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BACKGROUND: The self-renewal and multi-directional differentiation of pluripotent stem cells possess the potential to revolutionize people’s understanding of biology, medicine, development, and disease. Stem cells play an important role in the early stage of embryonic development, and the study of them could be beneficial to understanding of the basic principles of biological development and tissue or organ formation, exploring the potential mechanisms of various diseases, studying the repair and regeneration of damaged tissues or organs, and promoting drug discovery and personalized treatment. OBJECTIVE: To review the research progress of pluripotent stem cells, summarize and categorize the fundamental types of pluripotent stem cells, and elucidate the lineage situations of various types of pluripotent stem cells in common mammals. METHODS: PubMed, Web of Science, CNKI, and WanFang databases were searched systematically, with the keywords “pluripotent stem cells; embryonic stem cells; induced pluripotent stem cells; expanded potential stem cells; livestock pluripotent stem cells” in English and Chinese. The 99 articles related to mammalian pluripotent stem cells were systematically screened according to inclusion and exclusion criteria, and then reviewed. RESULTS AND CONCLUSION: (1) According to classical theory in mouse embryonic stem cell research, the pluripotent state of stem cells is divided into two forms: naïve and primed. Naïve state corresponds to the inner cell mass of pre-implantation embryos before attachment to the uterine wall, while primed state corresponds to the epiblast after implantation. These two states exhibit significant differences in epigenetic features, transcriptional activity, external signal dependency, and metabolic phenotype. It is later discovered that there is an intermediate state between naïve and primed called formative pluripotency. Therefore, the pluripotency of pluripotent stem cells is a continuous developmental process rather than a unique cell state. (2) In addition to obtaining pluripotent stem cells from the inner cell mass, there are various methods and lineages for acquiring pluripotent stem cells, including embryonic germ cells established using primitive germ cells from mouse embryos, induced pluripotent stem cells created by the dedifferentiation of adult mouse and human fibroblasts with four factors-Oct3/4, Sox2, c-Myc, and Klf4; embryonic stem cell-like cell lines cultured from somatic cell nuclear transfer, parthenogenesis, neonatal or adult testicular or ovarian tissue, very small embryonic-like stem cells derived from various adult tissues and expanded pluripotent stem cells derived from pre-implantation stages. These pluripotent stem cells all share the common characteristics of continuous self-renewal, expressing core pluripotency factors and possessing the ability to differentiate into the three primary germ layers. (3) Currently, pluripotent stem cells are being used for disease modeling to study the mechanisms of various diseases and develop new drugs. Simultaneously, scientists are attempting to use pluripotent stem cells to cultivate various tissues and organs, offering new possibilities for regenerative medicine and transplantation. However, the clinical application of pluripotent stem cells faces safety challenges, including issues of cell mutations and immune rejection. Continual improvement in the methods of generating pluripotent stem cells will make them safer and more efficient for clinical applications. (4) Based on the methods of obtaining and lineage establishment of pluripotent stem cells in mice and humans, various types of pluripotent stem cells have been established in livestock, including embryonic stem cells, induced pluripotent stem cells, germ lineages of pluripotent stem cells, and expanded potential stem cells. Research on livestock pluripotent stem cells opens up new avenues for animal reproduction, breeding, genetic engineering, disease modeling, drug screening, and the conservation of endangered wildlife. [ABSTRACT FROM AUTHOR]

BACKGROUND: The small-diameter blood vessel prosthesis faces the problem of lumen stenosis or even occlusion due to thrombogenesis and intimal hyperplasia after transplantation. The application of stem cells as seed cells to achieve endothelialization of blood vessel prosthesis helps to improve the long-term patency rate after vascular graft transplantation. OBJECTIVE: To summarize the research progress of the application of stem cells in the endothelialization of small-diameter blood vessel prosthesis. METHODS: The relevant articles published on PubMed and WanFang databases from 2013 to 2023 were retrieved by the first author. Chinese and English search terms included “vascular graft, tissue-engineered blood vessel/vascular tissue engineering, endothelialization, stem cells, endothelial progenitor cells, mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells.” The relative articles in the domestic and overseas about the application of stem cells in the endothelialization of small-diameter blood vessel prosthesis in the past 10 years were retrieved. A total of 552 articles were initially found and we finally selected 81 articles to review according to inclusion and exclusion criteria. RESULTS AND CONCLUSION: (1) The low long-term patency rate restricts the application of small-diameter blood vessel prosthesis in clinic. The main causes of low long-term patency rate are thrombogenesis and intimal hyperplasia. The endothelium of native vessels has the function of anti-thrombogenesis and intimal hyperplasia. Endothelialization can simulate the function of native vessels, which is an effective way to improve long-term patency rate. (2) The small-diameter blood vessel prosthesis will undergo in vivo endothelialization after being implanted, but it is difficult to form complete endothelium. Stem cells have the potential to differentiate into endothelial cells. Recruiting stem cells in vivo or seeding them on the inner surface of blood vessel prosthesis in vitro is research strategy to achieve endothelialization. (3) The long-term patency rate of small-diameter blood vessel prosthesis has been improved to a certain extent through seeding endothelial progenitor cells, mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells. Each has its own advantages. Endothelial progenitor cells are easy to obtain and can be directly used for seeding. Mesenchymal stem cells come from a wide range of sources and have the function of paracrine and immunological regulation. Induced pluripotent stem cells are rich in sources and the immunogenicity can be eliminated. Embryonic stem cells have a strong proliferative ability and can differentiate into many cells. (4) The application of stem cells in blood vessel prosthesis has not yet been transformed into clinic. Further researches are needed to promote clinical translation. [ABSTRACT FROM AUTHOR]

The development of in vitro gametogenesis (IVG) in the mouse opened up unforeseen possibilities for assisted reproduction. The development of this technology to be used in cattle production could accelerate the rate of genetic selection by dramatically reducing the generation interval, while decreasing the environmental impact of livestock production as the need to grow animals in the process of genetic selection would be reduced or even eliminated. Although several steps of the process of IVG such as in vitro oocyte maturation and fertilization, and embryo production are already routinely performed in cattle, other steps of the system such as in vitro follicle and oocyte development are still rudimentary. The stable derivation of bovine pluripotent stem cells is the starting point without which IVG cannot be realized. However, producing a primordial germ cell and taking this cell through oogenesis and folliculogenesis in a dish will require a more detailed understanding of the milestones that need to be accomplished in vivo before they can be recapitulated in vitro. In particular, understanding the regulatory circuitry of germ cell specification in the embryo, the timing and events related to development of the germ cell program, and the factors necessary to make a competent egg, will need to be uncovered. Here, we review the process of IVG and provide a brief description of the current advances and bottlenecks related to in vitro oogenesis and folliculogenesis in cattle. Finally, we provide a brief comparison between mice and cows in this regard. Producing eggs and sperm in the laboratory is a novel technology that has the potential to reduce the impact of animal agriculture on our planet and accelerate the genetic improvement of cattle. However, there are many pieces of information that we still need to learn about how cows reproduce before we can take advantage of this technology. Recent scientific advances are helping fill these gaps and should lead us toward more efficient and sustainable agriculture. Image by Anna C. Denicol. This article belongs to the Collection: Proceedings of the Annual Conference of the International Embryo Technology Society, Fort Worth, TX, USA, 18–22 January 2025. [ABSTRACT FROM AUTHOR]

Introduction: The rising number of cats as pets and the growing interest in animal welfare have led to an increased need for the latest treatments in feline veterinary medicine. Among these, veterinary regenerative medicine using pluripotent stem cells is gaining significant attention. However, there have been no reports on establishing feline embryonic stem cell (ESC) lines that possess the pluripotent potential and the ability to differentiate into three germ layers. Methods: In this study, we isolated three inner cell masses from feline in vitro-derived blastocysts and subcultured them in a chemically defined medium (StemFit AK02N). We assessed the expression of undifferentiated markers, the ability to differentiate into the three germ layers, and the karyotype structure. Results: We established three feline ESC lines. Feline ESCs exhibited positive staining for alkaline phosphatase. RT-qPCR analysis revealed that these cells express undifferentiated marker genes in vitro. Immunostaining and flow cytometry analysis demonstrated that feline ESCs express undifferentiated marker proteins in vitro. In the KSR/FBS medium with or without Activin A, feline ESCs differentiated into all three germ layers (ectoderm, endoderm, and mesoderm), expressing specific marker genes and proteins for each germ layer, as evidenced by RT-qPCR, immunostaining, and flow cytometry. Furthermore, we confirmed that feline ESCs formed teratomas comprising all three germ layers in mouse testes, demonstrating de novo pluripotency in vivo. We also verified that the feline ESCs maintained a normal karyotype. Conclusions: We successfully established three feline ESC lines, each possessing pluripotent potential and capable of differentiating into all three germ layers, derived from the inner cell masses of blastocysts produced in vitro.

Our understanding of β-cell differentiation from pluripotent stem cells (PSCs) is rapidly evolving. Although progress has been made, challenges remain, particularly in achieving glucose-stimulated insulin secretion (GSIS). Human embryonic stem cells (hESCs) are valuable due to their pluripotent ability. A fixed protocol targeting master regulatory genes initiates stem cells into pancreatic lineage commitment. Due to the observations that a single stem cell can differentiate into multiple cell types depending on various factors and conditions, non-linear differentiation pathways exist. Co-expression of key factors remains essential for successful β-cell differentiation. The mature β-cell marker MAFA plays a critical role in maintaining the differentiation state and preventing dedifferentiation. Recapitulating pancreatic islet clustering enhances physiological responses, offering potential avenues for diabetes treatment. On the other hand, several enhanced differentiation protocols from induced pluripotent stem cells (iPSCs) have improved the functional insulin producing β-cells generated. These findings, with their potential to revolutionize diabetes treatment, highlight the complexity of β-cell differentiation and guide further advancements in regenerative medicine.

Abstract The discovery of embryonic stem cell (ESC) mediating tumoricidal activity revealed the intimate relationship between ESCs and tumor cells, but the functional role of ESCs in tumor progression is poorly understood. To further investigate tumor cell and ESC interactions, we co-cultured mouse ESCs with mouse pancreatic cancer Pan02 cells or mouse melanoma B16-F10 cells in Transwell, and found that tumor cell invasion was significantly inhibited by ESCs. Application of ESCs to tumor-bearing mice resulted in significant inhibition of tumor metastasis in vivo. RNA-Seq analyses of tumor cell and ESC co-cultures identified TP53 and related signalling as major pathways involved in ESC-mediated inhibition of tumor cell invasion and metastasis, which indicated the potential clinical application of ESCs to treat cancer.

The mammalian p57 protein is a member of the CIP/KIP family of cyclin‐dependent kinase inhibitors and plays an essential role in the development of multiple tissues during embryogenesis as well as in the maintenance of tissue stem cells in adults. Although several transcription factors have been implicated in regulating the p57 gene, cis‐elements such as enhancers that regulate its expression have remained ill‐defined. Here we identify a candidate enhancer for the mouse p57 gene (Cdkn1c) within an intron of the Kcnq1 locus by 4C‐seq analysis in mouse embryonic stem cells (mESCs). Deletion of this putative enhancer region with the CRISPR‐Cas9 system or its suppression by CRISPR interference resulted in a marked attenuation of Cdkn1c expression in differentiating mESCs. Our results thus suggest that this region may serve as an enhancer for the p57 gene during early mouse embryogenesis. [ABSTRACT FROM AUTHOR]

The discovery of embryonic stem cell (ESC) mediating tumoricidal activity revealed the intimate relationship between ESCs and tumor cells, but the functional role of ESCs in tumor progression is poorly understood. To further investigate tumor cell and ESC interactions, we co-cultured mouse ESCs with mouse pancreatic cancer Pan02 cells or mouse melanoma B16-F10 cells in Transwell, and found that tumor cell invasion was significantly inhibited by ESCs. Application of ESCs to tumor-bearing mice resulted in significant inhibition of tumor metastasis in vivo. RNA-Seq analyses of tumor cell and ESC co-cultures identified TP53 and related signalling as major pathways involved in ESC-mediated inhibition of tumor cell invasion and metastasis, which indicated the potential clinical application of ESCs to treat cancer. [ABSTRACT FROM AUTHOR]

Stem cell‐derived cardiac spheroids are promising models for cardiac research and drug testing. However, generating contracting cardiac spheroids remains challenging because of the laborious experimental procedure. Here, we present a microfluidic hanging‐heart chip (HH‐chip) that uses a microchannel and flow‐driven system to facilitate cell loading and culture medium replacement operations to reduce the laborious manual handling involved in the generation of a large quantity of cardiac spheroids. The effectiveness of the HH‐chip was demonstrated by simultaneously forming 50 mouse embryonic stem cell‐derived embryonic bodies, which sequentially differentiated into 90% beating cardiac spheroids within 15 days of culture on the chip. A comparison of our HH‐chip method with traditional hanging‐drop and low‐attachment plate methods revealed that the HH‐chip could generate higher contracting proportions of cardiac spheroids with higher expression of cardiac markers. Additionally, we verified that the contraction frequencies of the cardiac spheroids generated from the HH‐chip were sensitive to cardiotoxic drugs. Overall, our results suggest that the microfluidic hanging drop chip‐based approach is a high‐throughput and highly efficient method for generating contracting mouse embryonic stem cell‐derived cardiac spheroids for cardiac toxicity and drug testing applications. [ABSTRACT FROM AUTHOR]

Advances in cell-based therapy, particularly CAR-T cell therapy, have transformed the treatment of hematological malignancies. Although an important step forward for the field, autologous CAR-T therapies are hindered by high costs, manufacturing challenges, and limited efficacy against solid tumors. With ongoing progress in gene editing and culture techniques, engineered stem cells and their application in cell therapy are poised to address some of these challenges. Here, we review stem cell-based immunotherapy approaches, stem cell sources, gene engineering and manufacturing strategies, therapeutic platforms, and clinical trials, as well as challenges and future directions for the field.

Producing chimaeras constitutes the most reliable method of verifying the pluripotency of newly established cells. Moreover, forming chimaeras by injecting genetically modified embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into the embryo is part of the procedure for generating transgenic mice, which are used for understanding gene function. Conventional methods for generating transgenic mice, including the breeding of chimaeras and tetraploid complementation, are time-consuming and cost-inefficient, with significant limitations that hinder their effectiveness and widespread applications. In the present study, we modified the traditional method of chimaera generation to significantly speed up this process by generating mice exclusively derived from ESCs. This study aimed to assess whether fully ESC-derived mice could be obtained by modulating fibroblast growth factor 4 (FGF4) levels in the culture medium and changing the direction of cell differentiation in the chimaeric embryo. We found that exogenous FGF4 directs all host blastomeres to the primitive endoderm fate, but does not affect the localisation of ESCs in the epiblast of the chimaeric embryos. Consequently, all FGF4-treated chimaeric embryos contained an epiblast composed exclusively of ESCs, and following transfer into recipient mice, these embryos developed into fully ESC-derived newborns. Collectively, this simple approach could accelerate the generation of ESC-derived animals and thus optimise ESC-mediated transgenesis and the verification of cell pluripotency. Compared to traditional methods, it could speed up functional studies by several weeks and significantly reduce costs related to maintaining and breeding chimaeras. Moreover, since the effect of stimulating the FGF signalling pathway is universal across different animal species, our approach can be applied not only to rodents but also to other animals, offering its utility beyond laboratory settings. • FGF4-treated ESC chimaeric embryos contain an epiblast composed exclusively of ESCs. • After transfer into recipient mice, embryos develop into fully ESC-derived newborns. • Supplementing culture medium with FGF4 can accelerate the generation of ESC-derived mice. • FGF4 supplementation can optimise the procedure of verifying cell pluripotency. [ABSTRACT FROM AUTHOR]

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

BACKGROUND: Autoimmune regulator gene (Aire) and Wnt signaling pathway play an important role in the maintenance and differentiation of mouse embryonic stem cell pluripotency. However, whether the Wnt signal and Aire are involved in the differentiation of embryonic stem cells to thymic epithelial progenitor cells remains poorly understood. OBJECTIVE: To investigate the relationship of the Wnt signaling pathway and Aire with the differentiation of embryonic stem cells. METHODS: A two-step differentiation method was used to induce mouse embryonic stem cells to differentiate into endoderm and then into thymic epithelial progenitor cells. Mouse embryonic stem cells were infected with Aire shRNA lentivirus, and monoclonal stable strains were screened by puromycin. Mouse embryonic stem cells were collected on days 0, 3 and 10 of the directed induction of differentiation after the induced differentiation by the two-step differentiation method. Cellular immunofluorescence, flow cytometry, western blot assay, and real-time qPCR were used to detect the expression changes of related genes and proteins. RESULTS AND CONCLUSION: (1) Immunofluorescence staining showed positive expression of SSEA1 and OCT4 on day 0 of targeted induction of differentiation. (2) Immunofluorescence staining showed double-positive expression of SOX17 and FOXA2 on day 3 of targeted induction of differentiation. (3) Flow cytometry results showed positive expression of EPCAM1, K5 and K8 on day 10 of targeted induction of differentiation. (4) Compared with undifferentiated mouse embryonic stem cells, the expressions of Wnt7a, β-catenin, and Gsk-3β proteins were elevated, and the expression level of Aire protein was decreased in induced differentiated thymic epithelial progenitor cells. (5) Compared with undifferentiated mouse embryonic stem cells, the expressions of Wnt7a, β-catenin, Gsk-3β and Aire mRNA were elevated in thymic epithelial progenitor cells. (6) Compared with normal cultured mouse embryonic stem cells and their ultimately differentiated thymic epithelial progenitor cells, the expression levels of Wnt7a, β-catenin and Gsk-3β proteins were reduced in mouse embryonic stem cells with knockdown of Aire genes and their final differentiated thymic epithelial progenitor cells. In conclusion, the Wnt signaling pathway and Aire are jointly involved in the process of targeted induction of differentiation of mouse embryonic stem cells into mouse thymic epithelial progenitor cells. [ABSTRACT FROM AUTHOR]

Cell therapy is one of the promising approaches used against type 1 diabetes. Efficient generation of human embryonic stem cell (hESC)-derived pancreatic progenitors (PPs) is of great importance. Since signaling pathways underlying human pancreas development are not yet fully understood, various differentiation protocols are conducted, each considering variable duration, timing, and concentrations of growth factors and small molecules. Therefore, we compared two PP differentiation protocols in static suspension culture. We tested modified protocols developed by Pagliuca et al. (protocol 1) and Royan researchers (protocol 2) until early PP stage. The morphological changes of hESC aggregates during differentiation, and also gene and protein expression after differentiation, were evaluated. Different morphological structures were formed in each protocol. Quantitative gene expression analysis, flow cytometry, and immunostaining revealed a high level of PDX1 expression on day 13 of Royan's differentiation protocol compared to protocol 1. Our data showed that using protocol 2, cells were further differentiated until day 16, showing higher efficiency of early PPs. Moreover, protocol 2 is able to produce hESCs-PPs in a static suspension culture. Since protocol 2 is inexpensive in terms of media, growth factors, and chemicals, it can be used for massive production of PPs using static and dynamic suspension cultures. [ABSTRACT FROM AUTHOR]

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing. [ABSTRACT FROM AUTHOR]

As the most prevalent and reversible internal epigenetic modification in eukaryotic mRNAs, N6-methyladenosine (m6A) post-transcriptionally regulates the processing and metabolism of mRNAs involved in diverse biological processes. m6A modification is regulated by m6A writers, erasers, and readers. Emerging evidence suggests that m6A modification plays essential roles in modulating the cell-fate transition of embryonic stem cells. Mechanistic investigation of embryonic stem cell maintenance and differentiation is critical for understanding early embryonic development, which is also the premise for the application of embryonic stem cells in regenerative medicine. This review highlights the current knowledge of m6A modification and its essential regulatory contribution to the cell fate transition of mouse and human embryonic stem cells.

Abstract Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8+ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8+ T cell responses and increasing the number of CD8+ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. Graphical Abstract

Abstract In recent years, extracellular vesicles (EVs) have attracted significant attention as carriers in intercellular communication. The vast array of information contained within EVs is critical for various cellular activities, such as proliferation and differentiation of multiple cell types. Moreover, EVs are being employed in disease diagnostics, implicated in disease etiology, and have shown promise in tissue repair. Recently, a phenomenon has been discovered in which cellular phenotypes, including the progression of differentiation, are synchronized among cells via EVs. This synchronization could be prevalent in widespread different situations in embryogenesis and tissue organization and maintenance. Given the increasing research on multi-cellular tissues and organoids, the role of EV-mediated intercellular communication has become increasingly crucial. This review begins with fundamental knowledge of EVs and then discusses recent findings, various modes of information transfer via EVs, and synchronization of cellular phenotypes.

Abstract Background Humans’ nervous system has a limited ability to repair nerve cells, which poses substantial challenges in treating injuries and diseases. Stem cells are identified by the potential to renew their selves and develop into several cell types, making them ideal candidates for cell replacement in injured neurons. Neuronal differentiation of embryonic stem cells in modern medicine is significant. Nanomaterials have distinct advantages in directing stem cell function and tissue regeneration in this field. We attempted in this systematic review to collect data, analyze them, and report results on the effect of nanomaterials on neuronal differentiation of embryonic stem cells. Methods International databases such as PubMed, Scopus, ISI Web of Science, and EMBASE were searched for available articles on the effect of nanomaterials on neuronal differentiation of embryonic stem cells (up to OCTOBER 2023). After that, screening (by title, abstract, and full text), selection, and data extraction were performed. Also, quality assessment was conducted based on the STROBE checklist. Results In total, 1507 articles were identified and assessed, and then only 29 articles were found eligible to be included. Nine studies used 0D nanomaterials, ten used 1D nanomaterials, two reported 2D nanomaterials, and eight demonstrated the application of 3D nanomaterials. The main biomaterial in studies was polymer-based composites. Three studies reported the negative effect of nanomaterials on neural differentiation. Conclusion Neural differentiation is crucial in neurological regenerative medicine. Nanomaterials with different characteristics, particularly those cellular regulating activities and stem cell fate, have much potential in neural tissue engineering. These findings indicate a new understanding of potential applications of physicochemical cues in nerve tissue engineering. Graphical Abstract

Cardiac regenerative therapy using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is expected to become an alternative to heart transplantation for severe heart failure. It is now possible to produce large numbers of human pluripotent stem cells (hPSCs) and eliminate non-cardiomyocytes, including residual undifferentiated hPSCs, which can cause teratoma formation after transplantation. There are two main strategies for transplanting hPSC-CMs: injection of hPSC-CMs into the myocardium from the epicardial side, and implantation of hPSC-CM patches or engineered heart tissues onto the epicardium. Transplantation of hPSC-CMs into the myocardium of large animals in a myocardial infarction model improved cardiac function. The engrafted hPSC-CMs matured, and microvessels derived from the host entered the graft abundantly. Furthermore, as less invasive methods using catheters, injection into the coronary artery and injection into the myocardium from the endocardium side have recently been investigated. Since transplantation of hPSC-CMs alone has a low engraftment rate, various methods such as transplantation with the extracellular matrix or non-cardiomyocytes and aggregation of hPSC-CMs have been developed. Post-transplant arrhythmias, imaging of engrafted hPSC-CMs, and immune rejection are the remaining major issues, and research is being conducted to address them. The clinical application of cardiac regenerative therapy using hPSC-CMs has just begun and is expected to spread widely if its safety and efficacy are proven in the near future. • hPSC-CM transplantation improves cardiac function of infarcted animal hearts. • Several administration methods using catheters or injection devices were developed. • Post-transplant arrhythmia is one of the critical hurdles in hPSC-CM transplantation therapy. • Cardiac regenerative therapy using hPSC-CMs has recently been applied in humans. [ABSTRACT FROM AUTHOR]

Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies. Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400. Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46. Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks. [ABSTRACT FROM AUTHOR]

Embryonic stem cell (ESC)-derived epitopes can act as therapeutic tumor vaccines against different types of tumors Jin (Adv Healthc Mater 2023). However, these epitopes have poor immunogenicity and stimulate insufficient CD8+ T cell responses, which motivated us to develop a new method to deliver and enhance their effectiveness. Bacterial outer membrane vesicles (OMVs) can serve as immunoadjuvants and act as a delivery vector for tumor antigens. In the current study, we engineered a new OMV platform for the co-delivery of ESC-derived tumor antigens and immune checkpoint inhibitors (PD-L1 antibody). An engineered Staphylococcal Protein A (SpA) was created to non-specifically bind to anti-PD-L1 antibody. SpyCatcher (SpC) and SpA were fused into the cell outer membrane protein OmpA to capture SpyTag-attached peptides and PD-L1 antibody, respectively. The modified OMV was able to efficiently conjugate with ESC-derived TAAs and PD-L1 antibody (SpC-OMVs + SpT-peptides + anti-PD-L1), increasing the residence time of TAAs in the body. The results showed that the combination therapy of ESC-based TAAs and PD-L1 antibody delivered by OMV had significant inhibitory effects in mouse tumor model. Specifically, it was effective in reducing tumor growth by enhancing IFN-γ-CD8+ T cell responses and increasing the number of CD8+ memory cells and antigen-specific T cells. Overall, the new OMV delivery system is a versatile platform that can enhance the immune responses of ESC-based TAA cancer vaccines. [ABSTRACT FROM AUTHOR]

Conventional cell biology studies focus on cellular responses to chemical signals, but cells also react to mechanical cues like density, size, and substrate rigidity, activating specific gene expression. Embryo development leads to the formation of a gastrula, establishing body structure and germ layers (endoderm, ectoderm, mesoderm) via diverse mechanisms. In humans, gastrulation begins with the Primitive Streak (PS) and T gene expression, guiding epiblast cell migration. Self-regulation occurs in gastruloid models, derived from human embryonic stem cells, capable of differentiation. Mediators like YAP/TAZ and PIEZO1 link density to cellular responses, with ETV4 serving as a link between mechanical environment and gene expression. This research employed a systematic literature review to synthesize relevant studies. Inspired by stem cell advancements, particularly ETV4's role, searches on PubMed yielded three articles meeting inclusion criteria. ES cells maintain undifferentiated states via ETV4 and ETV5. Rapid cell growth deactivates ETV4, prompting differentiation, influenced by mechanical cues. ETV4, ETV5, and SPRY4 regulate the FGF/ERK pathway, modulating sensitivity. High density initiates neuroectodermal cell formation, impacting integrin-actomyosin and FGFR pathways, via ETV4. Fluctuations in density dictate lineage fate, with ETV4 as a key sensor, linking density shifts to lineage determination via the ERK pathway. [ABSTRACT FROM AUTHOR]

As an important posttranslational modification, ubiquitination plays an important role in regulating protein homeostasis in eukaryotic cells. In our previous studies, both the transcriptome and proteome suggested that ubiquitination is involved in the formation of chicken primordial germ cells (PGCs). Here, affinity enrichment combined with liquid chromatography–tandem mass spectrometry (MS/MS) was used to analyze the ubiquitome during the differentiation from embryonic stem cells to PGCs, and we identify that 724 lysine ubiquitinated sites were up-regulated in 558 proteins and 138 lysine ubiquitinated sites were down-regulated in 109 proteins. Furthermore, GO and KEGG enrichment analysis showed that ubiquitination regulates key proteins to participate in the progression of key events related to PGC formation and the transduction of key signals such as Wnt, MAPK, and insulin signals, followed by the detailed explanation of the specific regulatory mechanism of ubiquitination through the combined proteome and ubiquitome analysis. Moreover, both the activation and inhibition of neddylation were detrimental to the maintenance of the biological characteristics of PGCs, which also verified the importance of ubiquitination. In conclusion, this study provides a global view of the ubiquitome during the formation of PGCs by label‐free quantitative ubiquitomics, which lays a theoretical foundation for the formation mechanism and specific application of chicken PGCs. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine condition among women with pleiotropic sequelae possessing reproductive, metabolic, and psychological characteristics. Although the exact origin of PCOS is elusive, it is known to be a complex multigenic disorder with a genetic, epigenetic, and environmental background. However, the pathogenesis of PCOS, and the role of genetic variants in increasing the risk of the condition, are still unknown due to the lack of an appropriate study model. Since the debut of induced pluripotent stem cell (iPSC) technology, the ability of reprogrammed somatic cells to self-renew and their potential for multidirectional differentiation have made them excellent tools to study different disease mechanisms. Recently, researchers have succeeded in establishing human in vitro PCOS disease models utilizing iPSC lines from heterogeneous PCOS patient groups (iPSCPCOS). The current review sets out to summarize, for the first time, our current knowledge of the implications and challenges of iPSC technology in comprehending PCOS pathogenesis and tissue-specific disease mechanisms. Additionally, we suggest that the analysis of polygenic risk prediction based on genome-wide association studies (GWAS) could, theoretically, be utilized when creating iPSC lines as an additional research tool to identify women who are genetically susceptible to PCOS. Taken together, iPSCPCOS may provide a new paradigm for the exploration of PCOS tissue-specific disease mechanisms. [ABSTRACT FROM AUTHOR]

Background: Coating a culture plate with molecules that aid in cell adhesion is a technique widely used to produce animal cell cultures. Extracellular matrix (ECM) is known for its efficiency in promoting adhesion, survival, and proliferation of adherent cells. Gelatin, a cost-effective type of ECM, is widely used in animal cell cultures including feeder-free embryonic stem (ES) cells. However, the optimal concentration of gelatin is a point of debate among researchers, with no studies having established the optimal gelatin concentration. Methods: In this study, we coated plastic plates with gelatin in a concentrationdependent manner and assessed Young’s modulus using atomic force microscopy (AFM) to investigate the microstructure of the surface of each plastic plate. The adhesion, proliferation, and differentiation of the ESCs were compared and analyzed revealing differences in surface microstructure dependent on coating concentration. Results: According to AFM analysis, there was a clear difference in the microstructure of the surface according to the presence or absence of the gelatin coating, and it was confirmed that there was no difference at a concentration of 0.5% or more. ES cell also confirmed the difference in cell adhesion, proliferation, and differentiation according to the presence or absence of gelatin coating, and also it showed no difference over the concentration of 0.5%. Conclusions: The optimum gelatin-coating for the maintenance and differentiation of ES cells is 0.5%, and the gelatin concentration-mediated microenvironment and ES cell signaling are closely correlated.

Abstract Background Prenatal infection has been implicated in the development of neuropsychiatric disorders in children. We hypothesised that exposure to lipopolysaccharide during prenatal development could induce anxiety-like behaviour and sensorineural hearing loss in offspring, as well as disrupt neural differentiation during embryonic neural development. Methods We simulated prenatal infection in FVB mice and mouse embryonic stem cell (ESC) lines, specifically 46C and E14Tg2a, through lipopolysaccharide treatment. Gene expression profiling analyses and behavioural tests were utilized to study the effects of lipopolysaccharide on the offspring and alterations in toll-like receptor (TLR) 2-positive and TLR4-positive cells during neural differentiation in the ESCs. Results Exposure to lipopolysaccharide (25 µg/kg) on gestation day 9 resulted in anxiety-like behaviour specifically in male offspring, while no effects were detected in female offspring. We also found significant increases in the expression of GFAP and CNPase, as well as higher numbers of GFAP + astrocytes and O4+ oligodendrocytes in the prefrontal cortex of male offspring. Furthermore, increased scores for genes related to oligodendrocyte and lipid metabolism, particularly ApoE, were observed in the prefrontal cortex regions. Upon exposure to lipopolysaccharide during the ESC-to-neural stem cell (NSC) transition, Tuj1, Map2, Gfap, O4, and Oligo2 mRNA levels increased in the differentiated neural cells on day 14. In vitro experiments demonstrated that lipopolysaccharide exposure induced inflammatory responses, as evidenced by increased expression of IL1b and ApoB mRNA. Conclusions Our findings suggest that prenatal infection at different stages of neural differentiation may result in distinct disturbances in neural differentiation during ESC—NSC transitions. Furthermore, early prenatal challenges with lipopolysaccharide selectively induce anxiety-like behaviour in male offspring. This behaviour may be attributed to the abnormal differentiation of astrocytes and oligodendrocytes in the brain, potentially mediated by ApoB/E signalling pathways in response to inflammatory stimuli.

Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies.Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400.Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46.Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks.

Background: Humans' nervous system has a limited ability to repair nerve cells, which poses substantial challenges in treating injuries and diseases. Stem cells are identified by the potential to renew their selves and develop into several cell types, making them ideal candidates for cell replacement in injured neurons. Neuronal differentiation of embryonic stem cells in modern medicine is significant. Nanomaterials have distinct advantages in directing stem cell function and tissue regeneration in this field. We attempted in this systematic review to collect data, analyze them, and report results on the effect of nanomaterials on neuronal differentiation of embryonic stem cells. Methods: International databases such as PubMed, Scopus, ISI Web of Science, and EMBASE were searched for available articles on the effect of nanomaterials on neuronal differentiation of embryonic stem cells (up to OCTOBER 2023). After that, screening (by title, abstract, and full text), selection, and data extraction were performed. Also, quality assessment was conducted based on the STROBE checklist. Results: In total, 1507 articles were identified and assessed, and then only 29 articles were found eligible to be included. Nine studies used 0D nanomaterials, ten used 1D nanomaterials, two reported 2D nanomaterials, and eight demonstrated the application of 3D nanomaterials. The main biomaterial in studies was polymer-based composites. Three studies reported the negative effect of nanomaterials on neural differentiation. Conclusion: Neural differentiation is crucial in neurological regenerative medicine. Nanomaterials with different characteristics, particularly those cellular regulating activities and stem cell fate, have much potential in neural tissue engineering. These findings indicate a new understanding of potential applications of physicochemical cues in nerve tissue engineering. [ABSTRACT FROM AUTHOR]

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the threedimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in derepression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability. [ABSTRACT FROM AUTHOR]

Fetal development is one of the most sensitive windows to methylmercury (MeHg) toxicity. Laboratory and epidemiological studies have shown a dose–response relationship between fetal MeHg exposure and neuro performance in different life stages from infants to adults. In addition, MeHg exposure has been reported to be associated with disorders in endoderm-derived organs, such as morphological changes in liver cells and pancreatic cell dysfunctions. However, the mechanisms of the effects of MeHg on non-neuronal organs or systems, especially during the early development of endoderm-derived organs, remain unclear. Here we determined the effects of low concentrations of MeHg exposure during the differentiation of definitive endoderm (DE) cells from human embryonic stem cells (hESCs). hESCs were exposed to MeHg (0, 10, 100, and 200 nM) that covers the range of Hg concentrations typically found in human maternal blood during DE cell induction. Transcriptomic analysis showed that sub-lethal doses of MeHg exposure could alter global gene expression patterns during hESC to DE cell differentiation, leading to increased expression of endodermal genes/proteins and the over-promotion of endodermal fate, mainly through disrupting calcium homeostasis and generating ROS. Bioinformatic analysis results suggested that MeHg exerts its developmental toxicity mainly by disrupting ribosome biogenesis during early cell lineage differentiation. This disruption could lead to aberrant growth or dysfunctions of the developing endoderm-derived organs, and it may be the underlying mechanism for the observed congenital diseases later in life. Based on the results, we proposed an adverse outcome pathway for the effects of MeHg exposure during human embryonic stem cells to definitive endoderm differentiation. [ABSTRACT FROM AUTHOR]

Gene editing in farm animals has produced a range of genetically sterilised hosts for complementation with elite donor cells. The most promising editable target gene is NANOS2 , which allows efficient breeding of a continuous supply of sterile male hosts. Competing complementation approaches have been used to restore the missing germlines by transplanting either spermatogonial stem cells (SSCs) into the testis or embryonic stem cells (ESCs) into early embryos. Both approaches are designed to produce animals that exclusively transmit the elite male donor genotype, but not the germline-disabling mutation, to their offspring. Additional biological barriers to testis colonisation and germline restoration after SSC transplantation were recently identified in mice. Pluripotent ESCs from different livestock species show extensive proliferation, gene editing potential, and a contribution to somatic but not germline chimaeras. Animal breeding drives genetic progress mainly through the male germline. This process is slow to respond to rapidly mounting environmental pressures that threaten sustainable food security from animal protein production. New approaches promise to accelerate breeding by producing chimaeras, which comprise sterile host and fertile donor genotypes, to exclusively transmit elite male germlines. Following gene editing to generate sterile host cells, the missing germline can be restored by transplanting either: (i) spermatogonial stem cells (SSCs) into the testis; or (ii) embryonic stem cells (ESCs) into early embryos. Here we compare these alternative germline complementation strategies and their impact on agribiotechnology and species conservation. We propose a novel breeding platform that integrates embryo-based complementation with genomic selection, multiplication, and gene modification. [ABSTRACT FROM AUTHOR]

Abstract Background Post-translational modifications of proteins are crucial to the regulation of their activity and function. As a newly discovered acylation modification, crotonylation of non-histone proteins remains largely unexplored, particularly in human embryonic stem cells (hESCs). Methods We investigated the role of crotonylation in hESC differentiation by introduce crotonate into the culture medium of GFP tagged LTR7 primed H9 cell and extended pluripotent stem cell lines. RNA-seq assay was used to determine the hESC transcriptional features. Through morphological changes, qPCR of pluripotent and germ layer-specific gene markers and flow cytometry analysis, we determined that the induced crotonylation resulted in hESC differentiating into the endodermal lineage. We performed targeted metabolomic analysis and seahorse metabolic measurement to investigate the metabolism features after crotonate induction. Then high-resolution tandem mass spectrometry (LC–MS/MS) revealed the target proteins in hESCs. In addition, the role of crotonylated glycolytic enzymes (GAPDH and ENOA) was evaluated by in vitro crotonylation and enzymatic activity assays. Finally, we used knocked-down hESCs by shRNA, wild GAPDH and GAPDH mutants to explore potential role of GAPDH crotonylation in regulating human embryonic stem cell differentiation and metabolic switch. Result We found that induced crotonylation in hESCs resulted in hESCs of different pluripotency states differentiating into the endodermal lineage. Increased protein crotonylation in hESCs was accompanied by transcriptomic shifts and decreased glycolysis. Large-scale crotonylation profiling of non-histone proteins revealed that metabolic enzymes were major targets of inducible crotonylation in hESCs. We further discovered GAPDH as a key glycolytic enzyme regulated by crotonylation during endodermal differentiation from hESCs. Conclusions Crotonylation of GAPDH decreased its enzymatic activity thereby leading to reduced glycolysis during endodermal differentiation from hESCs.

Summary: Stem cell technologies provide new opportunities for modeling cells in health and disease and for regenerative medicine. In both cases, developmental knowledge and defining the molecular properties and quality of the cell types is essential. In this study, we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into functional midbrain dopaminergic (mDA) neurons. We found that laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3β inhibition plus FGF8b, improved midbrain patterning. In addition, neurogenesis and differentiation were enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high-quality molecularly defined midbrain cell types, including mDA neurons. Our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly defined hESC-derived cell types in Parkinson disease.