Trophoblast Stem Cells Research Articles

SP6 controls human cytotrophoblast fate decisions and trophoblast stem cell establishment by targeting MSX2 regulatory elements

The commitment and differentiation of human placental progenitor cytotrophoblast (CT) cells are crucial for a successful pregnancy, but the underlying mechanism remains poorly understood. Here, we identified the transcription factor (TF), specificity protein 6 (SP6), as a human species-specific trophoblast lineage TF expressed in human placental CT cells. Using pluripotent stem cells as a model, we demonstrated that SP6 controls CT generation and the establishment of trophoblast stem cells (TSCs) and identified msh homeobox 2 (MSX2) as the downstream effector in these events. Mechanistically, we showed that SP6 interacts with histone acetyltransferase P300 to alter the landscape of H3K27ac at targeted regulatory elements, thereby favoring transcriptional activation and facilitating CT cell fate decisions and TSC maintenance. Our results established SP6 as a regulator of the human trophoblast lineage and implied its role in placental development and the pathogenies of placental diseases.

An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells.

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.

Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development

Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.

Human trophoblast stem cells restrict human cytomegalovirus replication.

Placental infection plays a central role in human cytomegalovirus (HCMV) pathogenesis during pregnancy, but the species specificity of HCMV and the limited availability and lifespan of primary trophoblasts have been persistent barriers to understanding how infection impacts this vital organ. Human trophoblast stem cells (TSCs) represent a new approach to modeling viral infection early in placental development. This study reveals that TSCs, like other stem cell types, restrict HCMV replication. However, infection perturbs the expression of genes involved in differentiation and cell fate determination, pointing to a mechanism by which HCMV could cause placental injury.

Quiescence enables unrestricted cell fate in naive embryonic stem cells

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation

The placenta serves as an essential organ for fetal growth throughout pregnancy. Histone modification is a crucial regulatory mechanism involved in numerous biological processes and development. Nevertheless, there remains a significant gap in our understanding regarding the epigenetic regulations that influence trophoblast lineage differentiation, a fundamental aspect of placental development. Here, through comprehensive mapping of H3K4me3, H3K27me3, H3K9me3, and H3K27ac loci during the differentiation of trophoblast stem cells (TSCs) into syncytiotrophoblasts (STs) and extravillous trophoblasts (EVTs), we reveal dynamic reconfiguration in H3K4me3 and H3K27ac patterns that establish an epigenetic landscape conducive to proper trophoblast lineage differentiation. We observe that broad H3K4me3 domains are associated with trophoblast lineage-specific gene expression. Unlike embryonic stem cells, TSCs lack robust bivalent domains. Notably, the repression of ST- and EVT-active genes in TSCs is primarily attributed to the weak H3K4me3 signal rather than bivalent domains. We also unveil the inactivation of TSC enhancers precedes the activation of ST enhancers during ST formation. Our results provide a comprehensive global map of diverse histone modifications, elucidating the dynamic histone modifications during trophoblast lineage differentiation.

TBX3 reciprocally controls key trophoblast lineage decisions in villi during human placenta development in the first trimester

Human trophoblastic lineage development is intertwined with placental development and pregnancy outcomes, but the regulatory mechanisms underpinning this process remain inadequately understood. In this study, based on single-nuclei RNA sequencing (snRNA-seq) analysis of the human early maternal-fetal interface, we compared the gene expression pattern of trophoblast at different developmental stages. Our findings reveal a predominant upregulation of TBX3 during the transition from villous cytotrophoblast (VCT) to syncytiotrophoblast (SCT), but downregulation of TBX3 as VCT progresses into extravillous trophoblast cells (EVT). Immunofluorescence analysis verified the primary expression of TBX3 in SCT, partial expression in MKi67-positive VCT, and absence in HLA-G-positive EVT, consistent with our snRNA-seq results. Using immortalized trophoblastic cell lines (BeWo and HTR8/SVneo) and human primary trophoblast stem cells (hTSCs), we observed that TBX3 knockdown impedes SCT formation through RAS-MAPK signaling, while TBX3 overexpression disrupts the cytoskeleton structure of EVT and hinders EVT differentiation by suppressing FAK signaling.In conclusion, our study suggests that the spatiotemporal expression of TBX3 plays a critical role in regulating trophoblastic lineage development via distinct signaling pathways. This underscores TBX3 as a key determinant during hemochorial placental development.

Single-cell assessment of primary and stem cell-derived human trophoblast organoids as placenta-modeling platforms

Human trophoblast stem cells (hTSCs) and related trophoblast organoids are state-of-the-art culture systems that facilitate the study of trophoblast development and human placentation. Using single-cell transcriptomics, we evaluate how organoids derived from freshly isolated first-trimester trophoblasts or from established hTSC cell lines reproduce developmental cell trajectories and transcriptional regulatory processes defined invivo. Although organoids from primary trophoblasts and hTSCs overall model trophoblast differentiation with accuracy, specific features related to trophoblast composition, trophoblast differentiation, and transcriptional drivers of trophoblast development show levels of misalignment. This is best illustrated by the identification of an expanded progenitor state in stem cell-derived organoids that is nearly absent invivo and transcriptionally shares both villous cytotrophoblast and extravillous trophoblast characteristics. Together, this work provides a comprehensive resource that identifies strengths and limitations of current trophoblast organoid platforms.

The GATA transcriptional program dictates cell fate equilibrium to establish the maternal–fetal exchange interface and fetal development

The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors, and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of transcription factors GATA2 and GATA3 in labyrinth trophoblast progenitors (LaTPs) have underdeveloped placenta and die by ~embryonic day 9.5. Single-cell RNA sequencing analysis revealed excessive accumulation of multipotent LaTPs upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate into matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promotes trophoblast progenitor-to-SynT commitment, ensuring establishment of the maternal-fetal exchange interface.

The transcriptional regulatory network modulating human trophoblast stem cells to extravillous trophoblast differentiation

During human pregnancy, extravillous trophoblasts play crucial roles in placental invasion into the maternal decidua and spiral artery remodeling. However, regulatory factors and their action mechanisms modulating human extravillous trophoblast specification have been unknown. By analyzing dynamic changes in transcriptome and enhancer profile during human trophoblast stem cell to extravillous trophoblast differentiation, we define stage-specific regulators, including an early-stage transcription factor, TFAP2C, and multiple late-stage transcription factors. Loss-of-function studies confirm the requirement of all transcription factors identified for adequate differentiation, and we reveal that the dynamic changes in the levels of TFAP2C are essential. Notably, TFAP2C pre-occupies the regulatory elements of the inactive extravillous trophoblast-active genes during the early stage of differentiation, and the late-stage transcription factors directly activate extravillous trophoblast-active genes, including themselves as differentiation further progresses, suggesting sequential actions of transcription factors assuring differentiation. Our results reveal stage-specific transcription factors and their inter-connected regulatory mechanisms modulating extravillous trophoblast differentiation, providing a framework for understanding early human placentation and placenta-related complications.

Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFβ), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)—a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.

Trophoblast stem cell-based organoid models of the human placental barrier

Human placental villi have essential roles in producing hormones, mediating nutrient and waste exchange, and protecting the fetus from exposure to xenobiotics. Human trophoblast organoids that recapitulate the structure of villi could provide an important in vitro tool to understand placental development and the transplacental passage of xenobiotics. However, such organoids do not currently exist. Here we describe the generation of trophoblast organoids using human trophoblast stem (TS) cells. Following treatment with three kinds of culture medium, TS cells form spherical organoids with a single outer layer of syncytiotrophoblast (ST) cells that display a barrier function. Furthermore, we develop a column-type ST barrier model based on the culture condition of the trophoblast organoids. The bottom membrane of the column is almost entirely covered with syndecan 1-positive ST cells. The barrier integrity and maturation levels of the model are confirmed by measuring transepithelial/transendothelial electrical resistance (TEER) and the amount of human chorionic gonadotropin. Further analysis reveals that the model can be used to derive the apparent permeability coefficients of model compounds. In addition to providing a suite of tools for the study of placental development, our trophoblast models allow the evaluation of compound transfer and toxicity, which will facilitate drug development.

Conserved and divergent features of trophoblast stem cells.

Trophoblast stem cells (TSCs) are a proliferative multipotent population derived from the trophectoderm of the blastocyst, which will give rise to all the functional cell types of the trophoblast compartment of the placenta. The isolation and culture of TSCs in vitro represent a robust model to study mechanisms of trophoblast differentiation into mature cells both in successful and diseased pregnancy. Despite the highly conserved functions of the placenta, there is extreme variability in placental morphology, fetal-maternal interface, and development among eutherian mammals. This review aims to summarize the establishment and maintenance of TSCs in mammals such as primates, including human, rodents, and nontraditional animal models with a primary emphasis on epigenetic regulation of their origin while defining gaps in the current literature and areas of further development. FGF signaling is critical for mouse TSCs but dispensable for derivation of TSCs in other species. Human, simian, and bovine TSCs have much more complicated requirements of signaling pathways including activation of WNT and inhibition of TGFβ cascades. Epigenetic features such as DNA and histone methylation as well as histone acetylation are dynamic during development and are expressed in cell- and gestational age-specific pattern in placental trophoblasts. While TSCs from different species seem to recapitulate some select epigenomic features, there is a limitation in the comprehensive understanding of TSCs and how well TSCs retain placental epigenetic marks. Therefore, future studies should be directed at investigating epigenomic features of global and placental-specific gene expression in primary trophoblasts and TSCs.

Efficient Reprogramming of Mouse Embryonic Stem Cells into Trophoblast Stem-like Cells via Lats Kinase Inhibition.

Mouse zygotes undergo multiple rounds of cell division, resulting in the formation of preimplantation blastocysts comprising three lineages: trophectoderm (TE), epiblast (EPI), and primitive endoderm (PrE). Cell fate determination plays a crucial role in establishing a healthy pregnancy. The initial separation of lineages gives rise to TE and inner cell mass (ICM), from which trophoblast stem cells (TSC) and embryonic stem cells (ESC) can be derived in vitro. Studying lineage differentiation is greatly facilitated by the clear functional distinction between TSC and ESC. However, transitioning between these two types of cells naturally poses challenges. In this study, we demonstrate that inhibiting LATS kinase promotes the conversion of ICM to TE and also effectively reprograms ESC into stable, self-renewing TS-like cells (TSLC). Compared to TSC, TSLC exhibits similar molecular properties, including the high expression of marker genes such as Cdx2, Eomes, and Tfap2c, as well as hypomethylation of their promoters. Importantly, TSLC not only displays the ability to differentiate into mature trophoblast cells in vitro but also participates in placenta formation in vivo. These findings highlight the efficient reprogramming of ESCs into TSLCs using a small molecular inducer, which provides a new reference for understanding the regulatory network between ESCs and TSCs.

Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells

IntroductionCultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1. MethodsHand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software. ResultsBaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2–4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44–47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation. DiscussionFirst, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.

VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification

In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.

ISRIB facilitates the co-culture of human trophoblast stem cells and embryonic stem cells.

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFβ signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFβ inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs.

Uncovering Phenotypes in Mutant Mice by Determining Embryo, Organ, Tissue, and Cell Developmental Potential.

The death of an embryo during gestation does not necessarily preclude the study of the mutant embryo or the developmental potential of its individual cells, tissues, or organs. Whole-embryo in vitro culture prior to the time of death will allow real-time observation of living embryos and direct comparisons with controls. Organ anlage can be removed from embryos and cultured in vitro beyond the time of death of the whole embryo. In both whole embryos and organ anlage culture, fluorescent protein reporters may be used productively to follow cell types or specific gene expression changes. Some cells, such as hematopoietic cells, and organ anlage, may be suitable for transplantation to wild-type hosts for further analysis of their potential. Additionally, cell lines, including embryonic stem (ES) cells, trophoblast stem (TS) cells, extraembryonic endoderm (XEN) stem cells, and epiblast-derived stem cells (EpiSC), can be derived from mutant embryos to reveal the potential of the mutant cells outside the context of the whole organism. Mutant stem cells or even whole mutant embryos can be used to test potential in chimeras or in teratomas.

CRISPR Activation in Mouse Trophoblast Stem Cells.

The placenta is a vital organ that regulates nutrient supply to the developing embryo during gestation. In mice, the placenta is composed of trophoblast lineage and mesodermal derivatives, which merge through the chorioallantoic fusion process in a critical event for the progression of placenta development. The trophoblast lineage is derived from self-renewing, multipotent cells known as mouse trophoblast stem cells (mTSCs). These cells are a valuable tool that allows scientists to comprehend the signals regulating major placental cell types' self-renewal and differentiation capacity. Recent advances in CRISPR-Cas9 genome editing applied in mTSCs have provided novel insights into the molecular networks involved in placentation. Here, we present a comprehensive CRISPR activation (CRISPRa) protocol based on the CRISPR/gRNA-directed synergistic activation mediator (SAM) method to overexpress specific target genes in mTSCs.

Generation of Knockout Mouse Trophoblast Stem Cells by CRISPR/Cas9.

The placenta is the organ that dictates the reproductive outcome of mammalian pregnancy by supplying nutrients and oxygen to the developing fetus to sustain its normal growth. During early mammalian development, trophoblast cells are the earliest cell type to differentiate with multipotent capacity to generate the trophoblast components of the placenta. The isolation and use of mouse trophoblast stem cells (mTSCs) to model in vitro trophoblast differentiation, in combination with CRISPR/Cas9 genome editing technology, has provided tremendous insight into the molecular mechanisms governing early mouse placentation. By knocking out a specific gene of interest in mTSCs, researchers are shedding light onto the molecular pathways involved in normal placental development and pregnancy disorders associated with abnormal placentation. In this chapter, we provide a detailed protocol for the genetic modification of mTSCs by using CRISPR/Cas9 genome editing system.