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In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.

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During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

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Endogenous retroviruses (ERVs), once thought to be mere remnants of ancient viral integrations in the mammalian genome, are now recognized for their critical roles in various physiological processes, including embryonic development, innate immunity, and tumorigenesis. Their impact on host organisms is significant driver of evolutionary changes, offering insight into evolutionary mechanisms. In our study, we explored the functionality of ERVs by examining single-cell transcriptomic profiles from human embryonic stem cells and urine cells. This led to the discovery of a unique ERVH48-1 expression pattern between these cell types. Additionally, somatic cell reprogramming efficacy was enhanced when ERVH48-1 was overexpressed in a urine cell-reprogramming system. Induced pluripotent stem cells (iPSCs) generated with ERVH48-1 overexpression recapitulated the traits of those produced by traditional reprogramming approaches, and the resulting iPSCs demonstrated the capability to differentiate into all three germ layers in vitro. Our research elucidated the role of ERVs in somatic cell reprogramming.

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Epidermal transplantation is a common and widely used surgical technique in clinical medicine. Derivatives of embryonic stem cells have the potential to serve as a source of transplantable cells. However, allograft rejection is one of the main challenges. To investigate the immunogenicity of keratinocytes derived from human embryonic stem cells (ESKCs), we conducted a series of in vivo and in vitro experiments. The results showed that ESKCs have low HLA molecule expression, limited antigen presentation capabilities, and a weak ability to stimulate the proliferation and secretion of inflammatory factors in allogeneic PBMCs in vitro. In humanized immune mouse models, ESKCs elicited weak transplant rejection responses in the host. Overall, we found that ESKCs have low immunogenicity and may have potential applications in the field of regenerative medicine.

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DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18-24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes.

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RNA sequencing (RNA-Seq) has become a widely adopted technique for studying gene expression. However, conventional RNA-Seq analyses rely on gene expression (GE) values that aggregate all the transcripts produced under a single gene identifier, overlooking the complexity of transcript variants arising from different transcription start sites or alternative splicing. Transcript variants may encode proteins with diverse functional domains, or noncoding RNAs. This study explored the implications of neglecting transcript variants in RNA-Seq analyses. Among the 1334 transcription factor (TF) genes expressed in mouse embryonic stem (ES) or trophoblast stem (TS) cells, 652 were differentially expressed in TS cells based on GE values (365 upregulated and 287 downregulated, ≥absolute 2-fold changes, false discovery rate (FDR) p-value ≤ 0.05). The 365 upregulated genes expressed 883 transcript variants. Further transcript expression (TE) based analyses identified only 174 (<20%) of the 883 transcripts to be upregulated. The remaining 709 transcripts were either downregulated or showed no significant changes. Meanwhile, the 287 downregulated genes expressed 856 transcript variants and only 153 (<20%) of the 856 transcripts were downregulated. The other 703 transcripts were either upregulated or showed no significant change. Additionally, the 682 insignificant TF genes (GE values < absolute 2-fold changes and/or FDR p-values > 0.05) between ES and TS cells expressed 2215 transcript variants. These included 477 (>21%) differentially expressed transcripts (276 upregulated and 201 downregulated, ≥absolute 2-fold changes, FDR p-value ≤ 0.05). Hence, GE based RNA-Seq analyses do not represent accurate expression levels due to divergent transcripts expression from the same gene. Our findings show that by including transcript variants in RNA-Seq analyses, we can generate a precise understanding of a gene's functional and regulatory landscape; ignoring the variants may result in an erroneous interpretation.

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Embryonic stem cells are crucial for studying developmental biology due to their self-renewal and pluripotency capabilities. This research investigates the differentiation of mouse ESCs into adipocytes, offering insights into obesity and metabolic disorders. Using a monolayer differentiation approach over 30 days, lipid accumulation and adipogenic markers, such as Cebpb, Pparg, and Fabp4, confirmed successful differentiation. RNA sequencing revealed extensive transcriptional changes, with over 15,000 differentially expressed genes linked to transcription regulation, cell cycle, and DNA repair. This study utilized Robust Rank Aggregation to identify critical regulatory genes like PPARG, CEBPA, and EP300. Network analysis further highlighted Atf5, Ccnd1, and Nr4a1 as potential key players in adipogenesis and its mature state, validated through RT-PCR. While key adipogenic factors showed plateaued expression levels, suggesting early differentiation events, this study underscores the value of ESCs in modeling adipogenesis. These findings contribute to our understanding of adipocyte differentiation and have significant implications for therapeutic strategies targeting metabolic diseases.

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Genomic imprinting involves differential DNA methylation and gene expression between homologous paternal and maternal loci. It remains unclear, however, whether DNA replication also shows parent-of-origin-specific patterns at imprinted or other genomic regions. Here, we investigate genome-wide asynchronous DNA replication utilizing uniparental human embryonic stem cells containing either maternal-only (parthenogenetic) or paternal-only (androgenetic) DNA. Four clusters of imprinted genes exhibited differential replication timing based on parent of origin, while the remainder of the genome, 99.82%, showed no significant replication asynchrony between parental origins. Active alleles in imprinted gene clusters replicated earlier than their inactive counterparts. At the Prader-Willi syndrome locus, replication asynchrony spanned virtually the entirety of S phase. Replication asynchrony was carried through differentiation to neuronal precursor cells in a manner consistent with gene expression. This study establishes asynchronous DNA replication as a hallmark of large imprinted gene clusters.

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Background: FOXR2-activated central nervous system (CNS) neuroblastoma (CNS NB-FOXR2) is a recently identified subtype of brain tumor characterized by the elevated expression of the transcription factor FOXR2 mainly due to genomic rearrangements. However, the precise pathogenic mechanisms, including the cell type of origin, remain elusive. Methods: A gene expression analysis of patient tumors was performed to identify putative cell types of origin. Based on this prediction, a new human embryonic stem cell-based model was developed to validate the origin and to examine the molecular and cellular mechanisms underlying the formation of CNS NB-FOXR2. Results: Our data showed that CNS NB-FOXR2 tumors express a high level of lineage marker genes associated with the medial ganglionic eminence (MGE), a transient structure located in the developing ventral forebrain. Our model confirmed the cell-type-specific effect of FOXR2 on the proliferation and in vivo tumorigenicity. Additionally, we found that FOXR2 overexpression activated the MEK/ERK signaling pathway through a suppression of the endogenous RAS inhibitor DIRAS3. The MEK inhibitor trametinib suppressed the proliferation of FOXR2-expressing MGE progenitors more than nonexpressing cells. Conclusions: Our study collectively demonstrates that MGE progenitors are the cell of origin of CNS NB-FOXR2 and that FOXR2 activates the MEK/ERK signaling pathway, providing a potential therapeutic target.

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Individuals are exposed to a wide arrays of hazardous chemicals on a daily basis through various routes, many of which have not undergone comprehensive toxicity assessments. While traditional developmental toxicity tests involving pregnant animals are known for their reliability, they are also associated with high costs and time requirements. Consequently, there is an urgent demand for alternative, cost-efficient, and rapid in vitro testing methods. This study aims to address the challenges related to automating and streamlining the screening of early developmental toxicity of chemicals by introducing a mouse embryoid body test (EBT) model in a 384-ultra low attachment well format. Embryoid bodies (EBs) generated in this format were characterized by a spontaneous differentiation trajectory into cardiac mesoderm by as analyzed by RNA-seq. Assessing prediction accuracy using reference compounds suggested in the ICH S5(R3) guideline and prior studies resulted in the establishment of the acceptance criteria and applicability domain of the EBT model. The results indicated an 84.38% accuracy in predicting the developmental toxicity of 23 positive and 9 negative reference compounds, with an optimized cutoff threshold of 750 µM. Overall, the developed EBT model presents a promising approach for more rapid, high-throughput chemical screening, thereby facilitating well-informed decision-making in environmental management and safety assessments.

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Bioactive lipids like sphingosine-1-phosphate (S1P) and lysophosphatidic acid have gained significant attention as signaling molecules with regulatory roles in stem cell proliferation and differentiation. The novel chemically synthesized sphingosine metabolite O-cyclic phytosphingosine-1-phosphate (cP1P) is derived from phytosphingosine-1-phosphate (P1P) and shares structural similarities with S1P. Previously, the role of cP1P in regulating ALK3/BMPR signaling during cardiomyocyte differentiation from human embryonic stem cells (hESCs) was demonstrated. In this study, the applicability of cP1P for endothelial cells (ECs) differentiation from hESCs was investigated an efficient method to obtain a high yield of functional ECs over several passages was standardized. The ECs derived from hESCs showed cellular and molecular characteristics similar to the native ECs. Thus, the results of this study open avenues for further research into cP1P-based stem cell differentiation for regenerative therapies.

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

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Gastrulation is a key milestone in the development of an organism. It is a period of cell proliferation and coordinated cellular rearrangement, that creates an outline of the body plan. Our current understanding of mammalian gastrulation has been improved by embryo culture, but there are still many open questions that are difficult to address because of the intrauterine development of the embryos and the low number of specimens. In the case of humans, there are additional difficulties associated with technical and ethical challenges. Over the last few years, pluripotent stem cell models are being developed that have the potential to become useful tools to understand the mammalian gastrulation. Here we review these models with a special emphasis on gastruloids and provide a survey of the methods to produce them robustly, their uses, relationship to embryos, and their prospects as well as their limitations.

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Selection strategies are performed post-fertilization when the random combination of paternal and maternal genomes has already occurred. It would be greatly advantageous to eliminate meiotic uncertainty by selecting genetically superior gametes before fertilization. To achieve this goal, haploid embryonic cells and embryonic stem cell lineages could be derived, genotyped, and used to substitute gametes. On the paternal side, androgenetic development can be achieved by removing the maternal chromosomes from the oocyte before or after fertilization. We have shown that once developed into an embryo, haploid cells can be removed for genotyping and, if carrying the selected genome, be used to replace sperm at fertilization. A similar strategy can be used on the maternal side by activating the oocyte parthenogenetically and using some embryonic cells for genotyping while the remaining are used to produce diploid embryos by fertilization. Placed together, both androgenetic and parthenogenetic haploid cells that have been genotyped to identify optimal genomes can be used to produce offspring with predetermined genomes. Successes and problems in developing such a breeding platform to achieve this goal are described and discussed below.

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

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Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates.

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OBJECTIVE: There are ethical and technical challenges in studying human germ cell development. Therefore, the aim of the study is in vitro differentiation of human embryonic stem cells (hESCs), as pluripotent cells, to the germ cells which is a valuable tool for studying molecular and cellular aspects of gametogenesis and understanding causes of infertility. MATERIALS AND METHODS: In this experimental study, two different complete media [Dulbecco's Modified Eagle Medium (DMEM)+20% fetal bovine serum (FBS) and embryoid bodies (EBs) medium; KOSR/HES without basic fibroblast growth factor (bFGF)] were used in the both of test groups using testicular cells derived conditioned medium (TCCM) and control groups spontaneously differentiated (SD). Thereby, EBs from hESCs (Yazd2; 46XY) were cultured in different conditions EB medium; EB medium and conditioned EB medium; EB medium, DMEM, and FBS without conditioning; EB medium, conditioned DMEM, and FBS medium. EBs were collected after 4, 7, and 14 days and their gene expression profiles were assessed and compared to hESCs, as day 0, using IF and relative reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: An increase in the gametogenesis gene expression level in TCCM groups was showed in comparison with SD groups. Additionally, immunostaining of differentiated cells in all groups showed in vitro gametogenesis (IVG). CONCLUSION: Our findings showed that human TCCM could be used as a natural niche for in vitro male and female germ cell development. However, further studies are needed to define the factors and metabolites within the human TCCM.

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Investigating the sevoflurane-induced perturbation in the differentiation of mouse embryonic stem cells (mESCs) into neural stem cells (mNSCs), our study delineates a novel SIRT1/PRRX1/DRD2/PKM2/NRF2 axis as a key player in this intricate process. Sevoflurane treatment hindered mESC differentiation, evidenced by altered expression patterns of pluripotency and neural lineage markers. Mechanistically, sevoflurane downregulated Sirt1, setting in motion a signaling cascade. Sevoflurane may inhibit PKM2 dimerization and NRF2 signaling pathway activation by inhibiting the expression of SIRT1 and its downstream genes Prrx1 and DRD2, ultimately inhibiting mESCs differentiation into mNSCs. These findings contribute to our understanding of the molecular basis of sevoflurane-induced neural toxicity, presenting a potential avenue for therapeutic intervention in sevoflurane-induced perturbation in the differentiation of mESCs into mNSCs by modulating the SIRT1/PRRX1/DRD2/PKM2/NRF2 axis.

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The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

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Embryonic stem cells (ESCs) are remarkable for the high activity level of ubiquitin-proteasome system-the molecular machinery of protein degradation in the cell. Various forms of the proteasome complexes comprising different subunits and interacting regulators are responsible for the substrate selectivity and degradation. Immunoproteasomes are amongst these forms which play an important role in antigen presentation; however, a body of recent evidence suggests their functions in pluripotent stem cells. Previous studies have established three consecutive phases of pluripotency, featured by epiblast cells and their cultured counterparts: naïve, formative, and primed phase. In this work, we report that immunoproteasomes and their chaperone co-regulators are suppressed in the naïve state but are readily upregulated in the formative phase of the pluripotency continuum, featured by epiblast-like cells (EpiLCs). Our data lay ground for the further investigation of the biological functions of immunoproteasome in the regulation of proteostasis during early mammalian development.

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