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Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

While an association between Parkinsons disease (PD) and viral infections has been recognized, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on PD progression remains unclear. Here, we demonstrate that SARS-CoV-2 infection heightens the risk of PD using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons and a human angiotensin-converting enzyme 2 (hACE2) transgenic (Tg) mouse model. Our findings reveal that SARS-CoV-2 infection exacerbates PD susceptibility and cellular toxicity in DA neurons pre-treated with human preformed fibrils (hPFFs). Additionally, nasally delivered SARS-CoV-2 infects DA neurons in hACE2 Tg mice, aggravating the damage initiated by hPFFs. Mice infected with SARS-CoV-2 display persisting neuroinflammation even after the virus is no longer detectable in the brain. A comprehensive analysis suggests that the inflammatory response mediated by astrocytes and microglia could contribute to increased PD susceptibility associated with SARS-CoV-2. These findings advance our understanding of the potential long-term effects of SARS-CoV-2 infection on the progression of PD.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Three Prime Repair Exonuclease 1 (TREX1) gene mutations have been associated with Aicardi-Goutières Syndrome (AGS) - a rare, severe pediatric autoimmune disorder that primarily affects the brain and has a poorly understood etiology. Microglia are brain-resident macrophages indispensable for brain development and implicated in multiple neuroinflammatory diseases. However, the role of TREX1 - a DNase that cleaves cytosolic nucleic acids, preventing viral- and autoimmune-related inflammatory responses - in microglia biology remains to be elucidated. Here, we leverage a model of human embryonic stem cell (hESC)-derived engineered microglia-like cells, bulk, and single-cell transcriptomics, optical and transmission electron microscopy, and three-month-old assembloids composed of microglia and oligodendrocyte-containing organoids to interrogate TREX1 functions in human microglia. Our analyses suggest that TREX1 influences cholesterol metabolism, leading to an active microglial morphology with increased phagocytosis in the absence of TREX1. Notably, regulating cholesterol metabolism with an HMG-CoA reductase inhibitor, FDA-approved atorvastatin, rescues these microglial phenotypes. Functionally, TREX1 in microglia is necessary for the transition from gliogenic intermediate progenitors known as pre-oligodendrocyte precursor cells (pre-OPCs) to precursors of the oligodendrocyte lineage known as OPCs, impairing oligodendrogenesis in favor of astrogliogenesis in human assembloids. Together, these results suggest routes for therapeutic intervention in pathologies such as AGS based on microglia-specific molecular and cellular mechanisms.

Spinal cord injury (SCI) is a devastating injury that significantly impairs patients' quality of life. To date, there is no effective treatment to mitigate nerve tissue damage and restore neurological function. Neural stem cells (NSCs) derived from human embryonic stem cells (hESCs) are considered an important cell source for reconstructing damaged neural circuits and enabling axonal regeneration. Recent preclinical studies have shown that NSCs are potential therapeutic cell sources for neuroprotection and neuroregeneration in SCI animal models. NSCs can be derived from different sources and the spinal cord‐specific NSCs have a higher potential for the regeneration of SCI. However, the long‐term therapeutic efficacy of spinal cord‐specific NSCs remains unproven. Here, we generated human spinal cord NSCs (hSCNSCs) and investigated the effects of transplanted hSCNSCs on the repair of the SCI model rats for 60 days. The transplanted hSCNSCs improved BBB scores, reduced the lesion area and promoted an increase in the number of Nestin‐positive cells in the spinal cord compared to the model rats. Meanwhile, hSCNSC transplantation promoted the expression of synaptophysin, a synaptic signature protein and MBP, a protein associated with remyelination. Interestingly, BAF45D, a chromatin remodelling factor that contributes to the induction of hSCNSCs with region‐specific spinal cord identity, were increased by the hSCNSC transplantation. In addition, conditioned medium derived from the hSCNSCs also promoted regenerative repair of the injured spinal cord. These results demonstrate that hSCNSCs may play a critical role in the regenerative repair of SCI. [ABSTRACT FROM AUTHOR]

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although intensive studies and therapeutic approaches, there are limited restorative treatments till now. Human embryonic stem cell (hESCs)-derived cortical neural progenitors have shown great potentials in ischemic stroke in adult brain. However, it is unclear whether they are feasible for cortical reconstruction in immature brain with hypoxic-ischemic encephalopathy. Methods: By using embryonic body (EB) neural differentiation method combined with DAPT pre-treatment and quantitative cell transplantation, human cortical neuroblasts were obtained and transplanted into the cortex of hypoxic-ischemic injured brain with different dosages 2 weeks after surgery. Then, immunostaining, whole-cell patch clamp recordings and behavioral testing were applied to explore the graft survival and proliferation, fate commitment of cortical neuroblasts in vitro, neural circuit reconstruction and the therapeutic effects of cortical neuroblasts in HIE brain. Results: Transplantation of human cortical neural progenitor cells (hCNPs) in HIE-injured cortex exhibited long-term graft overgrowth. DAPT pre-treatment successfully synchronized hCNPs from different developmental stages (day 17, day 21, day 28) to deep layer cortical neuroblasts which survived well in HIE injured brain and greatly prevented graft overgrowth after transplantation. Importantly, the cortical neuroblasts primarily differentiated into deep-layer cortical neurons and extended long axons to their projection targets, such as the cortex, striatum, thalamus, and internal capsule in both ipsilateral and contralateral HIE-injured brain. The transplanted cortical neurons established synapses with host cortical neurons and exhibited spontaneous excitatory or inhibitory post-synaptic currents (sEPSCs or sIPSCs) five months post-transplantation. Rotarod and open field tests showed greatly improved animal behavior by intra-cortex transplantation of deep layer cortical neuroblasts in HIE injured brain. Conclusions: Transplanted hESCs derived cortical neuroblasts survive, project to endogenous targets, and integrate into host cortical neural circuits to rescue animal behavior in the HIE-injured brain without graft overgrowth, providing a novel and safe cell replacement strategy for the future treatment of HIE. [ABSTRACT FROM AUTHOR]

Taxanes and platinum molecules, specifically paclitaxel and carboplatin, are widely used anticancer drugs that induce cell death and serve as first‐line chemotherapy for various cancer types. Despite the efficient effect of both drugs on cancer cell proliferation, many tumours have innate resistance against paclitaxel and carboplatin, which leads to inefficient treatment and poor survival rates. Haploid human embryonic stem cells (hESCs) are a novel and robust platform for genetic screening. To gain a comprehensive view of genes that affect or regulate paclitaxel and carboplatin resistance, genome‐wide loss‐of‐function screens in haploid hESCs were performed. Both paclitaxel and carboplatin screens have yielded selected plausible gene lists and pathways relevant to resistance prediction. The effects of mutations in selected genes on the resistance to the drugs were demonstrated. Based on the results, an algorithm that can predict resistance to paclitaxel or carboplatin was developed. Applying the algorithm to the DNA mutation profile of patients' tumours enabled the separation of sensitive versus resistant patients, thus, providing a prediction tool. As the anticancer drugs arsenal can offer alternatives in case of resistance to either paclitaxel or carboplatin, an early prediction can provide a significant advantage and should improve treatment. The algorithm assists this unmet need and helps predict whether a patient will respond to the treatment and may have an immediate clinically actionable application. [ABSTRACT FROM AUTHOR]

Angelman syndrome (AS) and Prader-Willi syndrome (PWS), two distinct neurodevelopmental disorders, result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele, respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless, gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences, reprogramming methods, and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here, we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally, these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. [ABSTRACT FROM AUTHOR]

Introduction: The changing composition of non-cell autonomous circulating factors in blood as humans age is believed to play a role in muscle mass and strength loss. The mechanisms through which these circulating factors act in age-related skeletal muscle changes is not fully understood. In this study, we used human myogenic progenitors derived from human pluripotent stem cells to study non-cell autonomous roles of circulating factors during the process of myogenic differentiation. Methods: Myogenic progenitors from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were supplemented with serum samples fromaged or young Fischer 344 × Brown Norway F1-hybrid rats. The effect of aged or young serum supplementation on myogenic progenitor proliferation, myotube formation capacity, differentiation, and early transcriptomic profiles were analyzed. Results: We found that aged rat serum supplementation significantly reduced cell proliferation and increased cell death in both ESC- and iPSC-derived myogenic progenitors. Next, we found that the supplementation of aged rat serum inhibited myotube formation and maturation during terminal differentiation from progenitors to skeletal myocytes when compared to the cells treated with young adult rat serum. Lastly, we identified that gene expression profiles were affected following serum supplementation in culture. Discussion: Together, aged serum supplementation caused cellular and transcriptomic changes in human myogenic progenitors. The current data from our in vitro model possibly simulate non-cell autonomous contributions of blood composition to age-related processes in human skeletal muscle. [ABSTRACT FROM AUTHOR]

Spinal cord injury creates an inflammatory microenvironment that regulates the capacity of transplanted human Neural Stem Cells (hNSC) to migrate, differentiate, and repair injury. Despite similarities in gene expression and markers detected by immunostaining, hNSC populations exhibit heterogeneous therapeutic potential. This heterogeneity derives in part from the epigenetic landscape in the hNSC genome, specifically methylation (5mC) and hydroxymethylation (5hmC) state, which may affect the response of transplanted hNSC in the injury microenvironment and thereby modulate repair capacity. We demonstrate a significant up-regulation of methylcytosine dioxygenase 2 gene (TET2) expression in undifferentiated hNSC derived from human embryonic stem cells (hES-NSC), and report that this is associated with hES-NSC competence for differentiation marker expression. TET2 protein catalyzes active demethylation and TET2 upregulation could be a signature of pluripotent exit, while shaping the epigenetic landscape in hES-NSC. We determine that the inflammatory environment overrides epigenetic programming in vitro and in vivo by directly modulating TET2 expression levels in hES-NSC to change cell fate. We also report the effect of cell fate and microenvironment on differential methylation 5mC/5hmC balance. Understanding how the activity of epigenetic modifiers changes within the transplantation niche in vivo is crucial for assessment of hES-NSC behavior for potential clinical applications. [ABSTRACT FROM AUTHOR]

The rapidly evolving field of radiomics has shown that radiomic features are able to capture characteristics of both tumor and normal tissue that can be used to make accurate and clinically relevant predictions. In the present study we sought to determine if radiomic features can characterize the adverse effects caused by normal tissue injury as well as identify if human embryonic stem cell (hESC) derived extracellular vesicle (EV) treatment can resolve certain adverse complications. A cohort of 72 mice (n = 12 per treatment group) were exposed to X-ray radiation to the whole lung (3 × 8 Gy) or to the apex of the right lung (3 × 12 Gy), immediately followed by retro-orbital injection of EVs. Cone-Beam Computed Tomography images were acquired before and 2 weeks after treatment. In total, 851 radiomic features were extracted from the whole lungs and < 20 features were selected to train and validate a series of random forest classification models trained to predict radiation status, EV status and treatment group. It was found that all three classification models achieved significantly high prediction accuracies on a validation subset of the dataset (AUCs of 0.91, 0.86 and 0.80 respectively). In the locally irradiated lung, a significant difference between irradiated and unirradiated groups as well as an EV sparing effect were observed in several radiomic features that were not seen in the unirradiated lung (including wavelet-LLH Kurtosis, wavelet HLL Large Area High Gray Level Emphasis, and Gray Level Non-Uniformity). Additionally, a radiation difference was not observed in a secondary comparison cohort, but there was no impact of imaging machine parameters on the radiomic signature of unirradiated mice. Our data demonstrate that radiomics has the potential to identify radiation-induced lung injury and could be applied to predict therapeutic efficacy at early timepoints. [ABSTRACT FROM AUTHOR]

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of the squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Up-regulation of the major p63 isoform Np63a, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together, these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression. [ABSTRACT FROM AUTHOR]

Extraembryonic mesoderm cells (EXMCs) are involved in the development of multiple embryonic lineages and umbilical cord formation, where they subsequently develop into mesenchymal stem cells (MSCs). Although EXMCs can be generated from human naïve embryonic stem cells (ESCs), it is unclear whether human primed ESCs (hpESCs) can differentiate into EXMCs that subsequently produce MSCs. The present report described a three‐dimensional differentiation protocol to induce hpESCs into EXMCs by activating the Wnt pathway using CHIR99021. Single‐cell transcriptome and immunostaining analyses revealed that the EXMC characteristics were similar to those of post‐implantation embryonic EXMCs. Cell sorting was used to purify and expand the EXMCs. Importantly, these EXMCs secreted extracellular matrix proteins, including COL3A1 and differentiated into MSCs. Inconsistent with other MSC types, these MSCs exhibited a strong differentiation potential for chondrogenic and osteogenic cells and lacked adipocyte differentiation. Together, these findings provided a protocol to generate EXMCs and subsequent MSCs from hpESCs. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study.Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type. [ABSTRACT FROM AUTHOR]

The article explores the use of mesenchymal stem cells (MSCs) with enhanced antioxidant capacity in treating diabetic detrusor underactivity (DUA). The study investigates the mechanisms and optimal protocols of MSC transplantation in a preclinical model of diabetic DUA, highlighting the importance of the HGF-MET pathway and PD-L1 in muscle regeneration and immunomodulation. The research demonstrates the therapeutic efficacy of human umbilical-cord derived MSCs with enhanced antioxidant capacity in alleviating tissue injury, contributing to muscle regeneration and immunomodulation in diabetic DUA, offering a promising approach for stem cell therapies. [Extracted from the article]

Mutations in STAMBP have been well-established to cause congenital human microcephaly-capillary malformation (MIC-CAP) syndrome, a rare genetic disorder characterized by global developmental delay, severe microcephaly, capillary malformations, etc. Previous biochemical investigations and loss-of-function studies in mice have provided insights into the mechanism of STAMBP, however, it remains controversial how STAMBP deficiency leads to malformation of those affected tissues in patients. In this study, we investigated the function and underlying mechanism of STAMBP during neural differentiation of human embryonic stem cells (hESCs). We found that STAMBP is dispensable for the pluripotency maintenance or neural differentiation of hESCs. However, neural progenitor cells (NPCs) derived from STAMBP-deficient hESCs fail to be long-term maintained/expanded in vitro. We identified the anti-apoptotic protein CFLAR is down-regulated in those affected NPCs and ectopic expression of CFLAR rescues NPC defects induced by STAMBP-deficiency. Our study not only provides novel insight into the mechanism of neural defects in STAMBP mutant patients, it also indicates that the death receptor mediated apoptosis is an obstacle for long-term maintenance/expansion of NPCs in vitro thus counteracting this cell death pathway could be beneficial to the generation of NPCs in vitro. [ABSTRACT FROM AUTHOR]

The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development, we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together, they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally, we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together, ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency. Here the authors identify ZIC2 and ZIC3 as key regulators of human primed pluripotency which recruit BRG1 to open primed hESC-specific enhancers. [ABSTRACT FROM AUTHOR]

Neonatal diabetes mellitus (NDM), defined as diabetes with an onset during the first 6 months of life, is a rare form of monogenic diabetes. The initial publications on this condition began appearing in the second half of the 1990s and quite surprisingly, the search for new NDM genes is still ongoing with great vigor. Between 2018 and early 2024, six brand new NDM‐genes have been discovered (CNOT1, FICD, ONECUT1, PDIA6, YIPF5, ZNF808) and three genes known to cause different diseases were identified as NDM‐genes (EIF2B1, NARS2, KCNMA1). In addition, NDM cases carrying mutations in three other genes known to give rise to diabetes during childhood have been also identified (AGPAT2, BSCL2, PIK3R1). As a consequence, the list of NDM genes now exceeds 40. This genetic heterogeneity translates into many different mechanism(s) of disease that are being investigated with state‐of‐the‐art methodologies, such as induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESC) manipulated with the CRISPR technique of genome editing. This diversity in genetic causes and the pathophysiology of diabetes dictate the need for a variety of therapeutic approaches. The aim of this paper is to provide an overview on recent achievements in all aspects of this area of research. [ABSTRACT FROM AUTHOR]

Myocardial infarction leads to myocardial necrosis, and cardiomyocytes are non-renewable. Fatty acid-containing cardiomyocyte maturation medium promotes maturation of stem cell-derived cardiomyocytes.To study the effect palmitic acid on maturation of cardiomyocytes derived from human embryonic stem cells (hESCs) to optimize differentiation for potential treatment of myocardial infarction by hESCs.hESCs were differentiated into cardiomyocytes using standard chemically defined medium 3 (CDM3). Up to day 20 of differentiation, 200 Mm palmitic acid were added, and then the culture was continued for another 8 days to mimic the environment in which human cardiomyocytes mainly use fatty acids as the main energy source. Light microscopy, transmission electron microscopy, immunofluorescence, reverse transcription-polymerase chain reaction, and cellular ATP assays, were carried out to analyze the expression of relevant cardiomyocyte-related genes, cell morphology, metabolism levels, and other indicators cardiomyocyte maturity.Cardiomyocytes derived from hESCs under exogenous palmitic acid had an elongated pike shape and a more regular arrangement. Sarcomere stripes were clear, and the cells color was clearly visible. The cell perimeter and elongation rate were also increased. Myogenic fibers were abundant, myofibrillar z-lines were regularly, the numbers of mitochondria and mitochondrial cristae were higher, more myofilaments were observed, and the structure of round-like discs was occasionally seen. Expression of mature cardiomyocyte-associated genes TNNT2, MYL2 and MYH6, and cardiomyocyte-associated genes KCNJ4, RYR2,and PPARα, was upregulated (p < 0.05). Expression of MYH7, MYL7, KCND2, KCND3, GJA1 and TNNI1 genes was unaffected (p > 0.05). Expression of mature cardiomyocyte-associated sarcomere protein MYL2 was significantly increased (p < 0.05), MYH7 protein expression was unaffected (p > 0.05). hESC-derived cardiomyocytes exposed to exogenous palmitic acid produced more ATP per unit time (p < 0.05).Exogenous palmitic acid induced more mature hESC-CMs in terms of the cellular architecture, expression of cardiomyocyte maturation genes adnprotein, and metabolism. [ABSTRACT FROM AUTHOR]

Background: X chromosome inactivation (XCI) is a critical epigenetic event for dosage compensation of X-linked genes in female mammals, ensuring developmental stability. A robust in vitro model is required for mimicking XCI during the early stages of embryonic development. This methodology article introduces an advanced framework for the in-depth study of XCI using human pluripotent stem cells (hPSCs). By focusing on the transition between naive and primed pluripotent states, we highlight the role of long non-coding RNA X-inactive specific transcript (XIST) and epigenetic alterations in mediating XCI. Results: Our methodology enables the distinction between naive and primed hESCs based on XIST expression and the activity of X-linked reporters, facilitating the investigation of XCI initiation and maintenance. Through detailed experimental procedures, we demonstrate the utility of our hESC lines in modeling the process of human XCI, including the establishment of conditions for random XCI induction and the analysis of X chromosome reactivation. Methods: The study outlines a comprehensive approach for characterizing the X chromosome status in hPSCs, employing dual fluorescent reporter hESC lines. These reporter lines enable real-time tracking of XCI dynamics through differentiation processes. We detailed protocols for the induction of X chromosome reactivation and inactivation, as well as the X status characterization methods including cultivation of hESCs, flow cytometric analysis, RNA fluorescence in situ hybridization (FISH), and transcriptome sequencing, providing a step-by-step guide for researchers to investigate XCI mechanisms in vitro. Conclusions: This article provides a detailed, reproducible methodology for studying XCI mechanisms in vitro, employing hPSCs as a model system. It presents a significant advance in our ability to investigate XCI, offering potential applications in developmental biology, disease modeling, and regenerative medicine. By facilitating the study of XCI dynamics, this methodological framework paves the way for deeper understanding and manipulation of this fundamental biological process. [ABSTRACT FROM AUTHOR]

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated Tyramide Signal Amplification (TSA)-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in human embryonic stem cells (hESCs). Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology. In this study, TSA-seq was used to compare the distribution and association of genomic regions with the nuclear lamina, nucleoli and pericentric heterochromatin across four human cell lines. [ABSTRACT FROM AUTHOR]

(I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. METHODS: We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte--specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. RESULTS: We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell--derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte--specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/ mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. CONCLUSIONS: Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury. [ABSTRACT FROM AUTHOR]

Background: Cardiovascular progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) are considered valuable cell sources for investigating cardiovascular physiology in vitro. Meeting the diverse needs of this application requires the large-scale production of CPCs in an in vitro environment. This study aimed to use an effective culture system utilizing signaling factors for the large-scale expansion of hESC-derived CPCs with the potential to differentiate into functional cardiac lineage cells. Methods and results: Initially, CPCs were generated from hESCs using a 4-day differentiation protocol with a combination of four small molecules (CHIR99021, IWP2, SB-431542, and purmorphamine). These CPCs were then expanded and maintained in a medium containing three factors (bFGF, CHIR, and A83-01), resulting in a > 6,000-fold increase after 8 passages. These CPCs were successfully cryopreserved for an extended period in late passages. The expanded CPCs maintained their gene and protein expression signatures as well as their differentiation capacity through eight passages. Additionally, these CPCs could differentiate into four types of cardiac lineage cells: cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, demonstrating appropriate functionality. Furthermore, the coculture of these CPC-derived cardiovascular lineage cells in rat tail collagen resulted in cardiac microtissue formation, highlighting the potential of this 3D platform for studying cardiovascular physiology in vitro. Conclusion: In conclusion, expandable hESC-derived CPCs demonstrated the ability to self-renewal and differentiation into functional cardiovascular lineage cells consistently across passages, which may apply as potential cell sources for in vitro cardiovascular studies. [ABSTRACT FROM AUTHOR]

Background: Spinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study, we compared different isoforms of basic fibroblast growth factor 2 (FGF2), overexpressed in stably transfected Human embryonic stem cells (hESCs), following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB). Methods: In the present work, hESCs bioengineered to overexpress 18, 23, and 31 kD isoforms of FGF2, were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA, followed by Tukey's or Dunnett's multiple comparison tests. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Results: For the first set of experiments, rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival, glial reaction, and synaptic coverage, two weeks after the lesion, indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently, the second set of experiments was performed with that isoform, so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival, glial reaction, synaptic coverage, and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection, coupled with immunomodulation, attenuation of astrogliosis, and preservation of inputs to the rescued motoneurons. Behaviorally, the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery. Conclusion: Transgenic hESCs were an effective delivery platform for neurotrophic factors, rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury, while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery. [ABSTRACT FROM AUTHOR]

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

This document includes summaries of three articles related to medical research. The first article discusses organ preservation strategies for rectal cancer management in France. The study found that 1 in 5 patients with mid and low rectal cancer were offered organ preservation, with a preference for a selective approach that included both watch-and-wait and local excision. The second article compares different surgical approaches for anastomosis in colorectal surgery and their impact on quality of life. The study found that intracorporeal anastomosis, particularly when performed using robot-assisted surgery, resulted in improved quality of life compared to extracorporeal anastomosis. The third article presents interim results of a randomized controlled trial comparing different treatments for pilonidal sinus disease. The study found that patients treated with ActiGraft coupled with the Trephine procedure had a shorter time to complete healing compared to other treatment options. [Extracted from the article]

Background: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule. Methods: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses. Results: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations. Conclusions: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis. [ABSTRACT FROM AUTHOR]

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, which were depleted upon increased energy demands. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health. [ABSTRACT FROM AUTHOR]

Retinal pigment epithelial (RPE) cells are exclusive to the retina, critically multifunctional in maintaining the visual functions and health of photoreceptors and the retina. Despite their vital functions throughout lifetime, RPE cells lack regenerative capacity, rendering them vulnerable which can lead to degenerative retinal diseases. With advancements in stem cell technology enabling the differentiation of functional cells from pluripotent stem cells and leveraging the robust autocrine and paracrine functions of RPE cells, extracellular vesicles (EVs) secreted by RPE cells hold significant therapeutic potential in supplementing RPE cell activity. While previous research has primarily focused on the trophic factors secreted by RPE cells, there is a lack of studies investigating miRNA, which serves as a master regulator of gene expression. Profiling and defining the functional role of miRNA contained within RPE‐secreted EVs is critical as it constitutes a necessary step in identifying the optimal phenotype of the EV‐secreting cell and understanding the biological cargo of EVs to develop EV‐based therapeutics. In this study, we present a comprehensive profile of miRNA in small extracellular vesicles (sEVs) secreted during RPE maturation following differentiation from human embryonic stem cells (hESCs); early‐stage hESC‐RPE (20–21 days in culture), mid‐stage hESC‐RPE (30–31 days in culture) and late‐stage hESC‐RPE (60–61 days in culture). This exploration is essential for ongoing efforts to develop and optimize EV‐based intraocular therapeutics utilizing RPE‐secreted EVs, which may significantly impact the function of dysfunctional RPE cells in retinal diseases. [ABSTRACT FROM AUTHOR]

Harnessing the power of single-cell genomics technologies, single-cell Hi-C (scHi-C) and its derived technologies provide powerful tools to measure spatial proximity between regulatory elements and their target genes in individual cells. Using a global background model, we propose SnapHiC-G, a computational method, to identify long-range enhancer–promoter interactions from scHi-C data. We applied SnapHiC-G to scHi-C datasets generated from mouse embryonic stem cells and human brain cortical cells. SnapHiC-G achieved high sensitivity in identifying long-range enhancer–promoter interactions. Moreover, SnapHiC-G can identify putative target genes for noncoding genome-wide association study (GWAS) variants, and the genetic heritability of neuropsychiatric diseases is enriched for single-nucleotide polymorphisms (SNPs) within SnapHiC-G-identified interactions in a cell-type-specific manner. In sum, SnapHiC-G is a powerful tool for characterizing cell-type-specific enhancer–promoter interactions from complex tissues and can facilitate the discovery of chromatin interactions important for gene regulation in biologically relevant cell types. [ABSTRACT FROM AUTHOR]

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

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

This article discusses the safety, structure, and function of two patients who underwent human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) patch transplantation for acute neovascular age-related macular degeneration (AMD) with submacular hemorrhage. The study followed the patients for five years and found no evidence of uncontrolled RPE proliferation, tumor formation, or major ocular immune reactions. The patients experienced improved best corrected visual acuity (BCVA) at two years, although the BCVA decreased for one patient at five years. The study also observed patch pigmentation and areas of continuous hyper-reflective lines consistent with the hESC-RPE on imaging. The authors note the need for further studies to address remaining questions in the field. [Extracted from the article]

Neurodevelopmental disorders are mostly studied using mice as models. However, the mouse brain lacks similar cell types and structures as those of the human brain. In recent years, emergence of three‐dimensional brain organoids derived from human embryonic stem cells or induced pluripotent stem cells allows for controlled monitoring and evaluation of early neurodevelopmental processes and has opened a window for studying various aspects of human brain development. However, such organoids lack original anatomical structure of the brain during maturation, and neurodevelopmental maturation processes that rely on unique cellular interactions and neural network connections are limited. Consequently, organoids are difficult to be used extensively and effectively while modelling later stages of human brain development and disease progression. To address this problem, several methods and technologies have emerged that aim to enhance the sophisticated regulation of brain organoids developmental processes through bioengineering approaches, which may alleviate some of the current limitations. This review discusses recent advances and application areas of human brain organoid culture methods, aiming to generalize optimization strategies for organoid systems, improve the ability to mimic human brain development, and enhance the application value of organoids. [ABSTRACT FROM AUTHOR]

Abstract Background Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although intensive studies and therapeutic approaches, there are limited restorative treatments till now. Human embryonic stem cell (hESCs)-derived cortical neural progenitors have shown great potentials in ischemic stroke in adult brain. However, it is unclear whether they are feasible for cortical reconstruction in immature brain with hypoxic-ischemic encephalopathy. Methods By using embryonic body (EB) neural differentiation method combined with DAPT pre-treatment and quantitative cell transplantation, human cortical neuroblasts were obtained and transplanted into the cortex of hypoxic-ischemic injured brain with different dosages 2 weeks after surgery. Then, immunostaining, whole-cell patch clamp recordings and behavioral testing were applied to explore the graft survival and proliferation, fate commitment of cortical neuroblasts in vitro, neural circuit reconstruction and the therapeutic effects of cortical neuroblasts in HIE brain. Results Transplantation of human cortical neural progenitor cells (hCNPs) in HIE-injured cortex exhibited long-term graft overgrowth. DAPT pre-treatment successfully synchronized hCNPs from different developmental stages (day 17, day 21, day 28) to deep layer cortical neuroblasts which survived well in HIE injured brain and greatly prevented graft overgrowth after transplantation. Importantly, the cortical neuroblasts primarily differentiated into deep-layer cortical neurons and extended long axons to their projection targets, such as the cortex, striatum, thalamus, and internal capsule in both ipsilateral and contralateral HIE-injured brain. The transplanted cortical neurons established synapses with host cortical neurons and exhibited spontaneous excitatory or inhibitory post-synaptic currents (sEPSCs or sIPSCs) five months post-transplantation. Rotarod and open field tests showed greatly improved animal behavior by intra-cortex transplantation of deep layer cortical neuroblasts in HIE injured brain. Conclusions Transplanted hESCs derived cortical neuroblasts survive, project to endogenous targets, and integrate into host cortical neural circuits to rescue animal behavior in the HIE-injured brain without graft overgrowth, providing a novel and safe cell replacement strategy for the future treatment of HIE.

Abstract Background Lung injury and pulmonary fibrosis (PF), frequently arising as sequelae of severe and acute lung disease, currently face a dearth of effective therapeutic potions. Mesenchymal stem cells (MSCs) with immunomodulatory and tissue repair functions have immense potential to treat lung injury and PF. However, the optimal route of administration, timing, and frequency of dosing remain elusive. Human embryonic stem cell-derived immunity-and-matrix-regulatory cells (IMRCs) have shown therapeutic potential for lung injury and PF. Methods To ascertain the optimal therapeutic regimen for IMRCs in PF, we conducted an experimental study. Utilizing a mouse model of PF induced by bleomycin (BLM), IMRCs were administered via either a single or double intravenous (IV) or intratracheal (IT) injection on the first and seventh days post-BLM induction. Results Our findings revealed that IV infusion of IMRCs surpassed IT infusion in enhancing survival rates, facilitating body weight recovery, and optimizing Ashcroft and Szapiel scores among the model mice. Notably, IV administration exhibited a more profound ability to mitigate lung inflammation and fibrosis. Moreover, earlier and more frequent administrations of IMRCs were found to be advantageous in enhancing their therapeutic effects. Specifically, early administration with two IV infusions significantly improved body weight, lung organ coefficient, pulmonary ventilation and diffusion functions, and PF. This was accompanied by an increase in alveolar type I and II epithelial cells and a suppression of macrophage infiltration via CD24. Conclusion Collectively, these results suggested that IMRCs infusion ameliorated lung injury by promoting lung regeneration and inhibiting macrophage infiltration in a route, time, and frequency-dependent manner.

Abstract Background X chromosome inactivation (XCI) is a critical epigenetic event for dosage compensation of X-linked genes in female mammals, ensuring developmental stability. A robust in vitro model is required for mimicking XCI during the early stages of embryonic development. This methodology article introduces an advanced framework for the in-depth study of XCI using human pluripotent stem cells (hPSCs). By focusing on the transition between naive and primed pluripotent states, we highlight the role of long non-coding RNA X-inactive specific transcript (XIST) and epigenetic alterations in mediating XCI. Results Our methodology enables the distinction between naive and primed hESCs based on XIST expression and the activity of X-linked reporters, facilitating the investigation of XCI initiation and maintenance. Through detailed experimental procedures, we demonstrate the utility of our hESC lines in modeling the process of human XCI, including the establishment of conditions for random XCI induction and the analysis of X chromosome reactivation. Methods The study outlines a comprehensive approach for characterizing the X chromosome status in hPSCs, employing dual fluorescent reporter hESC lines. These reporter lines enable real-time tracking of XCI dynamics through differentiation processes. We detailed protocols for the induction of X chromosome reactivation and inactivation, as well as the X status characterization methods including cultivation of hESCs, flow cytometric analysis, RNA fluorescence in situ hybridization (FISH), and transcriptome sequencing, providing a step-by-step guide for researchers to investigate XCI mechanisms in vitro. Conclusions This article provides a detailed, reproducible methodology for studying XCI mechanisms in vitro, employing hPSCs as a model system. It presents a significant advance in our ability to investigate XCI, offering potential applications in developmental biology, disease modeling, and regenerative medicine. By facilitating the study of XCI dynamics, this methodological framework paves the way for deeper understanding and manipulation of this fundamental biological process.

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

Abstract YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.

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

Background: Chromosome stability is crucial for homeostasis of pluripotent stem cells (PSCs) and early-stage embryonic development. Chromosomal defects may raise carcinogenic risks in regenerative medicine when using PSCs as original materials. However, the detailed mechanism regarding PSCs chromosome stability maintenance is not fully understood. Methods: Mouse embryonic stem cells (line D3) and human embryonic stem cells (line H9) were cultured under standard conditions. To confirm the loading of RetSat protein on mitotic chromosomes of PSCs, immunostaining was performed in PSCs spontaneous differentiation assay and iPSC reprogramming assay from mouse embryonic fibroblasts (MEFs), respectively. In addition, qPCR, immunoprecipitation, LC-MS/MS and immunoblotting were used to study the expression of RetSat, and interactions of RetSat with cohesin/condensin components. RNA sequencing and teratoma formation assay was conducted to evaluate the carcinogenic risk of mouse embryonic stem cells with RetSat deletion. Results: We reported a PSC high-expressing gene, RetSat, plays key roles in chromosome stabilization. We identified RetSat protein localizing onto mitotic chromosomes specifically in stemness positive cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found dramatic chromosome instability, e.g. chromosome bridging, lagging and interphase micronuclei in mouse and human ESCs when down regulating RetSat. RetSat knock-out mouse ESCs upregulated cancer associated gene pathways, and displayed higher tumorigenic capacities in teratoma formation assay. Mechanistically, we confirmed that RetSat interacts with cohesin/condensin components Smc1a and Nudcd2. RetSat deletion impaired the chromosome loading dosage of Smc1a, Smc3 and Nudcd2. Conclusions: In summary, we reported RetSat to be a key stabilizer of chromosome condensation in pluripotent stem cells. This highlights the crucial roles of RetSat in early-stage embryonic development, and potential value of RetSat as an effective biomarker for assessing the quality of pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Nucleoli are fundamentally essential sites for ribosome biogenesis in cells and formed by liquid-liquid phase separation (LLPS) for a multilayer condensate structure. How the nucleoli integrity is maintained remains poorly understood. Here, we reveal that METTL3/METTL14, the typical methyltransferase complex catalyzing N6-methyladnosine (m6A) on mRNAs maintain nucleoli integrity in human embryonic stem cells (hESCs). METTL3/METTL14 deficiency impairs nucleoli and leads to the complete loss of self-renewal in hESCs. We further show that SUV39H1/H2 protein, the methyltransferases catalyzing H3K9me3 were dramatically elevated in METTL3/METTL14 deficient cells, which causes an accumulation and infiltration of H3K9me3 across the whole nucleolus and impairs the LLPS. Mechanistically, METTL3/METTL14 complex serves as an essential adapter for CRL4 E3 ubiquitin ligase targeting SUV39H1/H2 for polyubiquitination and proteasomal degradation and therefore prevents H3K9me3 accumulation in nucleoli. Together, these findings uncover a previously unknown role of METTL3/METTL14 to maintain nucleoli integrity by facilitating SUV39H1/H2 degradation in human cells. Nucleoli are essential cellular sites for ribosome biogenesis that are formed by liquid-liquid phase separation. Here the authors show that METTL3/METTL14 maintain nucleoli integrity through mediating SUV39H1/2 degradation in human embryonic stem cells. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hPSCs) are pivotal in regenerative medicine, yet their in vitro expansion often leads to genetic abnormalities, raising concerns about their safety in clinical applications. This study analyzed ten human embryonic stem cell lines across multiple passages to elucidate the dynamics of chromosomal abnormalities and single-nucleotide variants (SNVs) in 380 cancer-related genes. Prolonged in vitro culture resulted in 80% of the lines acquiring gains of chromosome 20q or 1q, both known for conferring an in vitro growth advantage. 70% of lines also acquired other copy number variants (CNVs) outside the recurrent set. Additionally, we detected 122 SNVs in 88 genes, with all lines acquiring at least one de novo SNV during culture. Our findings showed higher loads of both CNVs and SNVs at later passages, which were due to the cumulative acquisition of mutations over a longer time in culture, and not to an increased rate of mutagenesis over time. Importantly, we observed that SNVs and rare CNVs followed the acquisition of chromosomal gains in 1q and 20q, while most of the low-passage and genetically balanced samples were devoid of cancer-associated mutations. This suggests that recurrent chromosomal abnormalities are potential drivers for the acquisition of other mutations. [ABSTRACT FROM AUTHOR]

CAR‐NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue‐derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large‐scale and cryopreserved mesothelin (MSLN) CAR‐NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR‐expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR‐NK cells via an efficient organoid induction system. The MSLN CAR‐NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR‐NK cells show increased secretion of IFN‐γ and TNF‐α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR‐NK cells in liquid nitrogen using a clinical‐grade freezing medium (CS10) for more than 6 months to mimic an off‐the‐shelf CAR‐NK cell product. The thawed MSLN CAR‐NK cells immediately recovered after 48–72‐h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR‐NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour‐bearing mice. Our study provides insights into the clinical translation of hESC‐derived MSLN CAR‐NK cells as a promising off‐the‐shelf cell product. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells and human induced pluripotent stem cells may be used to create 3D tissues called brain organoids. They duplicate the physiological and pathological characteristics of human brain tissue more faithfully in terms of both structure and function, and they more precisely resemble the morphology and cellular structure of the human embryonic brain. This makes them valuable models for both drug screening and in vitro studies on the development of the human brain and associated disorders. The technical breakthroughs enabled by brain organoids have a significant impact on the research of different brain regions, brain development and sickness, the connections between the brain and other tissues and organs, and brain evolution. This article discusses the development of brain organoids, their use in diabetes research, and their progress. [ABSTRACT FROM AUTHOR]

A new system for delivery of anthracycline antibiotics based on chitosan-polyaminoxyls (CPA) was studied in a model of non-tumor (human embryonic mesenchymal stem cells) and tumor cells (human hepatocellular carcinoma) in vitro. The presence of CPA micelles considerably suppresses daunorubicin-induced ROS generation in normal cells without affecting this process in tumor cells. CPA micelles do not reduce the cytotoxic effect of daunorubicin and do not prevent its accumulation in cells. The use of CPA significantly increases accumulation of Nrf2 transcription factor in the nuclei of both normal and tumor cells in comparison with free daunorubicin. Increased nuclear translocation of Nrf2 leads to a significant increase in the expression of its target gene TXN1, but not the NQO1, GPX1, and HMOX1 genes, the increased expression of which can lead to the development of resistance to anthracycline antibiotics. Redox-active CPA micelles have great potential for the development of nanoparticles for the transport of anthracycline antibiotics in experimental tumor chemotherapy, and also as promising activators of Nrf2 transcription factor. [ABSTRACT FROM AUTHOR]

AIM: To investigate the reparative effect of extracellular vesicles (EVs) derived from H9 human embryonic stem cells (H9-hESCs) on endometrial injury. METHODS: EVs were isolated from the culture supernatant of H9-hESCs and characterized. A mouse model of endometrial injury was established, with bilateral uterine divisions into an EVs experimental group and a PBS control group. EVs and PBS were injected respectively. Histological changes in the endometrium were assessed using HE staining, and proliferating cell nuclear antigen (PCNA) expression was analyzed via immunohistochemistry. The impact of EVs on the proliferation of human endometrial stromal cells (hEndoSCs) was evaluated using EdU staining and Western blot. RESULTS: H9-hESCs-EVs exhibited a membrane-structured nanobody with a particle size of (144.7±2.1) nm and expressed characteristic proteins CD63 and TSG101. Compared to the PBS control group, the EVs group showed increased endometrial tissue morphology, thickness, and gland numbers. The average optical density of PCNA expression significantly increased in the EVs group compared to the PBS group (P<0. 05). Results from EdU staining and Western blot demonstrated that H9-hESCs-EVs promoted hEndoSC proliferation, with a positive correlation observed between H9-hESCs-EVs and EVs protein concentration (P<0. 05). CONCLUSION: H9-hESCs-EVs enhance the repair of endometrial injury by stimulating the proliferation of endometrial stromal cells. [ABSTRACT FROM AUTHOR]