Li-Fraumeni syndrome (LFS) is a rare autosomal dominant inherited genetic disorder that greatly increases the risk of developing several types of cancer, including young children and young adults. LFS is primarily caused by specific mutations in the tumor suppressor gene TP53. In this study, we successfully generated two human induced pluripotent stem cell (iPSC) lines derived from patients diagnosed with LFS, each carrying a distinct heterozygous mutation in the TP53 gene. These LFS patient-derived iPSC lines exhibited robust expression of key pluripotency markers, demonstrated the capacity to differentiate into all three germ layers (endoderm, mesoderm, and ectoderm), and maintained a normal karyotype. The establishment of these iPSC lines provides a valuable tool for modeling LFS in vitro, enabling researchers to investigate the underlying pathological mechanisms associated with the disease across various cell types and tissues.

BACKGROUND: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium.
METHODS: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions.
RESULTS: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions.
CONCLUSIONS: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

Cell fate determination is an intricate process which is orchestrated by multiple regulatory layers including signal pathways, transcriptional factors, epigenetic modifications, and metabolic rewiring. Among the sophisticated epigenetic modulations, the repressive mark H3K27me3, deposited by PRC2 (polycomb repressive complex 2) and removed by demethylase KDM6, plays a pivotal role in mediating the cellular identity transition through its dynamic and precise alterations. Herein, we overview and discuss how H3K27me3 and its modifiers regulate pluripotency maintenance and early lineage differentiation. We primarily highlight the following four aspects: 1) the two subcomplexes PRC2.1 and PRC2.2 and the distribution of genomic H3K27 methylation; 2) PRC2 as a critical regulator in pluripotency maintenance and exit; 3) the emerging role of the eraser KDM6 in early differentiation; 4) newly identified additional factors influencing H3K27me3. We present a comprehensive insight into the molecular principles of the dynamic regulation of H3K27me3, as well as how this epigenetic mark participates in pluripotent stem cell-centered cell fate determination.

Mammalian embryos are very vulnerable to environmental toxicants (ETs) exposure. Bisphenol A (BPA), one of the most diffused ETs, exerts endocrine-disrupting effects through estro-gen-mimicking and hormone-like properties, with detrimental health effects, including on reproduction. However, its impact during the peri-implantation stages is still unclear. This study, using gastruloids as a 3D stem cell-based in vitro model of embryonic development, showed that BPA exposure arrests their axial elongation when present during the Wnt/β-catenin pathway activation period by β-catenin protein reduction. Gastruloid reshaping might have been impeded by the downregulation of Snail, Slug and Twist, known to suppress E-cadherin expression and to activate the N-cadherin gene, and by the low expression of the N-cadherin protein. Also, the lack of gastruloids elongation might be related to altered exit of BPA-exposed cells from the pluripotency condition and their following differentiation. In conclusion, here we show that the inhibition of gastruloids\' axial elongation by BPA might be the result of the concomitant Wnt/β-catenin perturbation, reduced N-cadherin expression and Oct4, T/Bra and Cdx2 altered patter expression, which all together concur in the impaired development of mouse gastruloids.

Embryonic diapause is a reproductive adaptation that enables some mammalian species to halt the otherwise continuous pace of embryonic development. In this dormant state, the embryo exploits poorly understood regulatory mechanisms to preserve its developmental potential for prolonged periods of time. Here, using mouse embryos and single-cell RNA sequencing, we molecularly defined embryonic diapause at single-cell resolution, revealing transcriptional dynamics while the embryo seemingly resides in a state of suspended animation. Additionally, we found that the dormant pluripotent cells rely on integrin receptors to sense their microenvironment and preserve their viability via Yap/Taz-mediated prosurvival signaling.

OBJECTIVE: This study aimed to investigate the effect of acoustic vibration on the pluripotency of human embryonic stem cells (hESCs) and evaluate cell proliferation and self-renewal ability post-treatment.
METHODS: The human ES cell line H1 was used for the experiments. hESCs were treated with an acoustic vibration device. Their proliferative ability was subsequently detected using a colony formation assay, while the expression of pluripotency-related markers was detected via immunofluorescence staining. Finally, changes in gene expression levels were examined using quantitative polymerase chain reaction (qPCR) in the presence of appropriate primers.
RESULTS: Compared with normal cells in the control group, the morphology of experimental cells subjected to acoustic vibration did not significantly change. Contrastingly, the colony-forming efficiency of the experimental cells significantly increased. Immunofluorescence staining results showed the cells in experimental group were positive for the pluripotency markers NANOG, octamer-binding transcription factor 4 gene (OCT4), and SRY (sex determining region Y)-box 2 (SOX2). In addition, the expression levels of pluripotency genes NANOG, OCT4, SOX2, and Yes-associated protein (YAP)-related genes were up-regulated following acoustic vibration.
CONCLUSIONS: Our results revealed that acoustic vibration enhanced the proliferative ability of hESCs and increased the expression levels of NANOG, OCT4, SOX2, and YAP-related genes, indicating that acoustic vibration can optimize the self-renewal ability of hESCs and that the YAP signaling pathway may play a critical role in the functional process of acoustic vibration.

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.

In this review, we consider the multipotency of neural crest cells (NCCs), Schwann cell precursors (SCPs), and their role in embryogenesis base on genetic tracing and knock out model animals and single cell transcriptomic analysis. In particular, we summarize and analyze data on the contribution of NCCs and SCPs to the gland development and functions.

During implantation, embryos undergo an unpolarized-to-polarized transition to initiate postimplantation morphogenesis. However, the underlying molecular mechanism is unknown. Here, we identify a transient transcriptional activation governing embryonic morphogenesis and pluripotency transition during implantation. In naive pluripotent embryonic stem cells (ESCs), which represent preimplantation embryos, we find that the microprocessor component DGCR8 can recognize stem-loop structures within nascent mRNAs to sequester transcriptional coactivator FLII to suppress transcription directly. When mESCs exit from naive pluripotency, the ERK/RSK/P70S6K pathway rapidly activates, leading to FLII phosphorylation and disruption of DGCR8/FLII interaction. Phosphorylated FLII can bind to transcription factor JUN, activating cell migration-related genes to establish poised pluripotency akin to implanting embryos. Resequestration of FLII by DGCR8 drives poised ESCs into formative pluripotency. In summary, we identify a DGCR8/FLII/JUN-mediated transient transcriptional activation mechanism. Disruption of this mechanism inhibits naive-poised-formative pluripotency transition and the corresponding unpolarized-to-polarized transition during embryo implantation, which are conserved in mice and humans.