem cells.

Here, we report that a chemical cocktail (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimethinedene maleate [DiM], minocycline hydrochloride), previously developed for extended pluripotent stem cells (EPSCs) in mice and humans, enables de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model to study bovine placentation and early pregnancy failure.

Bovine blastocyst-like structures derived from stem cell cultures.

Understanding the mechanisms of blastocyst formation and implantation is critical for improving farm animal reproduction but is hampered by a limited supply of embryos. Here, we developed an efficient method to generate bovine blastocyst-like structures (termed blastoids) via assembling bovine trophoblast stem cells and expanded potential stem cells. Bovine blastoids resemble blastocysts in morphology, cell composition, single-cell transcriptomes, in vitro growth, and the ability to elicit maternal recognition of pregnancy following transfer to recipient cows.

Dissecting embryonic and extra-embryonic lineage crosstalk with stem cell co-culture.

Faithful embryogenesis requires precise coordination between embryonic and extraembryonic tissues. Although stem cells from embryonic and extraembryonic origins have been generated for several mammalian species(Bogliotti et al., 2018; Choi et al.

Epiblast-like stem cells established by Wnt/β-catenin signaling manifest distinct features of formative pluripotency and germline competence.

Different formative pluripotent stem cells harboring similar functional properties have been recently established to be lineage neutral and germline competent yet have distinct molecular identities. Here, we show that WNT/β-catenin signaling activation sustains transient mouse epiblast-like cells as epiblast-like stem cells (EpiLSCs). EpiLSCs display metastable formative pluripotency with bivalent cellular energy metabolism and unique transcriptomic features and chromatin accessibility.

Blastocyst-like structures generated from human pluripotent stem cells.

Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages.

Cross-species single-cell transcriptomic analysis reveals pre-gastrulation developmental differences among pigs, monkeys, and humans.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogeneic pluripotent stem cell (PSC) derivatives, which raises an intriguing possibility to generate functional human tissues/organs in an animal host. However, differences in embryo development between human and host species may constitute the barrier for efficient chimera formation. Here, to understand these differences we constructed a complete single-cell landscape of early embryonic development of pig, which is considered one of the best host species for human organ generation, and systematically compared its epiblast development with that of human and monkey.

Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring.

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz.

Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures.

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization.

Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells.

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries.