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Mammalian tail length is controlled by several genetic determinants, among which are Hox13 genes, whose function is to terminate the body axis. Accordingly, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing a high number of CTCF sites, potentially isolating Hoxb13 from the rest of the cluster and thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes. These defects were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant Embryonic Stem (ES) cells grown as pseudoembryos in vitro, which allowed us to examine in detail the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB, and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations were already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

The systems of intermolecular interactions, including hydrogen bonds, in the crystal structures of the cocrystal of L -ascorbic (L -Asc) and nicotinic (Nic) acids and individual coformers are considered, and the hemolytic and cytotoxic activities of these compounds are studied in vitro. The influence of conformational differences of L -Asc molecules (total effective charge) on the formation of a network of H-bonds in the supramolecular structure of pristine L -Asc is revealed. Calculation of regioselectivity descriptors shows that the conformer of the L -Asc molecule in the L -AscNic cocrystal has higher activity compared to conformer molecules of the pristine L -Asc. No hemolysis caused by the solutions of tested acids and L -AscNic is shown. Selective cytotoxicity (antimetabolic effect according to the MTT test) of solutions of L -AscNic cocrystals against a 72-hour culture of MCF-7 breast cancer cell line versus healthy cells (mesenchymal stem cells and embryonic fibroblasts) is revealed. The possible relationship between the initial conformational state of the L -Asc molecules in the composition/structure of the studied solid compounds and the biological activity of solutions of these compounds requires further studies. [ABSTRACT FROM AUTHOR]

Background: Chimeric antigen receptor (CAR) engineered natural killer (NK) cells exhibit advantages such as MHC-independent recognition and strong anti-tumor functions. However, allogeneic CAR-NK cells derived from human tissues are heterogeneous and susceptible to clearance by hosts. Methods: We generated a B2M knockout, HLA-E and CD19 CAR ectopic expressing embryonic stem cell (ESC) line, which differentiated normally and gave rise to homogeneous CD19 CAR-NK (CD19 CAR-UiNK) cells using an organoid aggregate induction method. The CD19 CAR-UiNK were co-cultured with T cells or NK cells derived from peripheral blood mononuclear cells (PBMC) with the mismatched HLA to evaluate the immunogenicity of CD19 CAR-UiNK cells. We further assessed the therapeutic effects of CD19 CAR-UiNK cells on CD19+ tumor cells through in vitro cytotoxicity assays and in vivo animal models. Results: The CD19 CAR-UiNK cells exhibited typical expression patterns of activating and inhibitory receptors, and crucial effector molecules of NK cells, similar to those of unmodified NK cells. In co-culture assays, the CD19 CAR-UiNK cells evaded allogeneic T cell response and suppressed allogeneic NK cell response. Functionally, the CD19 CAR-UiNK cells robustly secreted IFN-γ and TNF-α, and upregulated CD107a upon stimulation with Nalm-6 tumor cells. The CD19 CAR-UiNK cells effectively eliminated CD19+ tumor cells in vitro , including B-cell cancer cell lines and primary tumor cells from human B-cell leukemia and lymphoma. Further, the CD19 CAR-UiNK cells exhibited strong anti-tumor activity in xenograft animals. Conclusion: We offer a strategy for deriving homogeneous and hypoimmunogenic CD19 CAR-iNK cells with robust anti-tumor effects from ESCs. Our study has significant implications for developing hypoimmunogenic CD19 CAR-NK cell therapy using human ESC as an unlimited cell source. [ABSTRACT FROM AUTHOR]

H3.3 is a highly conserved nonreplicative histone variant. In mammalian cells, H3.3 is enriched in both active chromatin and suppressed heterochromatin. H3.3 is associated with development and cell fate maintenance. H3.3 is also involved in heterochromatin formation and silencing of developmental genes. Multiple H3.3 mutations cause cancer or neurodegeneration. H3.3 is a highly conserved nonreplicative histone variant. H3.3 is enriched in promoters and enhancers of active genes, but it is also found within suppressed heterochromatin, mostly around telomeres. Accordingly, H3.3 is associated with seemingly contradicting functions: It is involved in development, differentiation, reprogramming, and cell fate, as well as in heterochromatin formation and maintenance, and the silencing of developmental genes. The emerging view is that different cellular contexts and histone modifications can promote opposing functions for H3.3. Here, we aim to provide an update with a focus on H3.3 functions in early mammalian development, considering the context of embryonic stem cell maintenance and differentiation, to finally conclude with emerging roles in cancer development and cell fate transition and maintenance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Stem cell research is the product of cumulative, integrated effort between and within laboratories and disciplines. The many collaborative steps that lead to that special "Eureka moment", when something that has been a puzzle perhaps for years suddenly become clear, is among the greatest pleasures of a scientific career. In this essay, the serendipitous pathway from first acquaintance with pluripotent stem cells to advanced cardiovascular models that emerged from studying development and disease will be described. Perhaps inspiration for later generations of stem cell researchers simply to follow whatever they find interesting. [ABSTRACT FROM AUTHOR]

Pluripotent stem cell lines derived from preimplantation mouse embryos have opened opportunities for the study of early mammalian development and generation of genetically uncompromised material for differentiation into specific cell types. Murine embryonic stem cells are highly versatile and can be engineered and introduced into host embryos, transferred to recipient females, and gestated to investigate gene function at multiple levels as well as developmental mechanisms, including lineage segregation and cell competition. In this review, we summarize the biomedical motivation driving the incremental modification to culture regimes and analyses that have advanced stem cell research to its current state. Ongoing investigation into divergent mechanisms of early developmental processes adopted by other species, such as agriculturally beneficial mammals and birds, will continue to enrich knowledge and inform strategies for future in vitro models. [ABSTRACT FROM AUTHOR]

Hypertranscription occurs when cells upregulate gene expression across the majority of the transcriptome, including at housekeeping and other ubiquitously expressed genes. Although masked by standard normalization procedures, hypertranscription can be detected using modifications to sample preparation and normalization of sequencing data. Originally characterized in embryonic stem cells, recent advances have identified hypertranscription to be pervasive in stem cells across development, reprogramming, organ homeostasis, and tissue regeneration. These contexts share unified mechanistic features of hypertranscription, many of which are redeployed in hypertranscribing cancer cells. As a counterpart to the hypertranscriptive state, emerging evidence suggests that global downregulation of transcriptional output to enter hypotranscription is a key component of embryonic diapause and stem cell quiescence, possibly also being co-opted by dormant cancer cells to survive chemotherapy. Stem cells are the fundamental drivers of growth during development and adult organ homeostasis. The properties that define stem cells – self-renewal and differentiation – are highly biosynthetically demanding. In order to fuel this demand, stem and progenitor cells engage in hypertranscription, a global amplification of the transcriptome. While standard normalization methods in transcriptomics typically mask hypertranscription, new approaches are beginning to reveal a remarkable range in global transcriptional output in stem and progenitor cells. We discuss technological advancements to probe global transcriptional shifts, review recent findings that contribute to defining hallmarks of stem cell hypertranscription, and propose future directions in this field. [ABSTRACT FROM AUTHOR]

Targeted mouse mutants are a common tool used to investigate gene function. The International Knockout Mouse Consortium undertook a large-scale screen of mouse mutants, making use of the knockout-first allele design that contains the En2 splice acceptor sequence coupled to the lacZ reporter gene. Although the knockout-first allele was designed to interfere with splicing and thus disrupt gene function, the En2 sequence has been reported to be transcribed within the host gene mRNA due to a cryptic splice site within the En2 sequence which allows splicing to the next exon of the host gene. In some circumstances, this has the potential to permit translation of a mutant protein. Here, we describe our computational analysis of all the mouse protein-coding genes with established knockout-first embryonic stem cell lines, and our predictions of their transcription outcome should the En2 sequence be included. As part of the large-scale mutagenesis program, mutant mice underwent a broad phenotyping screen, and their phenotypes are available. No wide-scale effects on mouse phenotypes reported were found as a result of the predicted En2 insertion. However, the En2 insertion was found experimentally in the transcripts of 24 of 35 mutant alleles examined, including the five already described, two with evidence of readthrough. Splicing from the cryptic splice site also has the potential to disrupt expression of the lacZ reporter gene. It is recommended that mutant transcripts be checked for this insertion as well as for leaky transcription in studies involving knockout-first alleles. [ABSTRACT FROM AUTHOR]

Background: Polycomb proteins are conventionally known as global repressors in cell fate determination. However, recent observations have shown their involvement in transcriptional activation, the mechanisms of which need further investigation. Methods: Herein, multiple data from ChIP-seq, RNA-seq and HiChIP before or after RYBP depletion in embryonic stem cell (ESC), epidermal progenitor (EPC) and mesodermal cell (MEC) were analyzed. Results: We found that Polycomb protein RYBP occupies super-enhancer (SE) in ESCs, where core Polycomb group (PcG) components such as RING1B and EZH2 are minimally enriched. Depletion of RYBP results in impaired deposition of H3K27ac, decreased expression of SE-associated genes, and reducing the transcription of enhancer RNA at SE regions (seRNA). Regarding the mechanism of seRNA transcription, the Trithorax group (TrxG) component WDR5 co-localizes with RYBP at SEs, and is required for seRNA expression. RYBP depletion reduces WDR5 deposition at SE regions. In addition, TrxG-associated H3K4me3 tends to be enriched at SEs with high levels of seRNA transcription, and RYBP deficiency impairs the deposition of H3K4me3 at SEs. Structurally, RYBP is involved in both intra- and inter-SE interactions. Finally, RYBP generally localizes at SEs in both in vitro cell lines and in vivo tissue-derived cells, dysfunction of RYBP is associated with various cancers and developmental diseases. Conclusion: RYBP cooperates with TrxG component to regulate SE activity. Dysfunction of RYBP relates to various diseases. The findings provide new insights into the transcriptionally active function of Polycomb protein in cell fate determination. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) sourced from the inner cell mass of blastocysts, are akin to this tissue in function but lack the capacity to form all extraembryonic structures. mESCs are transient cell populations that express high levels of transcripts characteristic of 2-cell (2C) embryos and are identified as "2-cell-like cells" (2CLCs). Previous studies have shown that 2CLCs can contribute to both embryonic and extraembryonic tissues upon reintroduction into early embryos. Approximately 1% of mESCs dynamically transition from pluripotent mESCs into 2CLCs. Nevertheless, the scarcity of mammalian embryos presents a significant challenge to the molecular characterization of totipotent cells. To date, Previous studies have explored various methods for reprogramming pluripotent cells into totipotent cells. While there is a good understanding of the molecular regulatory network maintaining ES pluripotency, the process by which pluripotent ESCs reprogram into totipotent cells and the associated molecular mechanisms of totipotent regulation remain poorly understood. This review synthesizes recent insights into the regulatory pathways of ESC reprogramming into 2CLC, exploring molecular mechanisms modulated by transcriptional regulators, small molecules, and epigenetic changes. The objective is to construct a theoretical framework for the field of researchers. [ABSTRACT FROM AUTHOR]

Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations. Here the authors measure how the absence of certain DNA repair genes change the frequencies of unique mutational outcomes generated by Cas9 DSBs at synthetic target sequences in mouse embryonic stem cells: they use this to build predictive models of the mutagenic outcomes of Cas9 scission. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis plays a pivotal role in maintaining stem cell homeostasis, yet the precise regulatory mechanisms governing this process in mouse embryonic stem cells (mESCs) remain largely unknown. In this investigation, we ascertain that DEAD-box RNA helicase 10 (DDX10) is indispensable for upholding cellular homeostasis and the viability of mESCs. Positioned predominantly at the nucleolar dense fibrillar component (DFC) and granular component (GC), DDX10 predominantly binds to 45S ribosomal RNA (rRNA) and orchestrates ribosome biogenesis. Degradation of DDX10 prevents the release of U3 snoRNA from pre-rRNA, leading to perturbed pre-rRNA processing and compromised maturation of the 18S rRNA, thereby disrupting the biogenesis of the small ribosomal subunit. Moreover, DDX10 participates in the process of liquid-liquid phase separation (LLPS), which is necessary for efficient ribosome biogenesis. Notably, the NUP98-DDX10 fusion associated with acute myelocytic leukemia (AML) alters the cellular localization of DDX10 and results in loss of ability to regulate pre-rRNA processing. Collectively, this study reveals the critical role of DDX10 as a pivotal regulator of ribosome biogenesis in mESCs. Ribosome biogenesis is crucial for maintaining stem cell homeostasis. Here, authors reveal that DDX10 plays a critical role in ribosome biogenesis by regulating 18S rRNA maturation, which is vital for the proliferation and maintenance of mESCs. [ABSTRACT FROM AUTHOR]

A population of CD133+lin-CD45- and CD34+lin-CD45- very small embryonic-like stem cells (VSELs) has been identified in postnatal human tissues, including bone marrow (BM), mobilized peripheral blood (mPB) and umbilical cord blood (UCB). Under appropriate conditions, VSELs in vitro and in vivo differentiate into tissue-committed stem cells for all three germ layers. Molecular analysis of adult murine BM-purified VSELs revealed that these rare cells deposited during development in adult tissues (i) express a similar transcriptome as embryonic stem cells, (ii) share several markers characteristic for epiblast and migratory primordial germ cells (PGCs), (iii) highly express a polycomb group protein enhancer of zeste drosophila homolog 2 (Ezh2) and finally (iv) display a unique pattern of imprinting at crucial paternally inherited genes that promotes their quiescence. Here, by employing single-cell RNA sequencing we demonstrate for the first time that purified from UCB human VSELs defined by expression of CD34 or CD133 antigens and lack of lineage markers, including CD45 antigen express similar molecular signature as murine BM-derived VSELs. Specifically, unsupervised clustering revealed numerous subpopulations of VSELs including ones i) annotated to germline compartments, ii) regulated by parental imprinting, iii) responding to early developmental fate decisions, iv) transcription factors involved in differentiation and development, including homeobox family of genes, and v) expressing innate immunity and purinergic signaling genes. [ABSTRACT FROM AUTHOR]

The document is a correction to an article titled "In Vitro Gametogenesis: Towards Competent Oocytes" published in BioEssays. It provides additional references for Table 1, which summarizes key studies on reconstitution of mouse germ cell development. The correction includes reference numbers and details of studies involving the development of various cell types and strategies for oocyte and sperm production from different origins. The references cover a range of experiments and outcomes related to in vitro gametogenesis. [Extracted from the article]

Purpose: This study investigates the effects of hexagonal microtextured titanium sheets on fibroblast growth and collagen synthesis, crucial factors in anastomotic healing. Materials and methods: Hexagonal titanium sheets were fabricated using a laser microtextured machine. Mouse fibroblastic embryonic stem cells (NIH/3T3) were cultured on these sheets. Cell proliferation was assessed using a CCK8 assay, and expression of TGF-β/Smad pathway-related genes and collagen types I and III was evaluated through qRT-PCR and western blot. Results: Hexagonal titanium sheets significantly enhanced fibroblast growth and collagen synthesis. The 50–30 group, with the smallest contact angle (48 ± 2.3°), exhibited the highest cell growth rates by CCK8 assay. Gene expression analysis revealed that TGF-β1, Smad2, Smad3, Smad4, and COL1A1 were significantly upregulated in the 50–30 group on day 7. Meanwhile, type I collagen expression was significantly increased in the 50–30 group on day 7 by western blot analysis. Conclusion: Our findings demonstrate that laser-fabricated hexagonal microtextured titanium sheets enhance hydrophilicity and promote fibroblast growth, activating the TGF-β/Smad pathway to facilitate collagen synthesis. These results have important implications for tissue repair and regeneration. [ABSTRACT FROM AUTHOR]

Stem cell therapies have emerged as a promising approach in regenerative medicine, demonstrating potential in personalized medicine, disease modeling, and drug discovery. Therapies based on induced pluripotent stem cells (iPSCs) particularly stand out for their ability to differentiate into various cell types while avoiding ethical concerns. However, the development and application of these therapies are influenced by varying regulatory frameworks across countries. This study provides a comparative analysis of regulations and research on stem cell therapies in key regions: The European Union (EU), Switzerland, South Korea, Japan, and the United States. First, the study reviews the regulatory frameworks on stem cell therapies. The EU and Switzerland maintain rigorous guidelines that prioritize safety and ethical considerations, which can hinder innovation. In contrast, the United States adopts a more flexible regulatory stance, facilitating the rapid development of stem cell therapies. South Korea and Japan take a balanced approach by incorporating practices from both regimes. These regulatory differences reflect each country's unique priorities and impact the pace and scope of stem cell therapy development. Moreover, the study examines global trends in clinical trials on stem cell treatments based on data obtained from two sources: ClinicalTrials.gov and ICTRP. Findings indicate a significant growth in the number of clinical trials since 2008, particularly in that involving iPSCs. Therapeutic studies involving iPSCs predominantly target conditions affecting the cardiovascular and nervous systems which are considered vital. The results put emphasis on the safety of stem cell treatments. Meanwhile, the number of such trials also varies by country. The United States and Japan, where relatively flexible guidelines on stem cell research are adopted, are in a leading position. However, countries in the EU fall behind with rigorous regulations imposed. This reflects the need for more flexible regulatory guidance for active development of stem cell therapies. The findings underscore the importance of legal frameworks in facilitating innovation while ensuring safety. Regulatory agencies in different countries should collaborate to achieve a balanced global standard to ensure the safe and efficient advancement of stem cell therapies. Global regulatory convergence will promote international collaboration in research and the applicability of new treatments. [ABSTRACT FROM AUTHOR]

Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Here, we reproduce cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages with gastruloids from mouse embryonic stem cells. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm development and differentiation in gastruloids compared to mouse embryos, we present the evidence for skeletal myogenesis in gastruloids. We identify different subpopulations of cardiomyocytes and skeletal muscles, the latter of which most likely correspond to different states of myogenesis with "head-like" and "trunk-like" skeletal myoblasts. In this work, we unveil the potential of gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases. The cardiopharyngeal mesoderm gives rise to both heart and head musculature, though how these populations are specified remains less clear. Here they use mouse gastruloids to model cardiopharyngeal mesoderm specification and differentiation into both cardiac and skeletal muscle lineages, thus highlighting their potential for studying muscle development and congenital diseases. [ABSTRACT FROM AUTHOR]

The formation of transcription regulatory complexes by the association of Smad4 with Smad2 and Smad3 (Smad2/3) is crucial in the canonical TGFβ pathway. Although the central requirement of Smad4 as a common mediator is emphasized in regulating TGFβ signaling, it is not obligatory for all responses. The role of Smad2/3 independently of Smad4 remains understudied. Here, we introduce a stepwise paradigm in which Smad2/3 regulate the lineage priming and differentiation of mouse embryonic stem cells (mESCs) by collaboration with different effectors. During the naïve-to-primed transition, Smad2/3 upregulate DNA methyltransferase 3b (Dnmt3b), which establishes the proper DNA methylation patterns and, in turn, enables Smad2/3 binding to the hypomethylated centers of promoters and enhancers of epiblast marker genes. Consequently, in the absence of Smad2/3, Smad4 alone cannot initiate epiblast-specific gene transcription. When primed epiblast cells begin to differentiate, Dnmt3b becomes less actively engaged in global genome methylation, and Smad4 takes over the baton in this relay race, forming a complex with Smad2/3 to support mesendoderm induction. Thus, mESCs lacking Smad4 can undergo the priming process but struggle with the downstream differentiation. This work sheds light on the intricate mechanisms underlying TGFβ signaling and its role in cellular processes. The role of Smad2/3 in the TGFβ pathway, independent of Smad4, is understudied. Authors reveal distinct roles for Smad2/3 and Smad4 during mESC priming to differentiation, and uncover a collaboration of Smad2/3 with Dnmt3b in epiblast formation. [ABSTRACT FROM AUTHOR]

Embryonic stem cells possess the remarkable ability to self-organize into blastocyst-like structures upon induction. These stem cell-based embryo models serve as invaluable platforms for studying embryogenesis and therapeutic developments. Nevertheless, the specific intrinsic regulators that govern this potential for blastoid formation remain unknown. Here we demonstrate an intrinsic program that plays a crucial role in both blastoids and blastocysts across multiple species. We first establish metrics for grading the resemblance of blastoids to mouse blastocysts, and identify the differential activation of gene regulons involved in lineage specification among various blastoid grades. Notably, abrogation of nuclear receptor subfamily 1, group H, member 2 (Nr1h2) drastically reduces blastoid formation. Nr1h2 activation alone is sufficient to rewire conventional ESC into a distinct pluripotency state, enabling them to form blastoids with enhanced implantation capacity in the uterus and contribute to both embryonic and extraembryonic lineages in vivo. Through integrative multi-omics analyses, we uncover the broad regulatory role of Nr1h2 in the transcriptome, chromatin accessibility and epigenome, targeting genes associated with embryonic lineage and the transposable element SINE-B1. The Nr1h2-centred intrinsic program governs and drives the development of both blastoids and early embryos. Intrinsic regulators of stem cell-based embryo models are understudied. Here, the authors show that Nr1h2 activation allows ESCs to form blastoids with better implantation and higher blastocyst rates via coactivator Kdm1a and the Nr1h2-SINE B1 axis. [ABSTRACT FROM AUTHOR]

DNA methylation at non-CG dinucleotides (mCH, H=A, C, T) widely occurs and plays an important role in specific cell types, including pluripotent, neural, and germ cells. However, the functions and regulatory mechanisms of mCH, particularly in species other than humans and mice, remain inadequately explored. In this study, we analyzed the distribution of mCH across different bovine tissues, identifying significantly elevated mCH levels in bovine embryonic stem cells (bESCs), as well as brain, spleen, and ileum tissues compared to other tissues. Marked differences in mCH patterns between somatic cells and bESCs were observed, reflecting distinct base preferences and the differential expression of DNA methyltransferases. We also identified exon methylation in both CG and nonCG contexts, resembling gene-associated methylation patterns observed in plants. To characterize tissue-specific variations in mCH, we developed a novel method for differential mCH analysis. Results indicated that mCH is not randomly distributed but tends to be enriched in tissuespecific functional regions. Furthermore, regression models demonstrated a positional correlation between CG methylation and mCH. This study enhances our understanding of mCH distribution and function in bovine somatic and stem cells, providing new insights into its potential roles across species and tissues. These findings advance knowledge of epigenetic mechanisms, shedding light on the potential involvement of mCH in development and disease processes. [ABSTRACT FROM AUTHOR]

Thyroid gland diseases remain clinical challenges due to the lack of reliable in vitro models to examine molecular pathways of thyrocytes development, maturation, and functional maintenance. This study aimed to develop in vitro thyrocytes model using a stem cell culture, P19 embryonal carcinoma which requires no feeder layer, differentiation into mature and functional thyrocytes that allow molecular and genetic manipulation for studying thyroid diseases. The procedure utilizes Activin A and thyroid stimulating hormone (TSH) to first induce embryoid body endoderm formation enriched in thyrocyte progenitors. Following dissociating embryoid bodies, thyrocyte progenitors are plated in Matrigel as monolayer cultures that allows thyrocyte progenitors mature to functional thyrocytes. These thyrocytes further maturate to form follicle-like structures expressing and accumulating thyroglobulin that can be secreted into the medium upon TSH stimulation. Thyrocyte differentiation-maturation process is monitored by the expression of essential transcriptional factors and thyrocyte-specific functional genes. Further, the applicability of this system is validated by introducing a siRNA control. Following molecular manipulation, the system can still be guided to differentiate into mature and functional thyrocytes. This system spans a time frame of 14 days, suitable for detailed molecular studies to dissect pathways and molecular players in thyrocytes development and functional maintenance. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications. [ABSTRACT FROM AUTHOR]

Stem cell-based therapy has gained importance over the past decades due to huge advances in science and technology behind the generation and directed differentiation of pluripotent cells from embryos and adult cells. Preclinical proof-of-concept studies have been followed by clinical trials showing efficacy and safety of transplantation of stem cell-based therapy, which are beginning to establish this as a modality of treatment. Disease candidates of interest are primarily conditions that may benefit from replacing dead or dying cells, including advanced inherited retinal dystrophies and age-related macular degeneration, and predominantly seek to transplant either RPE or photoreceptors, although neurotrophic approaches have also been trialed. Whilst a consensus has yet to be reached about the best stage/type of cells for transplantation (stem cells, progenitor cells, differentiated RPE and photoreceptors) and the methods of implantation (sheet, suspension), several CTs have shown safety. There remain potential concerns regarding tumorigenicity and immune rejection; however, with ongoing improvements in cell generation, selection, and delivery, these can be minimized. Earlier studies showed efficacy with immunosuppressive drugs to prevent rejection, and recent donor-matched transplants have avoided the need for immunosuppression. Retinal regenerative medicine is a challenging field and is in a nascent stage but holds tremendous promise. This narrative review delves into the current understanding of stem cells and the latest clinical trials of retinal cell transplantation. [ABSTRACT FROM AUTHOR]

Background: Cellular metabolism regulates the pluripotency of embryonic stem cells (ESCs). Yet, how metabolism regulates the transition among different pluripotent states remains elusive. It has been shown that protein lactylation, which uses lactate, a metabolic product of glycolysis, as a substrate, plays a critical role in various biological events. Here we focused on that glycolysis regulates the conversion between ESCs and 2-cell-like cells (2CLCs) through protein lactylation. Methods: RNA-seq revealed the activation of 2-cell (2C) genes by suppression of Ldh. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition. And we focused on Hdac1. Lactylation of Hdac1 required for silencing 2C genes was proved by quantitative reverse-transcription PCR (qRT-PCR), immunofluorescence (IF), Western blot and chimeric embryos. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and in vitro deacetylation assay confirmed lactylation of Hdac1 promoting its binding at 2C genes and enhancing its deacetylase activity, thereby facilitating the removal of H3K27ac and the silencing of 2C genes. Results: We found that inhibition or depletion of Ldha, the enzyme converting pyruvate to lactate, leads to the activation of 2C genes, as well as reduced global lactylation in ESCs. To investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition, quantitative lactylome analysis was performed, and 1716 lactylated proteins were identified. We then focused on Hdac1, a histone deacetylase involved in the silencing of 2C genes. Lactylation of Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes. Conclusions: In summary, our study reveals a mechanistic link between cellular metabolism and pluripotency regulation through protein lactylation. Our research is the first time to reveal that quantitative lactylome analysis in mouse ESCs. We found that lactylated Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes. [ABSTRACT FROM AUTHOR]

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy. [ABSTRACT FROM AUTHOR]

In mammalian cells, primary miRNAs are cleaved at their hairpin structures by the Microprocessor complex, whose core is composed of DROSHA and DGCR8. Here, we show that 5′ flanking regions, resulting from Microprocessor cleavage, are targeted by the RNA exosome in mouse embryonic stem cells (mESCs). This is facilitated by a physical link between DGCR8 and the nuclear exosome targeting (NEXT) component ZCCHC8. Surprisingly, however, both biochemical and mutagenesis studies demonstrate that a variant NEXT complex, containing the RNA helicase MTR4 but devoid of the RNA-binding protein RBM7, is the active entity. This Microprocessor-NEXT variant also targets stem-loop-containing RNAs expressed from other genomic regions, such as enhancers. By contrast, Microprocessor does not contribute to the turnover of less structured NEXT substrates. Our results therefore demonstrate that MTR4-ZCCHC8 can link to either RBM7 or DGCR8/DROSHA to target different RNA substrates depending on their structural context. [Display omitted] • A variant NEXT complex of MTR4 and ZCCHC8 interacts with the Microprocessor complex • RBM7 is absent from the variant NEXT (vNEXT) complex • vNEXT-Microprocessor targets 5' flanks of pre-miRNAs • Structured RNAs, independent of miRNAs, are targeted by vNEXT-Microprocessor Imamura et al. reveal a physical and functional link between the Microprocessor complex, responsible for pre-miRNA processing, and a variant NEXT complex, involved in RNA decay. Together, these complexes degrade the 5' flanks of pre-miRNAs and other stem-loop-containing RNAs. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) regulate totipotency, pluripotency maintenance, and induction. The intricacies of how they modulate these processes through their interaction with RNAs remain to be elucidated. Here we employed Targets of RBPs Identified By Editing (TRIBE) with single-cell resolution (scTRIBE) to profile the mRNA targets of the key pluripotency regulator LIN28A in mouse embryonic stem cells (ESCs), 2-cell embryo-like cells (2CLCs), and somatic cell reprogramming. LIN28A is known to act by controlling the maturation of the let-7 microRNA, but, in addition, it binds to multiple mRNAs and influences their stability and translation efficiency. However, the mRNA targets of LIN28A in 2CLCs and reprogramming are unclear. Through quantitative single-cell analysis of the scTRIBE dataset, we observed a marked increase in the binding of LIN28A to mRNAs of ribosome biogenesis factors and a selected group of totipotency factors in 2CLCs within ESC cultures. Our results suggest that LIN28A extends the half-life of at least some of these mRNAs, providing new insights into its role in the totipotent state. We also uncovered the distinct trajectory-specific LIN28A-mRNA networks in reprogramming, helping explain how LIN28A facilitates the mesenchymal-to-epithelial transition and pluripotency acquisition. Our study not only clarifies the multifunctional role of LIN28A in these processes but also highlights the importance of decoding RNA-protein interactions at the single-cell level. [ABSTRACT FROM AUTHOR]

Background: The importance of embryonic stem cells (ESCs) in chickens is undeniable, as they can be applied across various fields, including animal modeling, developmental biology, cell fate research, drug screening, toxicity testing, and gene function studies. However, a widely applicable culture system for chicken ESCs has yet to be developed. Objectives: This study aimed to investigate the effects of different culture systems on the derivation and maintenance of chicken ESCs, with a focus on optimizing the selected culture conditions. Methods: To achieve this, we tested the effectiveness of various species-specific ESC media in the derivation and culture of chicken PGCs, while incorporating different small molecule compounds to optimize the process. The pluripotency and differentiation potential of the resulting ESC-like cells were also evaluated. Results: The combination of PD0325901, SB431542, and LIF (R2i+LIF system) was found to be effective in generating chicken ESC-like clones. Further experiments showed that enhancing the R2i+LIF system with cytokines such as SCF and FGF2 significantly extended the culture period and increased the passage number of chicken ESC-like cells. These ESC-like cells were characterized through positive alkaline phosphatase staining and the expression of pluripotency markers POUV, NANOG, and SOX2. Additionally, differentiation assays confirmed their ability to form the three germ layers. Conclusions: The newly developed culture system provides suitable conditions for the short-term culture of chicken ESCs. However, further optimization is required to establish a system that can sustain long-term maintenance. [ABSTRACT FROM AUTHOR]

The in vitro generation of spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs) offers a viable approach for addressing male infertility. A multitude of molecules participate in this intricate process, which requires additional elucidation. Despite the decline in SSCs in aged testes, SSCs are deemed immortal since they can multiply for three years with repeated transplantation. Nonetheless, the examination of aging is challenging due to the limited quantity and absence of precise indicators. Using a microarray, we assessed genome-wide transcripts (about 55,000 transcripts) of fibroblasts and SSCs. The WGCNA approach was then used to look for SSC-specific transcription factors (TFs) and hub SSC-specific genes based on ATAC-seq, DNase-seq, RNA-seq, and microarray data from the GEO databases as well as gene expression data (RNA-seq and microarray data). The microarray analysis of three human cases with different SSCs revealed that 6 genes were upregulated, and the expression of 23 genes was downregulated compared to the normal case in relation to aging genes. To reach these results, online assessments of Enrich Shiny GO, STRING, and Cytoscape were used to forecast the molecular and functional connections of proteins before identifying the master routes. The biological process and molecular function keywords of cell–matrix adhesion, telomerase activity, and telomere cap complex were shown to be significantly altered in upregulated differentially expressed genes (DEGs) by the functional enrichment analysis. According to our preliminary research, cell-specific TFs and TF-mediated GRNs are involved in the creation of SSCs. In order to maximize the induction efficiency of ESC differentiation into SSCs in vitro, hub SSC-specific genes and important SSC-specific TFs were identified, and sophisticated network regulation was proposed. According to our research, these genes and the hub proteins that they interact with may be able to shine a light on the pathophysiologies of infertility and aberrant germ cells. [ABSTRACT FROM AUTHOR]

Advances in the treatment of childhood cancer have significantly improved survival rates, with more than 80% of survivors reaching adulthood. However, gonadotoxic cancer treatments endanger future fertility, and prepubertal males have no option to preserve fertility by sperm cryopreservation. In addition, boys with cryptorchidism are at risk of compromised fertility in adulthood. To investigate current evidence for male fertility restoration strategies, explore barriers to clinical implementation, and outline potential steps to overcome these barriers, a scoping review was conducted. This knowledge synthesis is particularly relevant for prepubertal male cancer survivors and boys with cryptorchidism. The review was conducted after the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews criteria and previously published guidelines and examined studies using human testis tissue of prepubertal boys or healthy male adults. A literature search in PubMed was conducted, and 72 relevant studies were identified, including in vivo and in vitro approaches. In vivo strategies, such as testis tissue engraftment and spermatogonial stem cell transplantation, hold promise for promoting cell survival and differentiation. Yet, complete spermatogenesis has not been achieved. In vitro approaches focus on the generation of male germ cells from direct germ cell maturation in various culture systems, alongside human induced pluripotent stem cells and embryonic stem cells. These approaches mark significant advancements in understanding and promoting spermatogenesis, but achieving fully functional spermatozoa in vitro remains a challenge. Barriers to clinical implementation include the risk of reintroducing malignant cells and introduction of epigenetic changes. Male fertility restoration is an area in rapid development. On the basis of the reviewed studies, the most promising and advanced strategy for restoring male fertility using cryopreserved testis tissue is direct testis tissue transplantation. This review identifies persistent barriers to the clinical implementation of male fertility restoration. However, direct transplantation of frozen-thawed testis tissue remains a promising strategy that is on the verge of clinical application. [ABSTRACT FROM AUTHOR]

Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Miscarriages cause a greater loss‐of‐life than cardiovascular diseases, but knowledge about environmentally induced miscarriages is limited. Cultured naïve pluripotent embryonic stem cells (ESC) differentiate into extra‐embryonic endoderm/extraembryonic endoderm (XEN) or formative pluripotent ESC, during the period emulating maximal miscarriage of peri‐implantation development. In previous reports using small marker sets, hyperosmotic sorbitol, or retinoic acid (RA) decreased naïve pluripotency and increased XEN by FACS quantitation. Methods: Bulk and single cell (sc)RNAseq analyses of two cultured ESC lines was done, corroborated by qPCR. Transcriptomic responses were analyzed of cultured ESC stressed by Sorbitol, with Leukemia inhibitory factor (LIF + ; stemness growth factor), RA without LIF to control for XEN induction, and compared with normal differentiation (LIF − , ND). Results: Sorbitol and RA increase subpopulations of 2‐cell embryo‐like (2CEL) and XEN sub‐lineages; primitive, parietal, and visceral endoderm (VE) cells and suppress formative pluripotency, imbalancing alternate lineage choices of initial naïve pluripotent cultured ESC compared with ND. Although bulk RNAseq and gene ontology (GO) group analyses suggest that stress induces anterior VE‐head organizer and placental markers, scRNAseq reveals relatively few cells. But VE and placental markers/cells were in adjacent stressed cell clusters in the UMAP, like recent, normal UMAP of conceptuses. UMAPs show that dose‐dependent stress overrides stemness to force premature lineage imbalance. Conclusions: Hyperosmotic stress, and other toxicological stresses, like drugs with active ingredient RA, may cause premature, lineage imbalance, resulting in miscarriages or birth defects. [ABSTRACT FROM AUTHOR]

Teeth are comprised of epithelial and mesenchymal cells, and regenerative teeth rely on the regeneration of both cell types. Transcription factors play a pivotal role in cell fate determination. In this study, we establish fluorescence models based on transcription factors to monitor and analyze dental epithelial cells. Using Pitx2-P2A-copGFP mice, we observe that Pitx2+ epithelial cells, when combined with E14.5 dental mesenchymal cells, are sufficient for the reconstitution of teeth. Induced-Pitx2+ cells, directly isolated from the embryoid body that employs the Pitx2-GFP embryonic stem cell line, exhibit the capacity to differentiate into ameloblasts and develop into teeth when combined with dental mesenchymal cells. The regenerated teeth exhibit a complete structure, including dental pulp, dentin, enamel, and periodontal ligaments. Subsequent exploration via RNA-seq reveals that induced-Pitx2+ cells exhibit enrichment in genes associated with FGF receptors and WNT ligands compared with induced-Pitx2- cells. Our results indicate that both primary Pitx2+ and induced Pitx2+ cells possess the capability to differentiate into enamel-secreting ameloblasts and grow into teeth when combined with dental mesenchymal cells. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) sourced from the inner cell mass of blastocysts, are akin to this tissue in function but lack the capacity to form all extraembryonic structures. mESCs are transient cell populations that express high levels of transcripts characteristic of 2-cell (2C) embryos and are identified as "2-cell-like cells" (2CLCs). Previous studies have shown that 2CLCs can contribute to both embryonic and extraembryonic tissues upon reintroduction into early embryos. Approximately 1% of mESCs dynamically transition from pluripotent mESCs into 2CLCs. Nevertheless, the scarcity of mammalian embryos presents a significant challenge to the molecular characterization of totipotent cells. To date, Previous studies have explored various methods for reprogramming pluripotent cells into totipotent cells. While there is a good understanding of the molecular regulatory network maintaining ES pluripotency, the process by which pluripotent ESCs reprogram into totipotent cells and the associated molecular mechanisms of totipotent regulation remain poorly understood. This review synthesizes recent insights into the regulatory pathways of ESC reprogramming into 2CLC, exploring molecular mechanisms modulated by transcriptional regulators, small molecules, and epigenetic changes. The objective is to construct a theoretical framework for the field of researchers. [ABSTRACT FROM AUTHOR]

Cardiac arrhythmias stemming from abnormal sinoatrial node (SAN) function can lead to sudden death. Developing a biological pacemaker device for treating sick sinus syndrome (SSS) could offer a potential cure. Understanding SAN differentiation is crucial, yet its regulatory mechanism remains unclear. We reanalyzed published RNA‐seq data and identified Odz4 as a SAN‐specific candidate. In situ hybridization revealed Odz4 expression in the cardiac crescent and throughout the cardiac conduction system (CCS). To assess the role of Odz4 in CCS differentiation, we utilized a Tet‐Off inducible system for its intracellular domain (ICD). Embryonic bodies (EBs) exogenously expressing Odz4‐ICD exhibited an increased propensity to develop into pacemaker‐like cells with enhanced automaticity and upregulated expression of SAN‐specific genes. CellChat and GO analyses unveiled SAN‐specific enrichment of ligand–receptor sets, especially Ptn‐Ncl, and extracellular matrix components in the group exogenously expressing Odz4‐ICD. Our findings underscore the significance of Odz4 in SAN development and offer fresh insights into biological pacemaker establishment. [ABSTRACT FROM AUTHOR]

The pluripotent mouse embryonic stem cell (mESCs) can transit into the totipotent-like state, and the transcription factor DUX is one of the master regulators of this transition. Intriguingly, this transition in mESCs is accompanied by massive cell death, which significantly impedes the establishment and maintenance of totipotent cells in vitro, yet the underlying mechanisms of this cell death remain largely elusive. In this study, we found that the totipotency transition in mESCs triggered cell death through the upregulation of DUX. Specifically, R-loops are accumulated upon DUX induction, which subsequently lead to DNA replication stress (RS) in mESCs. This RS further activates p53 and PMAIP1, ultimately leading to Caspase-9/7-dependent intrinsic apoptosis. Notably, inhibiting this intrinsic apoptosis not only mitigates cell death but also enhances the efficiency of the totipotency transition in mESCs. Our findings thus elucidate one of the mechanisms underlying cell apoptosis during the totipotency transition in mESCs and provide a strategy for optimizing the establishment and maintenance of totipotent cells in vitro. [ABSTRACT FROM AUTHOR]

Anophthalmia, microphthalmia and coloboma (AMC) comprise a spectrum of developmental eye disorders, accounting for approximately 20% of childhood visual impairment. While non-coding regulatory sequences are increasingly recognised as contributing to disease burden, characterising their impact on gene function and phenotype remains challenging. Furthermore, little is known of the nature and extent of their contribution to AMC phenotypes. We report two families with variants in or near MAB21L2, a gene where genetic variants are known to cause AMC in humans and animal models. The first proband, presenting with microphthalmia and coloboma, has a likely pathogenic missense variant (c.338 G > C; p.[Trp113Ser]), segregating within the family. The second individual, presenting with microphthalmia, carries an ~ 113.5 kb homozygous deletion 19.38 kb upstream of MAB21L2. Modelling of the deletion results in transient small lens and coloboma as well as midbrain anomalies in zebrafish, and microphthalmia and coloboma in Xenopus tropicalis. Using conservation analysis, we identify 15 non-coding conserved elements (CEs) within the deleted region, while ChIP-seq data from mouse embryonic stem cells demonstrates that two of these (CE13 and 14) bind Otx2, a protein with an established role in eye development. Targeted disruption of CE14 in Xenopus tropicalis recapitulates an ocular coloboma phenotype, supporting its role in eye development. Together, our data provides insights into regulatory mechanisms underlying eye development and highlights the importance of non-coding sequences as a source of genetic diagnoses in AMC. Coding sequence variants in the MAB21L2 gene can cause human eye disorders. Here the authors show that a deletion upstream of MAB21L2 leads to similar eye anomalies in humans, zebrafish and frogs due to the disruption of evolutionarily conserved regulatory elements. [ABSTRACT FROM AUTHOR]

Sea urchins are basal deuterostomes that share key molecular components of innate immunity with vertebrates. They are a powerful model for the study of innate immune system evolution and function, especially during early development. Here we characterize the morphology and associated molecular markers of larval immune cell types in a newly developed model sea urchin, Lytechinus pictus. We then challenge larvae through infection with an established pathogenic Vibrio and characterize phenotypic and molecular responses. We contrast these to the previously described immune responses of the purple sea urchin Strongylocentrotus purpuratus. The results revealed shared cellular morphologies and homologs of known pigment cell immunocyte markers (PKS, srcr142) but a striking absence of subsets of perforin‐like macpf genes in blastocoelar cell immunocytes. We also identified novel patterning of cells expressing a scavenger receptor cysteine rich (SRCR) gene in the coelomic pouches of the larva (the embryonic stem cell niche). The SRCR signal becomes further enriched in both pouches in response to bacterial infection. Collectively, these results provide a foundation for the study of immune responses in L. pictus. The characterization of the larval immune system of this rapidly developing and genetically enabled sea urchin species will facilitate more sophisticated studies of innate immunity and the crosstalk between the immune system and development. [ABSTRACT FROM AUTHOR]

Background: Deer antler stem cells (AnSCs) exhibit properties of both embryonic and mesenchymal stem cells, with superior self-renewal and proliferation, which drive rapid antler growth and regeneration. AnSCs and their derived small extracellular vesicles (sEVs) hold promising potential for applications in regeneration medicine. Due to the restricted proliferative capacity inherent in primary cells, the production capacity of AnSCs and their sEVs are limited. Human telomerase reverse transcriptase (hTERT) is the most important telomerase subunit, hTERT gene insertion has been successfully employed in generating immortalized cell lines. Results: In this study, we successfully established immortalized AnSCs by transducing the hTERT gene using lentivirus. Compared to primary AnSCs, hTERTAnSCs demonstrated extended passage potential and accelerated proliferation rates while maintaining the mesenchymal stem cell surface markers CD44 and CD90. Additionally, hTERT-AnSCs retained the capacity for osteogenic, adipogenic, and chondrogenic differentiation. sEVs derived from hTERT-AnSCs exhibited a particle size distribution similar to that of AnSCs, both displaying a cup-shaped morphology and expressing CD81, ALIX, and TSG101, while notably lacking GM130 expression. Conclusion: We successfully isolated primary stem cells from deer antler and established the immortalized hTERT-AnSCs. Remarkably, this cell line maintains its stem cell characteristics even after 40 passages. The sEVs derived from these cells exhibit identical morphological and structural features to those of primary AnSCs. This research provides essential technical support for the application of AnSCs and their sEVs in regenerative medicine. [ABSTRACT FROM AUTHOR]

One of the two X-chromosomes in female mammals is epigenetically silenced in embryonic stem cells by X-chromosome inactivation. This creates a mosaic of cells expressing either the maternal or the paternal X allele. The X-chromosome inactivation ratio, the proportion of inactivated parental alleles, varies widely among individuals, representing the largest instance of epigenetic variability within mammalian populations. While various contributing factors to X-chromosome inactivation variability are recognized, namely stochastic and/or genetic effects, their relative contributions are poorly understood. This is due in part to limited cross-species analysis, making it difficult to distinguish between generalizable or species-specific mechanisms for X-chromosome inactivation ratio variability. To address this gap, we measure X-chromosome inactivation ratios in ten mammalian species (9531 individual samples), ranging from rodents to primates, and compare the strength of stochastic models or genetic factors for explaining X-chromosome inactivation variability. Our results demonstrate the embryonic stochasticity of X-chromosome inactivation is a general explanatory model for population X-chromosome inactivation variability in mammals, while genetic factors play a minor role. Stochastic or genetic effects can drive variability in X-chromosome inactivation (XCI) ratios among mammals, though their relative contributions are poorly understood. This study measures distributions of XCI ratios for 10 species and finds the embryonic stochasticity of XCI is a general explanatory model for mammalian XCI ratio variability. [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]

Aneuploidy, a deviation of the chromosome number from euploidy, is one of the hallmarks of cancer. High levels of aneuploidy are generally correlated with metastasis and poor prognosis in cancer patients. However, the causality of aneuploidy in cancer metastasis remains to be explored. Here we demonstrate that teratomas derived from aneuploid murine embryonic stem cells (ESCs), but not from isogenic diploid ESCs, disseminated to multiple organs, for which no additional copy number variations were required. Notably, no cancer driver gene mutations were identified in any metastases. Aneuploid circulating teratoma cells were successfully isolated from peripheral blood and showed high capacities for migration and organ colonization. Single-cell RNA sequencing of aneuploid primary teratomas and metastases identified a unique cell population with high stemness that was absent in diploid ESCs-derived teratomas. Further investigation revealed that aneuploid cells displayed decreased proteasome activity and overactivated endoplasmic reticulum (ER) stress during differentiation, thereby restricting the degradation of proteins produced from extra chromosomes in the ESC state and causing differentiation deficiencies. Noticeably, both proteasome activator Oleuropein and ER stress inhibitor 4-PBA can effectively inhibit aneuploid teratoma metastasis. Aneuploidy is associated with cancer metastasis, but the causal relationship between them is unclear. Here the authors show that aneuploid murine embryonic stem cells lead to teratomas that can spread to multiple organs without requiring additional driver gene mutations and identify unique cell populations with high stemness in aneuploid teratomas. [ABSTRACT FROM AUTHOR]

The interplay between G4s and R-loops are emerging in regulating DNA repair, replication, and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops -associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease. [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]

Background: Quiescent self-renewal of leukemia stem cells (LSCs) and resistance to conventional chemotherapy are the main factors leading to relapse of acute myeloid leukemia (AML). Alpha-enolase (ENO1), a key glycolytic enzyme, has been shown to regulate embryonic stem cell differentiation and promote self-renewal and malignant phenotypes in various cancer stem cells. Here, we sought to test whether and how ENO1 influences LSCs renewal and chemoresistance within the context of AML. Methods: We analyzed single-cell RNA sequencing data from bone marrow samples of 8 relapsed/refractory AML patients and 4 healthy controls using bioinformatics and machine learning algorithms. In addition, we compared ENO1 expression levels in the AML cohort with those in 37 control subjects and conducted survival analyses to correlate ENO1 expression with clinical outcomes. Furthermore, we performed functional studies involving ENO1 knockdown and inhibition in AML cell line. Results: We used machine learning to model and infer malignant cells in AML, finding more primitive malignant cells in the non-response (NR) group. The differentiation capacity of LSCs and progenitor malignant cells exhibited an inverse correlation with glycolysis levels. Trajectory analysis indicated delayed myeloid cell differentiation in NR group, with high ENO1-expressing LSCs at the initial stages of differentiation being preserved post-treatment. Simultaneously, ENO1 and stemness-related genes were upregulated and co-expressed in malignant cells during early differentiation. ENO1 level in our AML cohort was significantly higher than the controls, with higher levels in NR compared to those in complete remission. Knockdown of ENO1 in AML cell line resulted in the activation of LSCs, promoting cell differentiation and apoptosis, and inhibited proliferation. ENO1 inhibitor can impede the proliferation of AML cells. Furthermore, survival analyses associated higher ENO1 expression with poorer outcome in AML patients. Conclusions: Our findings underscore the critical role of ENO1 as a plausible driver of LSC self-renewal, a potential target for AML target therapy and a biomarker for AML prognosis. [ABSTRACT FROM AUTHOR]

Interspecies somatic cell nuclear transfer (iSCNT) has the capacity to rescue endangered species, ethically produce human embryonic stem cells, and allow for more specific analysis of pre‐implantation embryos. However, several obstacles negatively affect iSCNT efficiency.Over 100 iSCNT studies have been published between 1999 and the spring of 2023. This review details the possible benefits of iSCNT, describes the hypothesized issues that prevented the development of some embryos and/or live offspring from developing; and analyzes the cleavage, blastocyst, pregnancy, live birth, and iSCNT efficiency rates based on published data.Embryos produced by iSCNT may help with the rescue of endangered species and the production of human embryonic stem cells with the use of oocytes from domestic species, as well as in‐depth analyses of embryonic genome activation (EGA) and mRNA transcripts. But the combination of the cells of two species in a single embryo presents several challenges: lack of EGA synchronisation, presence of mitochondrial DNA (mtDNA) from both species in the embryo, requirement of cell and embryo culture optimization for each species combination, lack of embryo transfer data, and an extremely low overall efficiency in terms of producing live offspring. Additional research on the aforementioned topics is warranted to increase iSCNT efficiency. [ABSTRACT FROM AUTHOR]

Background/Objectives: Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are leading causes of vision loss, with AMD affecting older populations and RP being a rarer, genetically inherited condition. Both diseases result in progressive retinal degeneration, for which current treatments remain inadequate in advanced stages. This review aims to provide an overview of the retina's anatomy and physiology, elucidate the pathophysiology of AMD and RP, and evaluate emerging cell-based therapies for these conditions. Methods: A comprehensive review of the literature was conducted, focusing on cell therapy approaches, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells. Preclinical and clinical studies were analyzed to assess therapeutic potential, with attention to mechanisms such as cell replacement, neuroprotection, and paracrine effects. Relevant challenges, including ethical concerns and clinical translation, were also explored. Results: Cell-based therapies demonstrate potential for restoring retinal function and slowing disease progression through mechanisms like neuroprotection and cell replacement. Preclinical trials show promising outcomes, but clinical studies face significant hurdles, including challenges in cell delivery and long-term efficacy. Combination therapies integrating gene editing and biomaterials offer potential future advancements. Conclusions: While cell-based therapies for AMD and RP have made significant progress, substantial barriers to clinical application remain. Further research is essential to overcome these obstacles, improve delivery methods, and ensure the safe and effective translation of these therapies into clinical practice. [ABSTRACT FROM AUTHOR]