Endogenous retroviruses (ERVs) are the remnants of retroviral germline infections and are highly abundant in the genomes of vertebrates. At one time considered to be nothing more than inert \'junk\' within genomes, ERVs have been tolerated within host genomes over vast timescales, and their study continues to reveal complex co-evolutionary histories within their respective host species. For example, multiple instances have been characterized of ERVs having been \'borrowed\' for normal physiology, from single copies to ones involved in various regulatory networks such as innate immunity and during early development. Within the cell, the accessibility of ERVs is normally tightly controlled by epigenetic mechanisms such as DNA methylation or histone modifications. However, these silencing mechanisms of ERVs are reversible, and epigenetic alterations to the chromatin landscape can thus lead to their aberrant expression, as is observed in abnormal cellular environments such as in tumors. In this review, we focus on ERV transcriptional control and draw parallels and distinctions concerning the loss of regulation in disease, as well as their precise regulation in early development.

Transposable elements (TEs) comprise a substantial portion of the mammalian genome, with potential implications for both embryonic development and cancer. This study aimed to characterize the expression profiles of TEs in embryonic stem cells (ESCs), cancer cell lines, tumor tissues, and the tumor microenvironment (TME). We observed similarities in TE expression profiles between cancer cells and ESCs, suggesting potential parallels in regulatory mechanisms. Notably, four TE RNAs (HERVH, LTR7, HERV-Fc1, HERV-Fc2) exhibited significant downregulation across cancer cell lines and tumor tissues compared to ESCs, highlighting potential roles in pluripotency regulation. The strong up-regulation of the latter two TEs (HERV-Fc1, HERV-Fc2) in ESCs has not been previously demonstrated and may be a first indication of their role in the regulation of pluripotency. Conversely, tandemly repeated sequences (MSR1, CER, ALR) showed up-regulation in cancer contexts. Moreover, a difference in TE expression was observed between the TME and the tumor bulk transcriptome, with distinct dysregulated TE profiles. Some TME-specific TEs were absent in normal tissues, predominantly belonging to LTR and L1 retrotransposon families. These findings not only shed light on the regulatory roles of TEs in both embryonic development and cancer but also suggest novel targets for anti-cancer therapy. Understanding the interplay between cancer cells and the TME at the TE level may pave the way for further research into therapeutic interventions.

The discovery of mouse embryonic stem cells in 1981 transformed research in mammalian developmental biology and functional genomics. The subsequent generation of human pluripotent stem cells (PSCs) and the development of molecular reprogramming have opened unheralded avenues for drug discovery and cell replacement therapy. Here, I review the history of PSCs from the perspective that long-term self-renewal is a product of the in vitro signaling environment, rather than an intrinsic feature of embryos. I discuss the relationship between pluripotent states captured in vitro to stages of epiblast in the embryo and suggest key considerations for evaluation of PSCs. A remaining fundamental challenge is to determine whether naïve pluripotency can be propagated from the broad range of mammals by exploiting common principles in gene regulatory architecture.

Embryonic stem cells (ESCs) have proven to be a great in vitro model that faithfully recapitulates the events that occur during in vivo embryogenesis, making them a unique tool to study the cellular and molecular mechanisms that define tissue specification during embryonic development. Livestock ESCs are particularly attractive and have broad prospects including drug selection and human disease modeling, improvement of reproductive biotechniques and agriculture-related applications such as production of genetically modified animals. While mice and human ESCs have been established many years ago, no significant advances were made in livestock species until recently. Nowadays, livestock ESCs are available from cattle, pigs, sheep, horses and rabbits with different states of pluripotency. In this review, we summarize the current advances on livestock ESCs establishment and maintenance along with their present and future applications.

The homeostasis of human pluripotent stem cells (hPSCs) requires the signaling balance of extracellular factors. Exogenous regulators from cell culture medium have been widely reported, but little attention has been paid to the autocrine factor from hPSCs themselves. In this report, we demonstrate that extracellular signal-related kinase 5 (ERK5) regulates endogenous autocrine factors essential for pluripotency and differentiation. ERK5 inhibition leads to erroneous cell fate specification in all lineages even under lineage-specific induction. hPSCs can self-renew under ERK5 inhibition in the presence of fibroblast growth factor 2 (FGF2) and transforming growth factor β (TGF-β), although NANOG expression is partially suppressed. Further analysis demonstrates that ERK5 promotes the expression of autocrine factors such as NODAL, FGF8, and WNT3. The addition of NODAL protein rescues NANOG expression and differentiation phenotypes under ERK5 inhibition. We demonstrate that constitutively active ERK5 pathway allows self-renewal even without essential growth factors FGF2 and TGF-β. This study highlights the essential contribution of autocrine pathways to proper maintenance and differentiation.

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.

The expansion of pluripotent stem cells (PSCs)in vitroremains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markersOct4, Nanog, andSox2and the differentiation markerLmnAas well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1µm pillars (1µm spacing, square arrangement), 2µm wells (c-c (x, y) = 4, 4µm), and 5µm pillars (c-c (x, y) = 7.5, 7.5µm). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion.

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.