ESRRB Facilitates the Conversion of Trophoblast-Like Stem Cells From Induced Pluripotent Stem Cells by Directly Regulating CDX2

Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.

MSX2 safeguards syncytiotrophoblast fate of human trophoblast stem cells

Multiple placental pathologies are associated with failures in trophoblast differentiation, yet the underlying transcriptional regulation is poorly understood. Here, we discovered msh homeobox 2 (MSX2) as a key transcriptional regulator of trophoblast identity using the human trophoblast stem cell model. Depletion of MSX2 resulted in activation of the syncytiotrophoblast transcriptional program, while forced expression of MSX2 blocked it. We demonstrated that a large proportion of the affected genes were directly bound and regulated by MSX2 and identified components of the SWItch/Sucrose nonfermentable (SWI/SNF) complex as strong MSX2 interactors and target gene cobinders. MSX2 cooperated specifically with the SWI/SNF canonical BAF (cBAF) subcomplex and cooccupied, together with H3K27ac, a number of differentiation genes. Increased H3K27ac and cBAF occupancy upon MSX2 depletion imply that MSX2 prevents premature syncytiotrophoblast differentiation. Our findings established MSX2 as a repressor of the syncytiotrophoblast lineage and demonstrated its pivotal role in cell fate decisions that govern human placental development and disease.

Bioengineered embryoids mimic post-implantation development in vitro

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

Single cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment

The establishment and function of the human placenta is dependent on specialized cells called trophoblasts. Progenitor cytotrophoblasts (CTBs) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTBs committed to the villous pathway fuse with neighboring CTBs forming the outer multinucleated syncytiotrophoblast layer (SCT), while CTBs committed to the extravillous pathway develop into multi-layered cell columns that anchor the placenta to uterine tissue. At distal column sites, column CTBs differentiate into invasive extravillous trophoblasts (EVT) that facilitate uterine remodeling of spiral arteries and modulate the maternal immune response to fetal antigen. Unfortunately, little is known about the cellular and molecular processes controlling human trophoblast stem cell maintenance and differentiation into these critical trophoblast sub-lineages. To address this, our laboratory established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify molecular programs and cell states important in trophoblast progenitor establishment, renewal, and differentiation. Using this dataset, eight distinct trophoblast states were identified, representing progenitor cytotrophoblasts, intermediate column CTBs, syncytiotrophoblast progenitors, and terminally differentiated EVT. Lineage trajectory analysis identified a CTB origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where CTBs selected for high levels of surface BCAM expression generated larger clonally-derived organoids than did CTB counterparts expressing lower levels of BCAM. Together, this work resolves complex gene networks within human trophoblast progenitor cells, and identifies BCAM as marker that defines an up-stream progenitor origin.

Histone demethylase JMJD2B/KDM4B regulates transcriptional program via distinctive epigenetic targets and protein interactors for the maintenance of trophoblast stem cells

Trophoblast stem cell (TSC) is crucial to the formation of placenta in mammals. Histone demethylase JMJD2 (also known as KDM4) family proteins have been previously shown to support self-renewal and differentiation of stem cells. However, their roles in the context of the trophoblast lineage remain unclear. Here, we find that knockdown of Jmjd2b resulted in differentiation of TSCs, suggesting an indispensable role of JMJD2B/KDM4B in maintaining the stemness. Through the integration of transcriptome and ChIP-seq profiling data, we show that JMJD2B is associated with a loss of H3K36me3 in a subset of embryonic lineage genes which are marked by H3K9me3 for stable repression. By characterizing the JMJD2B binding motifs and other transcription factor binding datasets, we discover that JMJD2B forms a protein complex with AP-2 family transcription factor TFAP2C and histone demethylase LSD1. The JMJD2B–TFAP2C–LSD1 complex predominantly occupies active gene promoters, whereas the TFAP2C–LSD1 complex is located at putative enhancers, suggesting that these proteins mediate enhancer–promoter interaction for gene regulation. We conclude that JMJD2B is vital to the TSC transcriptional program and safeguards the trophoblast cell fate via distinctive protein interactors and epigenetic targets.