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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., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

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., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

The seamless transition through stages of pluripotency relies on a balance between transcription factor networks and epigenetic mechanisms. Here, we reveal the crucial role of the transgene activation suppressor (TASOR), a component of the human silencing hub (HUSH) complex, in maintaining cell viability during the transition from naive to primed pluripotency. TASOR loss in naive pluripotent stem cells (PSCs) triggers replication stress, disrupts H3K9me3 heterochromatin, and impairs silencing of LINE-1 (L1) transposable elements, with more severe effects in primed PSCs. Notably, the survival of Tasor knockout PSCs during this transition can be restored by inhibiting caspase or deleting the mitochondrial antiviral signaling protein (MAVS). This suggests that unscheduled L1 expression activates an innate immune response, leading to cell death specifically in cells exiting naive pluripotency. Our findings highlight the importance of epigenetic programs established in naive pluripotency for normal development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Minimal in vitro systems composed of embryonic stem cells (ESCs) have been shown to recapitulate the establishment of the anteroposterior (AP) axis. In contrast to the native embryo, ESC aggregates - such as gastruloids - can break symmetry, which is demarcated by polarization of the mesodermal marker T, autonomously without any localized external cues. However, associated earliest patterning events, such as the spatial restriction of cell fates and concomitant transcriptional changes, remain poorly understood. Here, we dissect the dynamics of AP axis establishment in mouse gastruloids, particularly before external Wnt stimulation. Through single-cell RNA sequencing, we identify key cell state transitions and the molecular signatures of T+ and T- populations underpinning AP polarization. We also show that this process is robust to modifications of aggregate size. Finally, transcriptomic comparison with the mouse embryo indicates that gastruloids develop similar mesendodermal cell types, despite initial differences in their primed pluripotent populations, which adopt a more mesenchymal state in lieu of an epiblast-like transcriptome. Hence, our findings suggest the possibility of alternate ESC states in vivo and in vitro that can converge onto similar cell fates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., Competing Interests: AB, LS, LD, AP, FS, EH, CE, HJ, HY, GF, DC No competing interests declared, EG Now works for Boehringer Ingelheim Pharma GmbH & Co KG, MP Board member of Tartan Cell Technologies Ltd, JS Board member of Tartan Cell Technologies Ltd and Glencoe Software Ltd, AL Board member of Tartan Cell Technologies Ltd and Platinum Informatics Ltd, (© 2024, Brenes et al.)

Cell fate decision is crucial in biological development and plays fundamental roles in normal development and functional maintenance of organisms. By identifying key regulatory interactions and molecules involved in these fate decisions, we can shed light on the intricate mechanisms underlying the cell fates. This understanding ultimately reveals the fundamental principles driving biological development and the origins of various diseases. In this study, we present an overarching framework which integrates pseudo-trajectory inference and differential analysis to determine critical regulatory interactions and molecules during cell fate transitions. To demonstrate feasibility and reliability of the approach, we employ the differentiation networks of hepatobiliary system and embryonic stem cells as representative model systems. By applying pseudo-trajectory inference to biological data, we aim to identify critical regulatory interactions and molecules during the cell fate transition processes. Consistent with experimental observations, the approach can allow us to infer dynamical cell fate decision processes and gain insights into the underlying mechanisms which govern cell state decisions., Competing Interests: Declarations Ethical approval This article does not contain any studies with human participants or animals performed by the authors. Conflict of interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Erk signaling is indispensable for the self-renewal and differentiation of mouse embryonic stem cells (ESCs), as well as telomere homeostasis. But how Erk regulates these biological processes remains unclear. We identified 132 Erk2 interacting proteins by co-immunoprecipitation and mass spectrometric analysis, and focused on Ddx39 as a potential Erk2 substrate. We demonstrated that Erk2 phosphorylates Ddx39 on Y132 and Y138. Ddx39 knockout (KO) ESCs are defective in differentiation, due to reduced H3K27ac level upon differentiation. Phosphorylation of Ddx39 promotes the recruitment of Hat1 to acetylate H3K27 and activate differentiation genes. In addition, Ddx39 KO leads to telomere elongation in ESCs. Ddx39 is recruited to telomeres by the telomere-binding protein Trf1, consequently disrupting the DNA loop formed by Trf1 and suppressing the alternative lengthening of telomeres (ALT). Phosphorylation of Ddx39 weakens its interaction with Trf1, releasing it from telomeres. Thus, ALT activity is enhanced, and telomeres are elongated. Altogether, our studies reveal an essential role of Ddx39 in the differentiation and telomere homeostasis of ESCs., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

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.

Serum response factor (SRF) is a transcription factor essential for cell proliferation, differentiation, and migration and is required for primitive streak and mesoderm formation in the embryo. The canonical roles of SRF are mediated by a diverse set of context-dependent cofactors. Here, we show that SRF physically interacts with CTCF and cohesin subunits at topologically associating domain (TAD) boundaries and loop anchors. SRF promotes long-range chromatin loop formation and contributes to TAD insulation. In embryonic stem cells (ESCs), SRF associates with SOX2 and NANOG and contributes to the formation of three-dimensional (3D) pluripotency hubs. Our findings reveal additional roles of SRF in higher-order chromatin organization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The transcription factors NANOG and POU5F1 (OCT4) play crucial roles in maintaining pluripotency in embryonic stem (ES) cells. While their functions have been well-studied, the specific interactions between NANOG and POU5F1 and their combined effects on pluripotency in ES-like and Epiblast cells remain less understood. Understanding these associations is vital for refining pluripotent stem cell characterization and advancing regenerative medicine. In this matter, we investigated the associations between NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells and how these interactions contribute to the distinct pluripotent states of these cells. In the present paper, we examined the pattern of NANOG expression by the immunocytochemical method in embryonic stem-like (ES-like) cells and compared it with its expression pattern in embryonic stem cells (ESCs). Similarly, we examined the expression pattern of POU5F1 in ES-like cells, ESCs, and epiblast cells and compared the expression pattern of these two genes with each other. On the other hand, using Fluidigm Biomark system analysis, we compared the amount of NANOG mRNA in these three cell lines and differentiated and undifferentiated Spermatogonial stem cells in several passages. Microscopic observations indicated the cytoplasmic expression of NANOG in the considered cells; moreover, they showed a similar expression pattern of NANOG with POU5F1 in the experimented cells. It has also been suggested that the more limited the cell's pluripotency, the lower the expression of these two genes. However, the decrease in NANOG expression is less than that of POU5F1. Fluidigm real-time RT-PCR analysis also confirmed these results. During the experimental process, protein-protein (PPI) network analysis shows a significant association of NANOG with other stem cell proteins, such as POU5F1. Our findings reveal distinct yet overlapping roles of NANOG and POU5F1 in maintaining pluripotency in ES-like and Epiblast cells. The differential binding patterns and functional interactions between these factors underscore the complexity of pluripotency regulation in different stem cell states. This study provides new insights into the molecular mechanisms governing pluripotency and highlights potential targets for enhancing stem cell-based therapies., (© 2024. The Author(s).)

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding., (© 2024. The Author(s).)

Transposable elements (TEs) have emerged as important factors in establishing the cell type-specific gene regulatory networks and evolutionary novelty of embryonic and placental development. Recently, studies on the role of TEs and their dysregulation in cancers have shed light on the transcriptional, transpositional, and regulatory activity of TEs, revealing that the activation of developmental transcriptional programs by TEs may have a role in the dedifferentiation of cancer cells to the progenitor-like cell states. This essay reviews the recent evidence of the cis-regulatory TEs (henceforth crTE) in normal development and malignancy as well as the key transcription factors and regulatory pathways that are implicated in both cell states, and presents existing gaps remaining to be studied, limitations of current technologies, and therapeutic possibilities., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types, making further research difficult. Here, we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells' morphology, doubling rate, ability to form colonies, surface marker analysis, and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs' morphology, growth kinetics, and surface marker characterization. In contrast, the serum-containing medium gives better potential for further MSC differentiation into osteogenic, chondrogenic, and adipogenic lineages.

It has been reported that human embryonic stem cells (hESCs) treated with BMP4 and inhibitors of TGFβ signaling (A83-01) and FGF signaling (PD173074), called BAP, can efficiently differentiate to extraembryonic (ExE) cells in vitro . Due to restricted access to human embryos, it is ethically impossible to test the developmental potential of ExE cells in vivo . Here, we demonstrate that most ExE cells expressed molecular markers for both trophoblasts (TBs) and amniotic cells (ACs). Following intra-uterine transplantation, ExE cells contributed to the mouse placenta. More interestingly, ExE cells could chimerize with the mouse blastocyst as, after injection into the blastocyst, they penetrated its trophectoderm. After implantation of the injected blastocysts into surrogate mice, human cells were found at E14 in placental labyrinth, junction zones, and even near the uterine decidua, expressed placental markers, and secreted human chorionic gonadotropin. Surprisingly, ExE cells also contributed to cartilages of the chimeric embryo with some expressing the chondrogenic marker SOX9, consistent with the mesodermal potential of TBs and ACs in the placenta. Deleting MSX2 , a mesodermal determinant, restricted the contribution of ExE cells to the placenta. Thus, we conclude that hESC-derived ExE cells can chimerize with the mouse blastocyst and contribute to both the placenta and cartilages of the chimera consistent with their heteogenious nature. Intra-uterus and intra-blastocyst injections are novel and sensitive methods to study the developmental potential of ExE cells., Competing Interests: Competing Interests: R.X. is a founder of ImStem Biotechnology, Inc., a stem cell company. The other authors declare no competing financial interests., (© The author(s).)

Inhalation anesthesia stands as a pivotal modality within clinical anesthesia practices. Beyond its primary anesthetic effects, inhaled anesthetics have non-anesthetic effects, exerting bidirectional influences on the physiological state of the body and disease progression. These effects encompass impaired cognitive function, inhibition of embryonic development, influence on tumor progression, and so forth. For many years, inhaled anesthetics were viewed as inhibitors of stem cell fate regulation. However, there is now a growing appreciation that inhaled anesthetics promote stem cell biological functions and thus are now regarded as a double-edged sword affecting stem cell fate. In this review, the effects of inhaled anesthetics on self-renewal and differentiation of neural stem cells (NSCs), embryonic stem cells (ESCs), and cancer stem cells (CSCs) were summarized. The mechanisms of inhaled anesthetics involving cell cycle, metabolism, stemness, and niche of stem cells were also discussed. A comprehensive understanding of these effects will enhance our comprehension of how inhaled anesthetics impact the human body, thus promising breakthroughs in the development of novel strategies for innovative stem cell therapy approaches.

In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development., (© 2024. The Author(s).)

Embryonic and adult stem cells enable development and regeneration. Embryonic cells, like adult stem cells, can enter dormancy as part of their lifecycle. Recent evidence suggests that this cellular transition to dormancy requires active rewiring of metabolism. The dormancy-induced metabolic switches in embryonic and adult stem cells are explored here., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Embryonic stem (ES) cells are pluripotent stem cells that can produce all cell types of an organism. ES cells proliferate rapidly and are thought to experience high levels of intrinsic replication stress. Here, by investigating replication fork dynamics in substages of S phase, we show that mammalian pluripotent stem cells maintain a slow fork speed and high active origin density throughout the S phase, with little sign of fork pausing. In contrast, the fork speed of non-pluripotent cells is slow at the beginning of S phase, accompanied by increased fork pausing, but thereafter fork pausing rates decline and fork speed rates accelerate in an ATR-dependent manner. Thus, replication fork dynamics within the S phase are distinct between ES and non-ES cells. Nucleoside addition can accelerate fork speed and reduce origin density. However, this causes miscoordination between the completion of DNA replication and cell cycle progression, leading to genome instability. Our study indicates that fork slowing in the pluripotent stem cells is an integral aspect of DNA replication., (© 2024. The Author(s).)

Repetitive sequences play an indispensable role in gene expression, transcriptional regulation, and chromosome arrangements through trans and cis regulation. In this review, focusing on recent advances, we summarize the epigenetic regulatory mechanisms of repetitive sequences in embryonic stem cells. We aim to bridge the knowledge gap by discussing DNA damage repair pathway choices on repetitive sequences and summarizing the significance of chromatin organization on repetitive sequences in response to DNA damage. By consolidating these insights, we underscore the critical relationship between the stability of repetitive sequences and early embryonic development, seeking to provide a deeper understanding of repetitive sequence stability and setting the stage for further research and potential therapeutic strategies in developmental biology and regenerative medicine.

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., (© 2024. The Author(s).)

Migrasomes, organelles crucial for cell communication, undergo distinct stages of nucleation, maturation, and expansion. The regulatory mechanisms of migrasome formation, particularly through biological cues, remain largely unexplored. This study reveals that calcium is essential for migrasome formation. Furthermore, we identify that Synaptotagmin-1 (Syt1), a well-known calcium sensor, is not only enriched in migrasomes but also indispensable for their formation. The calcium-binding ability of Syt1 is key to initiating migrasome formation. The recruitment of Syt1 to migrasome formation sites (MFS) triggers the swelling of MFS into unstable precursors, which are subsequently stabilized through the sequential recruitment of tetraspanins. Our findings reveal how calcium regulates migrasome formation and propose a sequential interaction model involving Syt1 and Tetraspanins in the formation and stabilization of migrasomes., (© 2024 Han and Yu.)

Nerve regeneration and circuit reconstruction remain a challenge following spinal cord injury (SCI). Corticospinal pyramidal neurons possess strong axon projection ability. In this study, human induced pluripotent stem cells (iPSCs) were differentiated into pyramidal neuronal precursors (PNPs) by addition of small molecule dorsomorphin into the culture. iPSC-derived PNPs were transplanted acutely into a rat contusion SCI model on the same day of injury. Following engraftment, the SCI rats showed significantly improved motor functions compared with vehicle control group as revealed by behavioral tests. Eight weeks following engraftment, the PNPs matured into corticospinal pyramidal neurons and extended axons into distant host spinal cord tissues, mostly in a caudal direction. Host neurons rostral to the lesion site also grew axons into the graft. Possible synaptic connections as a bridging relay may have been formed between host and graft-derived neurons, as indicated by pre- and post-synaptic marker staining and the regulation of chemogenetic regulatory systems. PNP graft showed an anti-inflammatory effect at the injury site and could bias microglia/macrophages towards a M2 phenotype. In addition, PNP graft was safe and no tumor formation was detected after transplantation into immunodeficient mice and SCI rats. The potential to reconstruct a neuronal relay circuitry across the lesion site and to modulate the microenvironment in SCI makes PNPs a promising cellular candidate for treatment of SCI., (© 2024. The Author(s).)

Vascular tissue engineering is a promising approach for regenerating damaged blood vessels and developing new therapeutic approaches for heart disease treatment. To date, different sources of cells have been recognized that offer assistance within the recovery of heart supply routes and veins with distinctive capacities and are compelling for heart regeneration. However, some challenges still remain that need to be overcome to establish the full potential application of these cells. In this paper, we review the different cell sources used for vascular tissue engineering, focusing on extraembryonic tissue-derived cells (ESCs), and elucidate their roles in cardiovascular disease. In addition, we highlight the intricate interplay between mechanical and biochemical factors in regulating mesenchymal stem cell (MSC) differentiation, offering insights into optimizing their application in vascular tissues., (© 2024. The Author(s).)

Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries., (© 2024. The Author(s).)

DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1-10 μl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections., (© 2024. The Author(s).)

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFβ signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFβ inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Background Aims: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, offer groundbreaking therapeutic potential for degenerative diseases and cellular repair. Despite their significance, a comprehensive bibliometric analysis in this field, particularly in relation to age-related macular degeneration (AMD), is yet to be conducted. This study aims to map the foundational and emerging areas in stem cell and AMD research through bibliometric analysis., Methods: This study analyzed articles and reviews on stem cells and AMD from 2000 to 2022, sourced from the Web of Science Core Collection. We used VOSviewer and CiteSpace for analysis and visualization of data pertaining to countries, institutions, authors, journals, references and key words. Statistical analyses were conducted using R language and Microsoft Excel 365., Results: In total, 539 publications were included, indicating an increase in global literature on stem cells and AMD from 2000 to 2022. The USA was the leading contributor, with 239 papers and the highest H-index, also the USA had the highest average citation rate per article (59.82). Notably, 50% of the top 10 institutions were from the USA, with the University of California system being the most productive. Key authors included Masayo Takahashi, Michiko Mandai, Dennis Clegg, Pete J. Coffey, Boris Stanzel, and Budd A. Tucker. Investigative Ophthalmology & Visual Science published the majority of relevant papers (n = 27). Key words like "clinical trial," "stem cell therapy," "retinal organoid," and "retinal progenitor cells" were predominant., Conclusions: Research on stem cells and AMD has grown significantly, highlighting the need for increased global cooperation. Current research prioritizes the relationship between "ipsc," "induced pluripotent stem cell," "cell culture," and "human embryonic stem cell." As stem cell culture and safety have advanced, focus has shifted to prognosis and complications post-transplantation, signifying the movement of stem cell research from labs to clinical settings., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

As the most well-studied modification in mRNA, m 6 A has been shown to regulate multiple biological processes, including RNA degradation, processing, and translation. Recent studies showed that m 6 A modification is enriched in chromatin-associated RNAs and nascent RNAs, suggesting m 6 A might play regulatory roles in chromatin contexts. Indeed, in the past several years, a number of studies have clarified how m 6 A and its modulators regulate different types of chromatin states. Specifically, in the past 2-3 years, several studies discovered the roles of m 6 A and/or its modulators in regulating constitutive and facultative heterochromatin, shedding interesting lights on RNA-dependent heterochromatin formation in mammalian cells. This review will summarize and discuss the mechanisms underlying m 6 A's regulation in different types of heterochromatin, with a specific emphasis on the regulation in mammalian embryonic stem cells, which exhibit distinct features of multiple heterochromatin marks., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S ( Janky ) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency., (© 2024 Shen et al.; Published by Cold Spring Harbor Laboratory Press.)

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated., Methods: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m 6 A RNA methylation in nanoparticles-treated cells., Results: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m 6 A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m 6 A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m 6 A level of Sox1 mRNA, enhancing its stability., Conclusion: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m 6 A RNA methylation modification of Sox1 ., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Bai et al.)

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing., (© 2024. The Author(s).)

Background: Mesenchymal stem cells (MSCs) have promising potential in clinical application, whereas their limited amount and sources hinder their bioavailability. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have become prominent options in regenerative medicine as both possess the ability to differentiate into MSCs., Methods: Recently, our research team has successfully developed human leukocyte antigen (HLA)-homozygous iPSC cell lines with high immune compatibility, covering 13.5% of the Taiwanese population. As we deepen our understanding of the differences between these ESCs and HLA-homozygous iPSCs, our study focused on morphological observations and flow cytometry analysis of specific surface marker proteins during the differentiation of ESCs and iPSCs into MSCs., Results: The results showed no significant differences between the two pluripotent stem cells, and both of them demonstrated the equivalent ability to further differentiate into adipose, cartilage, and bone cells., Conclusion: Our research revealed that these iPSCs with high immune compatibility exhibit the same differentiation potential as ESCs, enhancing the future applicability of highly immune-compatible iPSCs., Competing Interests: Conflicts of interest: Dr. Shih-Hwa Chiou, an editorial board member at Journal of the Chinese Medical Association , had no role in the peer review process of or decision to publish this article. The other authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article., (Copyright © 2024, the Chinese Medical Association.)

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI., (© 2024 AstraZeneca R&D and The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The convergence of organoid and organ-on-a-chip (OoC) technologies is urgently needed to overcome limitations of current 3D in vitro models. However, integrating organoids in standard OoCs faces several technical challenges, as it is typically laborious, lacks flexibility, and often results in even more complex and less-efficient cell culture protocols. Therefore, specifically adapted and more flexible microfluidic platforms need to be developed to facilitate the incorporation of complex 3D in vitro models. Here, a modular, tubeless fluidic circuit board (FCB) coupled with reversibly sealed cell culture bricks for dynamic culture of embryonic stem cell-derived thyroid follicles is developed. The FCB is fabricated by milling channels in a polycarbonate (PC) plate followed by thermal bonding against another PC plate. LEGO-like fluidic interconnectors allow plug-and-play connection between a variety of cell culture bricks and the FCB. Lock-and-play clamps are integrated in the organoid brick to enable easy (un)loading of organoids. A multiplexed perfusion experiment is conducted with six FCBs, where thyroid organoids are transferred on-chip within minutes and cultured up to 10 d without losing their structure and functionality, thus validating this system as a flexible, easy-to-use platform, capable of synergistically combining organoids with advanced microfluidic platforms., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

The neural crest is an embryonic stem cell population unique to vertebrates 1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia 2 . Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates 3-8 . Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine β-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Embryonic stem-like cells (ES-like cells) are promising for medical research and clinical applications. Traditional methods involve "Yamanaka" transcription (OSKM) to derive these cells from somatic cells in vitro. Recently, a novel approach has emerged, obtaining ES-like cells from spermatogonia stem cells (SSCs) in a time-related process without adding artificial additives to cell cultures, like transcription factors or small molecules such as pten or p53 inhibitors. This study aims to investigate the role of the Nanog in the conversion of SSCs to pluripotent stem cells through both in silico analysis and in vitro experiments. We used bioinformatic methods and microarray data to find significant genes connected to this derivation path, to construct PPI networks, using enrichment analysis, and to construct miRNA-lncRNA networks, as well as in vitro experiments, immunostaining, and Fluidigm qPCR analysis to connect the dots of Nanog significance. We concluded that Nanog is one of the most crucial differentially expressed genes during SSC conversion, collaborating with critical regulators such as Sox2 , Dazl , Pou5f1 , Dnmt3 , and Cdh1 . This intricate protein network positions Nanog as a pivotal factor in pathway enrichment for generating ES-like cells, including Wnt signaling, focal adhesion, and PI3K-Akt-mTOR signaling. Nanog expression is presumed to play a vital role in deriving ES-like cells from SSCs in vitro. Finding its pivotal role in this path illuminates future research and clinical applications.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research., (© 2024 The Author(s).)

Haploid cells are a kind of cells with only one set of chromosomes. Compared with traditional diploid cells, haploid cells have unique advantages in gene screening and drug-targeted therapy, due to their phenotype being equal to the genotype. Embryonic stem cells are a kind of cells with strong differentiation potential that can differentiate into various types of cells under specific conditions in vitro. Therefore, haploid embryonic stem cells have the characteristics of both haploid cells and embryonic stem cells, which makes them have significant advantages in many aspects, such as reproductive developmental mechanism research, genetic screening, and drug-targeted therapy. Consequently, establishing haploid embryonic stem cell lines is of great significance. This paper reviews the progress of haploid embryonic stem cell research and briefly discusses the applications of haploid embryonic stem cells., (© 2024. The Author(s).)

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

L1TD1 is a cytoplasmic RNA-binding protein specifically expressed in pluripotent stem cells and, unlike its mouse ortholog, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known protein-coding gene domesticated from a LINE-1 (L1) retroelement, the functional legacy of its ancestral protein, ORF1p of L1, and how it is manifested in L1TD1 are still unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs and localizes to high-density ribonucleoprotein (RNP) condensates. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. L1TD1 depletion promoted the formation of stress granules in embryonic stem cells. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain of L1TD1 facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatment of neurodegenerative diseases caused by disturbed stress granule dynamics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors-Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future., (© 2024 Federation of European Biochemical Societies.)

Methods in which patient-derived T cells are genetically modified in vitro and administered to patients have been demonstrated effective in the area of cancer immunotherapy. However, these methods have some unresolved issues such as cost, time, and unstable quality. Several groups have developed strategies to overcome these barriers by regenerating T cells from iPSCs. We have been developing a method in which specific TCR genes are introduced into iPSCs and T cells are regenerated from these iPSCs (TCR-iPSC method). We are now using starting iPSCs from the iPSC stock lines provided by CiRA-F, as the iPSC stock cells are less likely to be rejected. A study aimed at application to solid tumors demonstrated the therapeutic effect of regenerated T cells in a patient tissue xenograft model of WT1 antigen-positive renal cell carcinoma. This article will also discuss strategies by other groups to regenerate various types of T cells from iPSCs.

From generation of germ cells, fertilization, and throughout early mammalian embryonic development, the chromatin undergoes significant alterations to enable precise regulation of gene expression and genome use. Methylation of histone 3 lysine 4 (H3K4) correlates with active regions of the genome, and it has emerged as a dynamic mark throughout this timeline. The pattern and the level of H3K4 methylation are regulated by methyltransferases and demethylases. These enzymes, as well as their protein partners, play important roles in early embryonic development and show phenotypes in embryonic stem cell self-renewal and differentiation. The various roles of H3K4 methylation are interpreted by dedicated chromatin reader proteins, linking this modification to broader molecular and cellular phenotypes. In this review, we discuss the regulation of different levels of H3K4 methylation, their distinct accumulation pattern, and downstream molecular roles with an early embryogenesis perspective., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Gastruloids acquire their organization and shape through cell biochemical and mechanical activities. Such activities determine the physical forces and changes in material properties that transform simple spherical aggregates into organized tissues. In this Perspective, we discuss why the concepts and approaches of mechanobiology, a discipline that focuses on cell and tissue mechanics and its contribution to the organization and functions of living systems, are essential to the gastruloid field and, in turn, what gastruloids may teach us about mechanobiology., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes., (© 2023. The Author(s).)

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism 1 . However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)