There are few studies on the establishment of induced pluripotent stem cells (iPSCs) in cats. Although induction using heterologous reprogramming factors delivered via viral vectors has been reported, its safety and reprogramming efficiency still require improvement. In addition, the reprogramming mechanism needs further elucidation. In this study, we constructed a series of expression vectors for cat-derived reprogramming transcription factors based on the piggyBac transposon system and transfected various factor combinations into cat fetal fibroblasts (CFFs) under different electroporation conditions to generate cat iPSCs (ciPSCs). Additionally, the specific roles of these factors in reprogramming were investigated. The results showed that under the optimized electroporation conditions (DMEM/F12 buffer, 300 V, 10 ms pulse duration, 2 pulses, 25 μg plasmid DNA, and 4 mm cuvette), the survival rate and transfection efficiency of CFFs reached 64 % and 67.8 %, respectively. Based on this condition, a seven-factor combination (cOSKM + pNL + SV40 Large T) was confirmed as a better inducer for establishing ciPSCs. The obtained ciPSCs exhibit good pluripotency and passaging stability. They express stemness-related genes and proteins, and can form embryoid bodies (EBs) capable of differentiating into all three germ layers. OCT4 (O), SOX2 (S), KLF4 (K), and c-MYC (M) play important cooperative and synergistic roles in the mesenchymal-to-epithelial transition (MET) during the initial stages of reprogramming, while the supplement of NANOG (N) and LIN28 (L) can further promote MET and is important for successful reprogramming. It lays a foundation for the further breeding of cloned and genetically modified cats, and provides a tool for studying embryonic developmental diseases, screening drugs, and applying to tissue regeneration.

Exposure to Cadmium-based quantum dots (QDs) is becoming a growing threat to human health, necessitating a deeper understanding of their intracellular behavior and the associated toxic effects. Among the various domains of nanosafety assessment, the impact of these QDs on the nervous system is particularly critical; however, the potential effects on neurodevelopment and the underlying mechanisms remain largely unexplored. The current study explores the neural developmental toxicities associated with exposure to QDs made of cadmium selenide (CdSe) and encapsulated within a zinc sulfide (ZnS) shell using mouse embryonic stem cells (mESCs). Exposure to CdSe/ZnS QDs was found to impair the neural differentiation of mESCs via a novel mechanism of programmed cell death known as ferroptosis. Specifically, the CdSe/ZnS QDs were found to be internalized by cells, with a substantial fraction remaining within the cells even after a 24 h clearance period. Furthermore, nanoparticle internalization induced significant ROS/MDA elevation, mitochondrial depolarization and intracellular iron overload, collectively triggering ferroptosis and consequent tricarboxylic acid (TCA) cycle dysfunction. Importantly, the application of ferroptosis inhibitors was found to alleviate the disruption in the TCA cycle induced by CdSe/ZnS QDs and restore neural differentiation. Additionally, ferroptosis was established as a common form of cell death triggered by nanoparticles. These findings underscore the urgent need for further investigations into the safety profiles of CdSe/ZnS QDs in a neurological context, as an understanding of the underlying mechanisms can facilitate informed risk assessments and guide the development of safer nanomaterials for biomedical applications.

Clustering is a critical step in the analysis of single-cell data, enabling the discovery and characterization of cell types and states. However, most popular clustering tools do not subject results to statistical inference testing, leading to risks of overclustering or underclustering data and often resulting in ineffective identification of cell types with widely differing prevalence. To address these challenges, we present CHOIR (cluster hierarchy optimization by iterative random forests), which applies a framework of random forest classifiers and permutation tests across a hierarchical clustering tree to statistically determine clusters representing distinct populations. We demonstrate the performance of CHOIR through extensive benchmarking against 15 existing clustering methods across 230 simulated and five real single-cell RNA sequencing, assay for transposase-accessible chromatin sequencing, spatial transcriptomic and multi-omic datasets. CHOIR can be applied to any single-cell data type and provides a flexible, scalable and robust solution to the challenge of identifying biologically relevant cell groupings within heterogeneous single-cell data.

Ovarian cancer remains the most lethal gynecological malignancy, largely owing to its chemotherapy resistance and high recurrence rate. Emerging evidence has linked the aberrant expression of SOX2, a transcription factor that is important in the development and maintenance of stem cell state, with chemoresistance and poor prognosis of ovarian cancer patients. In this study, we aimed to elucidate the mechanisms that drive aberrant SOX2 expression in ovarian cancer cells. By examining multiple ovarian cancer cell lines and a panel of clinical tumor samples, we observed a broad overexpression of SOX2 in ovarian cancer cell lines and tumors. To identify signaling pathway(s) that drives SOX2 overexpression in ovarian cancer cells, we screened a set of small-molecule kinase inhibitors that target 30 major cellular kinases. Among the top hits identified are AKT inhibitors. We demonstrated that inhibition or knockdown of AKT1 can drastically downregulate SOX2 protein level, impairs the growth and stemness of SOX2-positive ovarian cancer cells, and markedly sensitize SOX2-positive ovarian cancer cells to platinum drugs. Mechanically, we found that AKT1 drives SOX2 overexpression primarily by enhancing its protein stability and does so by phosphorylating SOX2 at threonine 116. Altogether, our study reveals an underlying mechanism that drives SOX2 overexpression in ovarian cancer and underscores pharmacological inhibition of AKT1 as a potential therapeutic strategy to sensitize SOX2-positive ovarian cancer to platinum drugs.

Cell identity maintenance faces many challenges during mitosis, as most DNA-binding proteins are evicted from DNA and transcription is virtually abolished. How cells maintain their identity through division and faithfully re-initiate gene expression during mitotic exit is unclear. Here, we develop a novel reporter system enabling cell cycle synchronization-free separation of pluripotent stem cells in temporal bins of <30 min during mitotic exit. This allows us to quantify genome-wide reactivation of transcription, sequential changes in chromatin accessibility and transcription factor footprints, and re-binding of the pluripotency transcription factors OCT4, SOX2, and NANOG (OSN). We find that transcriptional activity progressively ramps up after mitosis and that OSN rapidly reoccupy the genome during the anaphase-telophase transition. We also demonstrate transcription factor-specific, dynamic relocation patterns and a hierarchical reorganization of the OSN binding landscape governed by OCT4 and SOX2. Our study sheds light on the dynamic orchestration of transcriptional reactivation after mitosis.

Sex-determining region Y box 2 (Sox2) is a critical transcription factor for embryogenesis and neural stem and progenitor cell (NSPC) maintenance. While distal enhancers control Sox2 in embryonic stem cells (ESCs), enhancers closer to the gene are implicated in Sox2 transcriptional regulation in neural development. We hypothesize that a downstream enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), regulates Sox2 transcription in neural stem cells and we investigate this in NSPCs derived from mouse ESCs. Using functional genomics and CRISPR-Cas9-mediated deletion analyses, we investigate the role of SRR2-18 in Sox2 regulation during neural differentiation. Transcriptome analyses demonstrate that the loss of even 1 copy of SRR2-18 disrupts the region-specific identity of NSPCs, reducing the expression of genes associated with more anterior regions of the embryonic nervous system. Homozygous deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which causes differentiation into posterior neural fates, specifically the caudal neural tube. This study highlights the importance of precise Sox2 regulation by SRR2-18 in neural differentiation.

Two main pluripotent cell lines can be established from the preimplantation and postimplantation mouse embryo as naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. Although the two pluripotent states are interconvertible, the molecular mechanism controlling the transition between naïve and primed pluripotency remains to be fully elucidated. Here, by performing a CRISPR-based loss-of-function screen in ESCs, we identify Sox2 involved in the repression of lineage-specification marker brachyury (T). Upon Sox2 ablation in ESCs, two populations of cells mutually exclusive for CDX2 (trophectoderm marker) and T expression can be observed. T-positive cells display features resembling the salient characteristics of EpiSCs including molecular and functional properties. By using genetic ablation approach, we show that acquisition and maintenance of primed pluripotency in Sox2 null T-positive cells heavily depend on fibroblast growth factor (Fgf) and Nodal, which is produced in an autocrine manner in these cells. We further demonstrate that Sox3 compensates for the absence of Sox2 in maintaining the primed state of Sox2-null pluripotent cells. Establishment of Sox2-deficient pluripotent cells will enable the elucidation of the mechanisms controlling the transition of cells between different states of pluripotency.

Currently, the efficiency of somatic cell nuclear transfer (SCNT) technology is relatively low, primarily owing to reprogramming abnormalities in donor cells or reconstructed embryos. Using histone deacetylase inhibitor (HDACi) to artificially alter the epigenetic modifications of donor cells and improve the reprogramming ability of reconstructed embryos is effective in improving nuclear transfer efficiency. In this study, we used Albas cashmere goat cells as donor cells, treated them with Scriptaid, and constructed embryos using SCNT. The results suggest that donor cell treatment with Scriptaid significantly increased the cellular histone acetylation modification level, perturbed the expression of the pluripotency molecule NANOG, altered the reprogramming ability of embryos, and increased the developmental rate of SCNT-reconstructed embryos. Scriptaid inhibited donor cell proliferation, induced apoptosis, and blocked the G0/G1 phase of the cell cycle. These results provide a new research direction for improving SCNT efficiency and a new perspective in the fields of regenerative medicine, agriculture, and animal husbandry.

The paper aims to investigate the biological role of microRNAs secreted by preimplantation embryo into the blastocoel fluid and to detect a distinctive molecular signature for identifying embryos with the highest implantation potential.

We carried on a multicenter retrospective study involving five European IVF centers. We collected 112 blastocoel fluid samples from embryos on day 5 post-fertilization, cultured individually, along with data on blastocyst grade and embryo transfer outcomes. Using a custom TLDA Array, we compared the expression levels of 89 miRNAs between 33 fluids from high-quality implanted embryos and 30 fluids from high-quality not-implanted embryos. Expression differences were assessed using SAM and t-test. Additionally, correlation and function enrichment analysis and network construction were conducted to identify the biological roles of deregulated microRNAs.

We identified six up-regulated microRNAs in the blastocoel fluid from implanted embryos, significantly and positively correlated across all samples (r ≥ 0.7; P ≤ 0.05). They could take part in pluripotency circuits, regulating and being regulated by transcription factors associated with stemness, cell growth, and embryo development. The ROC curve analysis confirmed the potential of these miRNAs as implantation classifiers.

The six miRNAs up-regulated in blastocoel fluid from implanted embryos may represent a functional molecular signature for evaluating blastocyst quality and identifying the most competent embryos. Their evaluation associated with non-invasive preimplantation genetic testing, integrating epigenetic and genomic analyses, could enhance implantation grade and allow for identification of the euploid embryo not able to implant.

The LIF signaling pathway and its regulation of internal factors like Sox2 is crucial for maintaining self-renewal and pluripotency in mESCs. However, the direct impact of LIF signaling on Sox2 transcriptional dynamics at the single-cell level remains elusive. Here, we employ PP7/PCP-mediated live imaging to analyze the transcriptional dynamics of Sox2 under perturbation of the LIF signaling pathway at single-cell resolution. Removal of the LIF ligand or addition of a JAK inhibitor heterogeneously affects the cell population, reducing the number of Sox2-active cells, rather than completely abolishing Sox2 expression. Moreover, Sox2-active cells under LIF perturbation exhibit significant reductions in mRNA production per cell. This reduction is characterized by decreased size and frequency of transcriptional bursting, resulting in shorter duration of Sox2 activity. Notably, cells with reduced or absent Sox2 expression demonstrate a significant loss in pluripotency, indicating that a reduction in Sox2 transcription (rather than a complete loss) is sufficient to trigger the transition from embryonic to an early differentiated state. In LIF-perturbed cells with Sox2 expression reduced to about 50% of non-perturbed levels, we observe a binary behavior, with cells either retaining or losing pluripotency-associated traits. Lastly, we find Sox2 expression is transcriptionally inherited across cell cycles, with Sox2-active mother cells more likely to reactivate Sox2 after mitosis compared to Sox2-inactive cells. This robust transcriptional memory is observed independent of LIF signaling perturbation. Our findings provide new insights into the transcriptional regulation of Sox2, advancing our understanding of the quantitative thresholds of gene expression required for pluripotency maintenance and highlighting the power of single-cell approaches to unravel dynamic regulatory mechanisms.

SOX17 directs the differentiation toward endoderm and acts as a human germline specifier. We previously found that the replacement of glutamate at position 57 of the high-mobility group (HMG) box with the basic lysine residue in SOX2 alters interactions with OCT4 and turns SOX17 into a pluripotency factor. Here, we systematically interrogated how mutations at this critical position affect the cellular reprogramming activity of SOX17 in mouse and human. We found that most mutations turn SOX17 into a pluripotency factor regardless of their biophysical properties except for acidic residues and proline. The conservative mutation to an aspartate allows the SOX17E57D protein to maintain a self-renewing endodermal state. We showed that only the glutamate in the wild-type protein blocks the formation of an SOX17/OCT4 dimer at composite DNA elements in pluripotency enhancers. Insights into how modifications of an ultra-conserved residue affect functions of developmental transcription factors provide avenues to advance cell fate engineering.

During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory toward the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.

Changes in epigenetic processes such as histone acetylation are proposed as key events influencing cancer cell function and the initiation and progression of pediatric brain tumors. Valproic acid (VPA) is an antiepileptic drug that acts partially by inhibiting histone deacetylases (HDACs) and could be repurposed as an epigenetic anticancer therapy. Here, we show that VPA reduced medulloblastoma (MB) cell viability and led to cell cycle arrest. These effects were accompanied by enhanced H3K9 histone acetylation (H3K9ac) and decreased expression of the MYC oncogene. VPA impaired the expansion of MB neurospheres enriched in stemness markers and reduced MYC while increasing TP53 expression in these neurospheres. In addition, VPA induced morphological changes consistent with neuronal differentiation and the increased expression of differentiation marker genes TUBB3 and ENO2. The expression of stemness genes SOX2, NES, and PRTG was differentially affected by VPA in MB cells with different TP53 status. VPA increased H3K9 occupancy of the promoter region of TP53. Among the genes regulated by VPA, the stemness regulators MYC and NES showed an association with patient survival in specific MB subgroups. Our results indicate that VPA may exert antitumor effects in MB by influencing histone acetylation, which may result in the modulation of stemness, neuronal differentiation, and the expression of genes associated with patient prognosis in specific molecular subgroups. Importantly, the actions of VPA in MB cells and neurospheres include a reduction in the expression of MYC and an increase in TP53.

Changes in epigenetic processes such as histone acetylation are proposed as key events influencing cancer cell function and the initiation and progression of pediatric brain tumors. Valproic acid (VPA) is an antiepileptic drug that acts partially by inhibiting histone deacetylases (HDACs) and could be repurposed as an epigenetic anticancer therapy. Here, we show that VPA reduced medulloblastoma (MB) cell viability and led to cell cycle arrest. These effects were accompanied by enhanced H3K9 histone acetylation (H3K9ac) and decreased expression of the MYC oncogene. VPA impaired the expansion of MB neurospheres enriched in stemness markers and reduced MYC while increasing TP53 expression in these neurospheres. In addition, VPA induced morphological changes consistent with neuronal differentiation and the increased expression of differentiation marker genes TUBB3 and ENO2. The expression of stemness genes SOX2, NES, and PRTG was differentially affected by VPA in MB cells with different TP53 status. VPA increased H3K9 occupancy of the promoter region of TP53. Among the genes regulated by VPA, the stemness regulators MYC and NES showed an association with patient survival in specific MB subgroups. Our results indicate that VPA may exert antitumor effects in MB by influencing histone acetylation, which may result in the modulation of stemness, neuronal differentiation, and the expression of genes associated with patient prognosis in specific molecular subgroups. Importantly, the actions of VPA in MB cells and neurospheres include a reduction in the expression of MYC and an increase in TP53.

The establishment of naive pluripotency is a continuous process starting with the generation of inner cell mass (ICM) that then differentiates into epiblast (EPI). Recent studies have revealed key transcription factors (TFs) for ICM formation, but which TFs initiate EPI specification remains unknown. Here, using a targeted rapid protein degradation system, we show that GABPA is not only a regulator of major ZGA, but also a master EPI specifier required for naive pluripotency establishment by regulating 47% of EPI genes during E3.5 to E4.5 transition. Chromatin binding dynamics analysis suggests that GABPA controls EPI formation at least partly by binding to the ICM gene promoters occupied by the pluripotency regulators TFAP2C and SOX2 at E3.5 to establish naive pluripotency at E4.5. Our study not only uncovers GABPA as a master pluripotency regulator, but also supports the notion that mammalian pluripotency establishment requires a dynamic and stepwise multi-TF regulatory network.

The establishment of naïve pluripotency is a continuous process starting with the generation of inner cell mass (ICM) which then differentiating into epiblast (EPI). Recent studies have revealed key transcription factors (TFs) for ICM formation, but which TFs initiate EPI specification remains unknown. Here, using a targeted rapid protein degradation system, we show that GABPA is not only a regulator of major ZGA, but also a master EPI specifier required for naïve pluripotency establishment by regulating 47% of EPI genes during E3.5 to E4.5 transition. Chromatin binding dynamics analysis suggests that GABPA controls EPI formation at least partly by binding to the ICM gene promoters occupied by the pluripotency regulators TFAP2C and SOX2 at E3.5 to establish naïve pluripotency at E4.5. Our study not only uncovers GABPA as a master pluripotency regulator, but also supports the notion that mammalian pluripotency establishment requires a dynamic and stepwise multi-TFs regulatory network.

Stem cells are a hallmark of animal multicellularity. Sox and POU transcription factors are associated with stemness and were believed to be animal innovations, reported absent in their unicellular relatives. Here we describe unicellular Sox and POU factors. Choanoflagellate and filasterean Sox proteins have DNA-binding specificity similar to mammalian Sox2. Choanoflagellate-but not filasterean-Sox can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSCs) through interacting with the mouse POU member Oct4. In contrast, choanoflagellate POU has a distinct DNA-binding profile and cannot generate iPSCs. Ancestrally reconstructed Sox proteins indicate that iPSC formation capacity is pervasive among resurrected sequences, thus loss of Sox2-like properties fostered Sox family subfunctionalization. Our findings imply that the evolution of animal stem cells might have involved the exaptation of a pre-existing set of transcription factors, where pre-animal Sox was biochemically similar to extant Sox, whilst POU factors required evolutionary innovations.

Embryonic stem cells (ESCs) hold great promise for regenerative medicine thanks to their ability to self-renew and differentiate into somatic cells and the germline. ESCs correspond to pluripotent epiblast - the tissue from which the following three germ layers originate during embryonic gastrulation: the ectoderm, mesoderm, and endoderm. Importantly, ESCs can be induced to differentiate toward various cell types by varying culture conditions, which can be exploited for in vitro modeling of developmental processes such as gastrulation. The classical model of gastrulation postulates that mesoderm and endoderm specification is made possible through the FGF-, BMP-, Wnt-, and Nodal-signaling gradients. Hence, it can be expected that one of these signals should direct ESC differentiation towards specific germ layers. However, ESC specification appears to be more complicated, and the same signal can be interpreted differently depending on the readout. In this research, using chemically defined culture conditions, homogeneous naïve ESCs as a starting cell population, and the Foxa2 gene-driven EGFP reporter tool, we established a robust model of definitive endoderm (DE) specification. This in vitro model features formative pluripotency as an intermediate state acquired by the epiblast in vivo shortly after implantation. Despite the initially homogeneous state of the cells in the model and high Activin concentration during endodermal specification, there remains a cell subpopulation that does not reach the endodermal state. This simple model developed by us can be used to study the origins of cellular heterogeneity during germ layer specification.

Nanog is a crucial regulatory factor in maintaining the self-renewal and pluripotency of embryonic stem cells. It is involved in various biological processes, such as early embryonic development, cell reprogramming, cell cycle regulation, the proliferation and migration of primordial germ cells. While research on this gene has primarily focused on mammals, there has been a growing interest in studying nanog in fish. However, there is a notable lack of comprehensive reviews regarding this gene in fish, which is essential for guiding future research. This review aims to provide a thorough summary of the gene's structure, expression patterns, functions and regulatory mechanisms in fish. The findings suggest that nanog probably has both conserved and divergent functions in regulating cell pluripotency, early embryonic development, and germ cell development in teleosts compared to other species, including mammals. These insights lay the foundation for future research and applications of the nanog gene, providing a new perspective for understanding the evolution and conserved charactristics of teleost nanog.

Yak is an excellent germplasm resource on the Tibetan Plateau and is able to live in high-altitude areas with hypoxic, cold, and harsh environments. Studies on induced pluripotent stem cells (iPSCs) in large ruminants commonly involve a combination strategy involving six transcription factors, Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 (OSKMNL). This strategy tends to utilize genes from the same species to optimize pluripotency maintenance. In this study, we cloned the six pluripotency genes (OSKMNL) from yak and constructed a multi-cistronic lentiviral vector carrying these genes. This vector efficiently delivered the genes into yak fibroblasts, aiming to promote the reprogramming process. We verified that the treated cells had several pluripotency characteristics, marking the first successful construction of a lentiviral system carrying yak pluripotency genes. This achievement lays the foundation for subsequent establishment of yak iPSCs and holds significant implications for yak-breed improvement and germplasm-resource conservation.

Transcription factors are essential DNA-binding proteins that regulate the transcription rate of several genes and control the expression of genes inside a cell. The prediction of transcription factors with high precision is important for understanding biological processes such as cell differentiation, intracellular signaling, and cell-cycle control. In this study, we developed a hybrid method that combines alignment-based and alignment-free methods for predicting transcription factors with higher accuracy. All models have been trained, tested, and evaluated on a large dataset that contains 19,406 transcription factors and 523,560 non-transcription factor protein sequences. To avoid biases in evaluation, the datasets were divided into training and validation/independent datasets, where 80% of the data was used for training, and the remaining 20% was used for external validation. In the case of alignment-free methods, models were developed using machine learning techniques and the composition-based features of a protein. Our best alignment-free model obtained an AUC of 0.97 on an independent dataset. In the case of the alignment-based method, we used BLAST at different cut-offs to predict the transcription factors. Although the alignment-based method demonstrated excellent performance, it was unable to cover all transcription factors due to instances of no hits. To combine the strengths of both methods, we developed a hybrid method that combines alignment-free and alignment-based methods. In the hybrid method, we added the scores of the alignment-free and alignment-based methods and achieved a maximum AUC of 0.99 on the independent dataset. The method proposed in this study performs better than existing methods. We incorporated the best models in the webserver/Python Package Index/standalone package of "TransFacPred" (https://webs.iiitd.edu.in/raghava/transfacpred).

Animals and plants have developed resilience mechanisms to effectively endure and overcome physical damage and environmental challenges throughout their life span. To sustain their vitality, both animals and plants employ mechanisms to replenish damaged cells, either directly, involving the activity of adult stem cells, or indirectly, via dedifferentiation of somatic cells that are induced to revert to a stem cell state and subsequently redifferentiate. Stem cell research has been a rapidly advancing field in animal studies for many years, driven by its promising potential in human therapeutics, including tissue regeneration and drug development. A major breakthrough was the discovery of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells by expressing a limited set of transcription factors. This discovery enabled the generation of an unlimited supply of cells that can be differentiated into specific cell types and tissues. Equally, a keen interest in the connection between plant stem cells and regeneration has been developed in the last decade, driven by the demand to enhance plant traits such as yield, resistance to pathogens, and the opportunities provided by CRISPR/Cas-mediated gene editing. Here we discuss how knowledge of stem cell biology benefits regeneration technology, and we speculate on the creation of a universal genotype-independent iPSC system for plants to overcome regenerative recalcitrance.

Cancer stem cells (CSCs), a small subset of cells in tumors that are characterized by self-renewal and continuous proliferation, lead to tumorigenesis, metastasis, and maintain tumor heterogeneity. Cancer continues to be a significant global disease burden. In the past, surgery, radiotherapy, and chemotherapy were the main cancer treatments. The technology of cancer treatments continues to develop and advance, and the emergence of targeted therapy, and immunotherapy provides more options for patients to a certain extent. However, the limitations of efficacy and treatment resistance are still inevitable. Our review begins with a brief introduction of the historical discoveries, original hypotheses, and pathways that regulate CSCs, such as WNT/β-Catenin, hedgehog, Notch, NF-κB, JAK/STAT, TGF-β, PI3K/AKT, PPAR pathway, and their crosstalk. We focus on the role of CSCs in various therapeutic outcomes and resistance, including how the treatments affect the content of CSCs and the alteration of related molecules, CSCs-mediated therapeutic resistance, and the clinical value of targeting CSCs in patients with refractory, progressed or advanced tumors. In summary, CSCs affect therapeutic efficacy, and the treatment method of targeting CSCs is still difficult to determine. Clarifying regulatory mechanisms and targeting biomarkers of CSCs is currently the mainstream idea.

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Embryonic stem cells (ESCs) are defined as stem cells with self-renewing and differentiation capabilities. These unique properties are tightly regulated and controlled by complex genetic and molecular mechanisms, whose understanding is essential for both basic and translational research. A large number of studies have mostly focused on understanding the molecular mechanisms governing pluripotency and differentiation of ESCs, while the regulation of proliferation has received comparably less attention. Here, we investigate the role of ZZZ3 (zinc finger ZZ-type containing 3) in human ESCs homeostasis. We found that knockdown of ZZZ3 negatively impacts ribosome biogenesis, translation, and mTOR signaling, leading to a significant reduction in cell proliferation. This process occurs without affecting pluripotency, suggesting that ZZZ3-depleted ESCs enter a "dormant-like" state and that proliferation and pluripotency can be uncoupled also in human ESCs.

The mammalian olfactory neuronal lineage is regenerative, and accordingly, maintains a population of pluripotent cells that replenish olfactory sensory neurons and other olfactory cell types during the life of the animal. Moreover, in response to acute injury, the early transit amplifying cells along the olfactory sensory neuronal lineage are able to de-differentiate to shift resources in support of tissue restoration. In order to further explore plasticity of various cellular stages along the olfactory sensory neuronal lineage, we challenged the epigenetic stability of two olfactory placode-derived cell lines that model immature olfactory sensory neuronal stages. We found that perturbation of the Ehmt2 chromatin modifier transformed the growth properties, morphology, and gene expression profiles towards states with several stem cell characteristics. This transformation was dependent on continued expression of the large T-antigen, and was enhanced by Sox2 over-expression. These findings may provide momentum for exploring inherent cellular plasticity within early cell types of the olfactory lineage, as well as potentially add to our knowledge of cellular reprogramming.

Discovering how epigenetic modifications influence olfactory neuronal lineage plasticity offers insights into regenerative potential and cellular reprogramming.

The remarkable diversity observed in the structure and development of the molluscan nervous system raises intriguing questions regarding the molecular mechanisms underlying neurogenesis in Mollusca. The expression of SoxB family transcription factors plays a pivotal role in neuronal development, thereby offering valuable insights into the strategies of neurogenesis.

In this study, we conducted gene expression analysis focusing on SoxB-family transcription factors during early neurogenesis in the gastropod Lymnaea stagnalis. We employed a combination of hybridization chain reaction in situ hybridization (HCR-ISH), immunocytochemistry, confocal microscopy, and cell proliferation assays to investigate the spatial and temporal expression patterns of LsSoxB1 and LsSoxB2 from the gastrula stage to hatching, with particular attention to the formation of central ring ganglia.

Our investigation reveals that LsSoxB1 demonstrates expanded ectodermal expression from the gastrula to the hatching stage, whereas expression of LsSoxB2 in the ectoderm ceases by the veliger stage. LsSoxB1 is expressed in the ectoderm of the head, foot, and visceral complex, as well as in forming ganglia and sensory cells. Conversely, LsSoxB2 is mostly restricted to the subepithelial layer and forming ganglia cells during metamorphosis. Proliferation assays indicate a uniform distribution of dividing cells in the ectoderm across all developmental stages, suggesting the absence of distinct neurogenic zones with increased proliferation in gastropods.

Our findings reveal a spatially and temporally extended pattern of SoxB1 expression in a gastropod representative compared to other lophotrochozoan species. This prolonged and widespread expression of SoxB genes may be interpreted as a form of transcriptional neoteny, representing a preadaptation to prolonged neurogenesis. Consequently, it could contribute to the diversification of nervous systems in gastropods and lead to an increase in the complexity of the central nervous system in Mollusca.

The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors-Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.

Understanding the genetic underpinnings of protein networks conferring stemness is of broad interest for basic and translational research.

We used multi-omics analyses to identify and characterize stemness genes, and focused on the zinc finger protein 982 (Zfp982) that regulates stemness through the expression of Nanog, Zfp42, and Dppa3 in mouse embryonic stem cells (mESC).

Zfp982 was expressed in stem cells, and bound to chromatin through a GCAGAGKC motif, for example near the stemness genes Nanog, Zfp42, and Dppa3. Nanog and Zfp42 were direct targets of ZFP982 that decreased in expression upon knockdown and increased upon overexpression of Zfp982. We show that ZFP982 expression strongly correlated with stem cell characteristics, both on the transcriptional and morphological levels. Zfp982 expression decreased with progressive differentiation into ecto-, endo- and mesodermal cell lineages, and knockdown of Zfp982 correlated with morphological and transcriptional features of differentiated cells. Zfp982 showed transcriptional overlap with members of the Hippo signaling pathway, one of which was Yap1, the major co-activator of Hippo signaling. Despite the observation that ZFP982 and YAP1 interacted and localized predominantly to the cytoplasm upon differentiation, the localization of YAP1 was not influenced by ZFP982 localization.

Together, our study identified ZFP982 as a transcriptional regulator of early stemness genes, and since ZFP982 is under the control of the Hippo pathway, underscored the importance of the context-dependent Hippo signals for stem cell characteristics.

Oxidative stress caused by light and high temperature arises during in vitro maturation (IVM), resulting in low-quality embryos compared with those obtained in vivo. To overcome this problem, we investigated the influence of piperine (PIP) treatment during maturation of porcine oocytes on subsequent embryo development in vitro. Porcine oocytes were cultured in IVM medium supplemented with 0, 50, 100, 200, or 400 μM PIP. After parthenogenetic activation, the blastocyst (BL) formation was significantly higher and the apoptosis rate was significantly lower using 200 μM PIP-treated oocytes (200 PIP). In the 200 PIP group, the level of reactive oxygen species at the metaphase II stage was decreased, accompanied by an increased level of glutathione and increased expression of antioxidant processes (Nrf2, CAT, HO-1, SOD1, and SOD2). Consistently, chromosome misalignment and aberrant spindle organization were alleviated and phosphorylated p44/42 mitogen-activated protein kinase activity was increased in the 200 PIP group. Expression of development-related (CDX2, NANOG, POU5F1, and SOX2), anti-apoptotic (BCL2L1 and BIRC5), and pro-apoptotic (BAK, FAS, and CASP3) processes was altered in the 200 PIP group. Ultimately, embryo development was improved in the 200 PIP group following somatic cell nuclear transfer. These findings suggest that PIP improves the quality of porcine oocytes by reducing oxidative stress, which inevitably arises via IVM. In-depth mechanistic studies of porcine oocytes will improve the efficiencies of assisted reproductive technologies.

Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate reactivation after mitotic gene silencing. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the biological significance and importance of their bookmarking function. Here we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover a large redundancy in mitotic binding among members of the protein super-family of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ESRRB is essential in maintaining pluripotency in mouse embryonic stem cells, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly those most efficiently reactivated in G1. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 μM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

Micro- and nanoplastics have recently been detected in human blood and placentas, indicating inevitable embryonic exposure to these particles. However, their influence on human embryogenesis and the underlying mechanisms are still unknown. In this study, the effects of polystyrene nanoplastics (PS-NPs) exposure on cardiac differentiation of human embryonic stem cells (hESCs) were evaluated. Uptake of PS-NPs not only caused cellular injury, but also regulated cardiac-related pathways as revealed by RNA-sequencing. Consequently, the efficiency of cardiomyocyte differentiation from hESCs was compromised, leading to immature of cardiomyocytes and smaller cardiac organoids with impaired contractility. Mechanistically, PS-NPs promoted mitochondrial oxidative stress, activated P38/Erk MAPK signaling pathway, blocked autophagy flux, and eventually reduced the pluripotency of hESCs. Consistently, in vivo exposure of PS-NPs from cleavage to gastrula period of zebrafish embryo led to reduced cardiac contraction and blood flow. Collectively, this study suggests that PS-NPs is a risk factor for fetal health, especially for heart development.

Due to self-renewal, differentiation, and limitless proliferation properties, Cancer Stem Cells (CSCs) increase the probability of tumor development. These cells are identified by using CSC markers, which are highly expressed proteins on the cell surface of CSCs. Recently, the therapeutic application of CSCs as novel biomarkers improved both the prognosis and diagnosis outcome of colorectal Cancer. In the present review, we focused on a specific panel of colorectal CSC markers, including LGR5, ALDH, CD166, CD133, and CD44, which offers a targeted and comprehensive analysis of their functions. The selection criteria for these markers cancer were based on their established significance in Colorectal Cancer (CRC) pathogenesis and clinical outcomes, providing novel insights into the CSC biology of CRC. Through this approach, we aim to elevate understanding and stimulate further research for developing effective diagnostic and therapeutic strategies in CRC.