Low concentrations of saracatinib promote definitive endoderm differentiation through inhibition of FAK-YAP signaling axis.

Optimizing the efficiency of definitive endoderm (DE) differentiation is necessary for the generation of diverse organ-like structures. In this study, we used the small molecule inhibitor saracatinib (SAR) to enhance DE differentiation of human embryonic stem cells and induced pluripotent stem cells. SAR significantly improved DE differentiation efficiency at low concentrations. The interaction between SAR and Focal Adhesion Kinase (FAK) was explored through RNA-seq and molecular docking simulations, which further supported the inhibition of DE differentiation by p-FAK overexpression in SAR-treated cells. In addition, we found that SAR inhibited the nuclear translocation of Yes-associated protein (YAP), a downstream effector of FAK, which promoted DE differentiation. Moreover, the addition of SAR enabled a significant reduction in activin A (AA) from 50 to 10 ng/mL without compromising DE differentiation efficiency. For induction of the pancreatic lineage, 10 ng/ml AA combined with SAR at the DE differentiation stage yielded a comparative number of PDX1+/NKX6.1+ pancreatic progenitor cells to those obtained by 50 ng/ml AA treatment. Our study highlights SAR as a potential modulator that facilitates the cost-effective generation of DE cells and provides insight into the orchestration of cell fate determination.

Assessment and Comparison of Early Developmental Toxicity of Six Per- and Polyfluoroalkyl Substances with Human Embryonic Stem Cell Models.

Per- and polyfluoroalkyl substances (PFAS) are extensively utilized in varieties of products and tend to accumulate in the human body including umbilical cord blood and embryos/fetuses. In this study, we conducted an assessment and comparison of the potential early developmental toxicity of perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid, perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate, and perfluorobutyric acid at noncytotoxic concentrations relevant to human exposure using models based on human embryonic stem cells in both three-dimensional embryoid body (EB) and monolayer differentiation configurations. All six compounds influenced the determination of cell fate by disrupting the expression of associated markers in both models and, in some instances, even led to alterations in the formation of cystic EBs. The expression of cilia-related gene IFT122 was significantly inhibited. Additionally, PFOS and PFOA inhibited ciliogenesis, while PFOA specifically reduced the cilia length. Transcriptome analysis revealed that PFOS altered 1054 genes and disrupted crucial signaling pathways such as WNT and TGF-ß, which play integral roles in cilia transduction and are critical for early embryonic development. These results provide precise and comprehensive insights into the potential adverse health effects of these six PFAS compounds directly concerning early human embryonic development.

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 TGFß 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 TGFß 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.

Differential Gene Regulation of the Human Blastocyst Trophectoderm and Inner Cell Mass by Progesterone.

Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.

Impact of benzo[a]pyrene, PCB153 and sex hormones on human ESC-Derived thyroid follicles using single cell transcriptomics.

INTRODUCTION: Endocrine disruptors are compounds of manmade origin able to interfere with the endocrine system and constitute an important environmental concern. Indeed, detrimental effects on thyroid physiology and functioning have been described. Differences exist in the susceptibility of human sexes to the incidence of thyroid disorders, like autoimmune diseases or cancer. METHODS: To study how different hormonal environments impact the thyroid response to endocrine disruptors, we exposed human embryonic stem cell-derived thyroid organoids to physiological concentrations of sex hormones resembling the serum levels of human females post-ovulation or males of reproductive age for three days. Afterwards, we added 10 µM benzo[a]pyrene or PCB153 for 24 h and analyzed the transcriptome changes via single-cell RNA sequencing with differential gene expression and gene ontology analysis. RESULTS: The sex hormones receptors genes AR, ESR1, ESR2 and PGR were expressed at low levels. Among the thyroid markers, only TG resulted downregulated by benzo[a]pyrene or benzo[a]pyrene with the "male" hormones mix. Both hormone mixtures and benzo[a]pyrene alone upregulated ribosomal genes and genes involved in oxidative phosphorylation, while their combination decreased the expression compared to benzo[a]pyrene alone. The "male" mix and benzo[a]pyrene, alone or in combination, upregulated genes involved in lipid transport and metabolism (APOA1, APOC3, APOA4, FABP1, FABP2, FABP6). The combination of "male" hormones and benzo[a]pyrene induced also genes involved in inflammation and NFkB targets. Benzo[a]pyrene upregulated CYP1A1, CYP1B1 and NQO1 irrespective of the hormonal context. The induction was stronger in the "female" mix. Benzo[a]pyrene alone upregulated genes involved in cell cycle regulation, response to reactive oxygen species and apoptosis. PCB153 had a modest effect in presence of "male" hormones, while we did not observe any changes with the "female" mix. CONCLUSION: This work shows how single cell transcriptomics can be applied to selectively study the in vitro effects of endocrine disrupters and their interaction with different hormonal contexts.

PPARgamma dependent PEX11beta counteracts the suppressive role of SIRT1 on neural differentiation of HESCs.

The membrane peroxisomal proteins PEX11, play a crucial role in peroxisome proliferation by regulating elongation, membrane constriction, and fission of pre-existing peroxisomes. In this study, we evaluated the function of PEX11B gene in neural differentiation of human embryonic stem cell (hESC) by inducing shRNAi-mediated knockdown of PEX11B expression. Our results demonstrate that loss of PEX11B expression led to a significant decrease in the expression of peroxisomal-related genes including ACOX1, PMP70, PEX1, and PEX7, as well as neural tube-like structures and neuronal markers. Inhibition of SIRT1 using pharmacological agents counteracted the effects of PEX11B knockdown, resulting in a relative increase in PEX11B expression and an increase in differentiated neural tube-like structures. However, the neuroprotective effects of SIRT1 were eliminated by PPAR inhibition, indicating that PPARÉ£ may mediate the interaction between PEX11B and SIRT1. Our findings suggest that both SIRT1 and PPARÉ£ have neuroprotective effects, and also this study provides the first indication for a potential interaction between PEX11B, SIRT1, and PPARÉ£ during hESC neural differentiation.