Stem Cells

Immune checkpoint inhibitor-induced myocarditis (ICIM) is a severe immune-related adverse event with heterogeneous clinical presentations and potential genetic susceptibility. Here, we established a human induced pluripotent stem cell (iPSC) line from an ICIM patient with an HLA-type distinct from previously reported line, who developed concurrent type I diabetes following ICI treatment. This line exhibited typical morphology, normal female karyotype, pluripotency, trilineage differentiation into all three germ layers, Sendai virus clearance, and no mycoplasma contamination. Given the fulminant nature and diverse clinical presentations of ICIM, expanding the repertoire of iPSC lines are critical for investigating ICIM heterogeneity and its underlying mechanisms.

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

Induced pluripotent stem cells (iPSCs) hold great promise for both allogeneic and autologous cellular therapies. However, broad application and clinical translation is hindered by fragmented, complex and time-intensive workflows, resulting in high manufacturing costs, poor standardization and increased risk of genomic aberrations in derived iPSCs. In this study we developed a standardizable, automatable and time- efficient process for the derivation of monoclonal iPSC lines straight from skin including a comprehensive and cascaded OC strategy. We generated monoclonal iPSC lines derived from human skin punch biopsies of ten donors (age 49-81) via mRNA-based reprogramming that subsequently underwent comprehensive and thorough characterization of phenotypic and genetic properties. The use of a combined mechanical and enzymatic fibroblast isolation protocol and a transient non-integrative reprogramming technology allowed us to obtain 78 monoclonal iPSC lines, ready for banking, molecular characterization and further differentiation within seven weeks from initial sample processing to passage four iPSC lines. The phenotypical characterization via flow cytometry-based pluripotency marker expression and 2D-directed differentiation into the three germ layers showed low intra- and inter-donor variability over all generated lines. A combination of SNP array based CNV analysis followed by whole exome sequencing proved to be the most efficient approach for assessment of genomic integrity. Proof-of-concept experiments for closed system processing revealed that a substantial part of the most error-prone and technically demanding steps can be transferred to semi- automated, closed systems. In conclusion, the described protocol allows for time- efficient, standardizable and automatable generation of high-quality monoclonal iPSC lines from human skin punch biopsies within seven weeks, thus moving the field of autologous iPSC manufacturing one step further towards cost-efficient clinical implementation.

Human pluripotent stem cells serve as a vital model for studying early human lineage specification, yet conventional assessments relying on endpoint canonical markers of the three germ layers may overlook transient intermediate states and broader cellular programs. Here we combined directed differentiation of human induced pluripotent stem cells toward neuroectodermal, cardiac mesodermal, and hepatic endodermal lineages with comparative transcriptomic profiling across timepoints. Our analyses revealed a transient primitive streak-like mesendodermal state shared by mesodermal and endodermal trajectories, followed by lineage-specific divergence characterized by distinct transcriptional, metabolic, proliferative, and chromatin remodeling dynamics. Notably, endodermal differentiation exhibited rapid definitive endoderm commitment with enriched oxidative metabolism, whereas cardiac mesoderm differentiation showed progressive transcriptional remodeling and cardiac progenitor activation. These findings demonstrate that comparative transcriptomics can resolve developmental intermediates and cellular-state dynamics during human germ layer specification, providing a framework for evaluating lineage commitment beyond endpoint canonical marker expression, and to inform strategies for optimizing or redirecting differentiation.

During development, endothelial cells (ECs) migrate into the brain and acquire blood-brain barrier (BBB) properties such as tight junctions, limited transcellular transport, and high electrical resistance. Although key signaling pathways that are active in vivo have been identified, factors critical in inducing brain EC differentiation in vitro remain unclear. Here, we describe conditions that promote brain EC-like gene expression in human pluripotent stem cell (hiPSC)-derived ECs. Activation of Wnt/{beta}-catenin signaling upregulates the brain EC marker GLUT1 (SLC2A1) while suppressing the peripheral EC marker PLVAP. Simultaneously, stimulation of STAT3 by CNTF together with TGF-{beta} inhibition increases CLDN5 expression. We further found that hiPSC-derived ECs secrete high levels of angiopoietin-2 (ANGPT2) and that razuprotafib (AKB-9778), a PTPRB (VE-PTP) inhibitor, inhibits ANGPT2 and improves monolayer integrity. These results suggest that combinatorial modulation of specific signaling pathways stimulates the differentiation of human brain ECs in vitro.

Vascular Endothelial Zinc Finger 1 (VEZF1) is essential for embryonic development, but its role in pluripotency exit remains unclear. Previous work showed that Vezf1-deficient ESCs exhibit impaired differentiation, reduced Dnmt3b expression, and genome-wide hypomethylation. Here, we show that Vezf1-/- ESCs fail to efficiently repress the pluripotency transcriptional program during differentiation, a defect that persists after ectopic Dnmt3b expression. Genome-wide analysis revealed VEZF1 occupancy at regulatory regions of genes involved in several developmental signaling pathways, including MAPK, WNT, and Hippo, as well as at some pluripotency-associated genes. Many VEZF1-bound MAPK genes showed reduced expression in undifferentiated Vezf1-/-ESCs, suggesting that VEZF1 activity contributes to transcriptional competence required for efficient pluripotency exit. VEZF1 loss also led to widespread acquisition of new CTCF sites associated with developmental signaling, a subset of which overlapped VEZF1-bound regulatory regions. CTCF depletion had only limited effects on the expression of the VEZF1-bound MAPK genes examined, indicating that increased CTCF occupancy alone is insufficient to explain their reduced expression in Vezf1-/- ESCs. Together, our findings identify a DNMT3B-independent function of VEZF1 in facilitating the exit from pluripotency and establishing transcriptional competence for differentiation, while revealing a potential role for VEZF1 in regulating CTCF occupancy during developmental state transitions.

Life-long production of blood requires the preservation of hematopoietic stem cell (HSCs) regenerative capacity during inflammation. The cytokine, Thrombopoietin (THPO), is essential for HSC maintenance yet its role during inflammatory stress remains incompletely understood. Long-term repopulating potential was rapidly depleted in THPO-deficient HSCs upon poly(I:C) administration through inflammatory pyroptosis. Transcriptomic and chromatin accessibility analyses revealed constitutive interferon (IFN) pathway activation in THPO-deficient HSCs, characterized by enhanced STAT1 signaling, increased accessibility of STAT and IRF motifs, and elevated expression of IFN-stimulated genes. Lipidomic profiling further identified selective shifts in sphingomyelin (SM) species and enrichment of features associated with increased bilayer rigidity. THPO-deficient HSCs displayed elevated membrane SM incorporation, impaired membrane fluidity and altered membrane ultrastructure. Genetic ablation of Stat1 normalized membrane lipid abnormalities and reduced pyroptotic activation and restored HSC survival and regenerative function under inflammatory stress. Together, these findings identify a STAT1 and SM metabolism as critical THPO downstream to protect HSCs from inflammatory pyroptosis. Our results reveal membrane lipid homeostasis as a fundamental mechanism through which cytokine signaling safeguards HSC function during stress.

Introductory ParagraphMultiple regulatory mechanisms govern cardiomyocyte proliferation including epigenetic modifications, metabolism and mechanical load. However, it is unclear whether such mechanisms can be pharmacologically targeted to induce cardiomyocyte proliferation without affecting other cell types. Here, we develop a dual-reporter (TNNT2eGFP; PCNAmScarlet-I) and a high-throughput image-based pipeline in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, with counter screening in non-myocytes, to identify compounds that selectively promote cardiomyocyte proliferation without affecting other cell types. We identify the PIM kinase inhibitor GDC-0339 as a cardiomyocyte-selective pro-proliferative compound. GDC-0339 induced proliferation of hiPSC-derived cardiomyocytes without activity in non-myocytes, non-cardiac fibroblasts or epithelial cells. Phosphoproteomic profiling of GDC-0339 in cardiomyocytes and non-cardiac fibroblasts revealed a cardiomyocyte-specific mechanism of action involving sarcomere disassembly via remodelling of the F-actin cytoskeleton and metabolic reprogramming to anaerobic metabolism via Pyruvate Dehydrogenase Kinases (PDKs). Thus, we uncover cardiomyocyte-specific mechanisms governing the cell cycle that are potentially druggable.

BackgroundCardiac fibrosis drives adverse myocardial remodelling through persistent fibroblast activation, ECM deposition, and impaired cardiac function. Current therapies offer limited protection against cardiac fibrosis progression. Elafibranor is a dual PPAR-/{delta} agonist approved for the treatment of liver disease. However, its effects in human models of cardiac fibrosis remain insufficiently explored. MethodsElafibranor was evaluated in complementary human in vitro TGF-{beta}1-induced cardiac fibrosis models: 2D primary fibroblasts, 3D fibroblast spheroids, spontaneously contracting 3D cardiac microtissues, and hiPSC-derived cardiomyocytes. Viability, apoptosis, fibroblast activation, ECM remodelling, mitochondrial respiration, nucleotide and NAD pools, calcium handling, contractility, and transcriptomic profiles were assessed. ResultsAt non-cytotoxic concentrations, elafibranor attenuated TGF-{beta}1-driven cardiac fibrosis responses. In 2D cardiac fibroblasts, it reduced myofibroblast differentiation, procollagen 11 secretion, and partially restored mitochondrial respiratory capacity. In 3D spheroids, it preserved viability, attenuated caspase-3/7 activation, and suppressed procollagen 11 release. In cardiac microtissues, elafibranor reduced ECM accumulation, shifted transcriptomic profiles toward redox-metabolic/cytoprotective pathways, altered adenine nucleotide and NAD pools, and partially recovered contraction parameters. In hiPSC-derived cardiomyocytes, elafibranor modulated calcium handling, contractility, and mitochondrial respiration. ConclusionsElafibranor mitigates TGF-{beta}1-driven cardiac fibrosis by suppressing fibroblast activation and ECM remodelling while promoting adaptive metabolic, redox, and bioenergetic responses, supporting balanced PPAR-/{delta} activation as a potential therapeutic strategy for cardiac fibrosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/745425v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1cbd94eorg.highwire.dtl.DTLVardef@27a44borg.highwire.dtl.DTLVardef@9354baorg.highwire.dtl.DTLVardef@9f9946_HPS_FORMAT_FIGEXP M_FIG C_FIG

Endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) are increasingly used to model vascular diseases and test therapeutic strategies. However, the efficiency and reproducibility of differentiation can vary depending on the culture medium and its supplemented factors and stages. Here, we directly compared two defined media, APEL and BPEL, for iPSC-to-ECs differentiation. iPSCs were differentiated over 10 days with sequential growth factor induction, followed by magnetic-activated cell sorting or flow cytometry for CD31+ cells. Both media produced ECs with similar morphology and marker expression, including CD31 and VE-cadherin. Functional assays demonstrated comparable tube formation, indicating equivalent endothelial functionality. Cost analysis indicated that APEL had a higher total reagent cost but generated a higher total cell yield, resulting in a comparable cost per 10 total cells, whereas BPEL was more cost-efficient for producing CD31/VE-cadherin endothelial-specific cells. Our results suggest that APEL and BPEL media are equally effective for generating iPSC-derived ECs, providing flexibility in method selection for vascular disease modeling and drug discovery applications.

Background and aimsAssisted reproductive technologies (ART), including in vitro fertilisation (IVF), account for over 10 million births worldwide. ART-conceived young offspring show altered cardiovascular phenotypes, including cardiac remodelling and raised blood pressure, but the mechanisms remain unclear. Mitochondrial disturbance during preimplantation development may link early ART exposure to later cardiac dysfunction. However, to our knowledge, no one has assessed mitochondrial function in adult offspring from IVF pregnancies. In this study, investigated the effects of IVF and embryo vitrification on blastocyst mitochondrial redox balance and metabolism, and determined whether these effects persisted into the adult heart. Methods and ResultsIGS-CD1 mouse blastocysts from naturally mated donors or IVF were transferred fresh or after vitrification-warming. IVF reduced blastocyst total, trophectoderm and inner cell mass cell number, while vitrification lowered the inner cell mass proportion and increased apoptosis. Both exposures depolarised mitochondrial membrane potential and depleted glutathione; reactive oxygen species rose with an interaction, being highest in vitrified IVF embryos. IVF reduced live birth rate and litter size. In the adult offspring, high-resolution respirometry of isolated mitochondria from left ventricle revealed reduced oxidative phosphorylation capacity with an increased H2O2 production, altered OXPHOS subunit abundance and reduced complex I, III and IV activities. ConclusionsIVF and vitrification impose distinct disturbance on preimplantation embryo redox states and bioenergetics, and this early disturbance is followed into adulthood with a reduced mitochondrial aerobic capacity and increased basal ROS production. These results have important implications for IVF practices and suggest that mitochondria may be permanently programmed by this procedure. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/744765v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1cd1bd7org.highwire.dtl.DTLVardef@ded6b8org.highwire.dtl.DTLVardef@1e2ddf7org.highwire.dtl.DTLVardef@15abc84_HPS_FORMAT_FIGEXP M_FIG C_FIG IVF and vitrification impose distinct and partly independent effects on the preimplantation embryo that persist into the adult offspring heart. At the blastocyst stage, IVF reduced cell number and vitrification altered lineage allocation, while both exposures lowered mitochondrial membrane potential ({Delta}{Psi}m) and glutathione (GSH) and raised reactive oxygen species (ROS); vitrification additionally increased apoptosis. After embryo transfer, IVF reduced live birth rate and litter size, whereas vitrification altered postnatal growth trajectory. In adult offspring, ventricular mitochondria, vitrification reduced OXPHOS capacity and IVF reduced LEAK respiration, while both exposures increased H2O2/ O2 flux, reduced respiratory chain enzyme activities and altered OXPHOS subunit abundance.

The blood-gonadal barrier protects the germline from parental exposures. A phenomenon known as intergenerational inheritance suggests that, exceptionally, this barrier can be surpassed with consequences for the subsequent generation. Specific diet regimes and early traumatic experiences have been among the chronic stressors shown to be able to lead to intergenerational inheritance in mammals. Less is known about how acute stress can affect the germline. Cardiac damage leads to several alterations in peripheral organs and, overall, affects blood flow, metabolism, and the immune response. Whether cardiac damage can also affect the reproductive system is not known and might offer new insights into the potential inheritance of cardiovascular disease. Here, we used zebrafish and mouse models to explore the intergenerational role of cardiac damage and repair. In the first week after a cardiac cryolesion, male zebrafish gonads and gametes activated responses associated with inflammation. In sperm, chromatin accessibility was found altered in response to cardiac cryolesion. Offspring of cryoinjured zebrafish males revealed changes in cardiac function and cardiac gene expression. Induction of systemic sterile inflammation in the paternal generation mimicked cardiac injury effects in the following generation, while anti-inflammatory treatments in the injured paternal generation partially recovered F1 cardiac features. Similar features were found in mouse testis after a neonatal injury, and in the hearts of their offspring, suggesting a conserved role of sterile inflammation as a vector for intergenerational transmission of cardiac injury.

Traumatic brain injury (TBI) produces persistent multidomain disability spanning motor, cognitive, emotional and sleep-wake function, with no approved restorative therapy. Here, we tested pd.S6.133.hNSC, a cryopreserved, GMP-like human neural stem cell (hNSC) product derived from Shef-6 and FACS-sorted on CD133+/CD34-, in a randomized dose-ranging study in common marmosets subjected to controlled cortical impact (n = 18). Seven weeks after injury, animals received MRI-guided stereotactic transplantation into perilesional cortex bilaterally under tacrolimus immunosuppression, with either vehicle or pd.S6.133.hNSC at 1 million (1e6) or 5 million (5e6) cell dose. At 3 months post-transplantation, 5e6 dosage improved executive and problem-solving performances (Object Retrieval Task with Barrier Detour), gait dynamics (CatWalk assay), anxiety-like behavior (Human Intruder Test), and actigraphy-derived sleep-wake and circadian rhythm measures relative to vehicle and 1e6 dose. Longitudinal 7T MRI demonstrated a dose-dependent reduction in lesion volume and preservation of corpus callosum white matter volume in the 5e6 group. Transplantation was well tolerated, with no observed adverse events across 1,197 cumulative post-transplant animal-days. Histopathology at 3 months post-transplantation in NHPs showed engraftment without tumor formation or abnormal tissue overgrowth. These findings support the safety and multidomain efficacy of a cryopreserved hNSC product in a nonhuman primate TBI model and inform translational development toward first-in-human testing with clinically aligned endpoints.

Uterine fibroids (leiomyomas) are highly prevalent benign tumors defined by excessive extracellular matrix (ECM) deposition, altered vascular structure, and progressive tissue stiffening, yet the cellular programs that coordinate these features remain poorly understood. Cellular senescence has been implicated in fibroid biology, but whether senescence represents a uniform state or distinct, functionally specialized cell identities within fibroids is unknown. Here, we identify the distinct heterogeneous populations of senescent cells ("senotypes") present in human fibroids and characterize their role in shaping the fibroid microenvironment. Using single-cell RNA sequencing (scRNA-seq) integrated with a senescence gene signature and protein-level validation, we identify senescent cells (SnC) distributed across fibroblast, mural, and endothelial compartments, each exhibiting distinct transcriptional programs. SnC endothelial cells (ECs) are enriched in fibroids relative to matched myometrium and activate TEAD4-associated mechanosensing, angiogenic, and immune signaling pathways, despite being associated with impaired vessel maturation in situ. In parallel, SnC fibroblast and mural populations in fibroids upregulated SRF-associated cytoskeletal and ECM programs, accompanied by increased COL6A3 expression and collagen VI deposition, consistent with tissue stiffening. Ligand-receptor and spatial analyses reveal that these SnC populations function as interconnected signaling hubs, coordinating immune cell recruitment and stromal remodeling. Importantly, analysis of human fibroids treated with collagenase demonstrated a reduction in both ECM density and SnC burden, supporting a reinforcing relationship between matrix mechanics and senescence. Together, these findings establish senescence in fibroids as a heterogeneous, mechanically reinforced, and network-driven process that links vascular dysfunction, immune signaling, and fibrosis, highlighting distinct SnC states as potential translational targets for non-surgical therapies.

Adipose-derived mesenchymal stem cells (gAD-MSCs) are promising candidates for veterinary regenerative medicine, yet the characterization of gAD-MSCs from locally adapted Indonesian goat breeds remains limited. This study aimed to isolate and characterize gAD-MSCs from Peranakan Ettawa (PE) goats using tissue explant culture. Subcutaneous adipose tissue was collected from the base of the tail of healthy PE goats (n=3). Primary cell outgrowth from explants was observed by Day 5, displaying characteristic fibroblast-like, spindle-shaped morphology and strong plastic adherence. Serial passaging to Passage 3 (P3) yielded a morphologically stable, homogeneous cell population. Assessment of cellular metabolic activity via the resazurin assay demonstrated sustained cell viability and a statistically significant increase in metabolic activity between Day 3 and Day 5 (p < 0.05). Furthermore, functional clonogenic capacity, evaluated using the colony-forming unit (CFU) assay, showed continuous temporal expansion of colonies over 14 days, yielding an average of 52.0 + - 4.1 colonies per dish. These findings confirm that expanded gAD-MSCs P3from PE goats maintain characteristic mesenchymal morphology, sustained metabolic activity, and clonogenic capacity. This work provides a baseline cellular profile of PE goat gAD-MSCs, supporting their potential use in veterinary regenerative medicine and tissue engineering.

Lipid droplets (LDs) are protein-coated organelles that store neutral lipids and regulate diverse cellular processes beyond energy metabolism. In neural stem/progenitor cells (NSPCs), LD abundance and morphology vary across cellular states, yet whether LD molecular composition is similarly state-dependent remains unknown. Here, we define the first endogenous LD proteome and lipidome atlas of NSPCs and their progeny. State-resolved analyses reveal extensive differences in both LD-associated proteins and stored lipids, allowing for identification of LD signatures that distinguish quiescent and proliferative states, and uncovering selective enrichment of numerous proteins on quiescent NSPC LDs. Functional interrogation of one such protein, CIDEB, showed that its knockdown alters LD morphology and induces senescence-associated transcriptional programs, implicating CIDEB in the maintenance of NSPC quiescence. These findings establish LDs as dynamically specialized organelles in NSPCs and their progeny and provide a resource for investigating LD-mediated regulation of stem cell state and lineage progression.

Hematopoietic stem cell (HSC) aging is associated with epigenetic remodeling, yet the molecular mechanisms driving these changes, their overlap with stress-induced alterations, and whether this course can be durably reset remain incompletely understood. Here, we show that transient induction of the Yamanaka factors OCT4, SOX2, KLF4, and MYC in young mice durably delays and partially reverses physiological and LPS-driven HSC aging in mice. Transient reprogramming improved hematopoietic reconstitution, reduced myeloid bias, and limited DNA damage. Multi-omic analyses revealed reduced chromatin accessibility at AP-1-enriched regulatory regions, attenuated age-associated AP-1 transcriptional programs, and repression of transposable elements (TEs). Pharmacological AP-1 inhibition prevented LPS-induced TE activation and loss of HSC clonogenicity. Reverse transcriptase inhibition in aged mice reduced DNA damage and improved HSC function, demonstrating a functional contribution of TE activity to HSC decline. Together, these findings identify AP-1-associated chromatin remodeling as a candidate mechanism linking inflammatory stress, TE activation and HSC aging.

Mammalian preimplantation development requires precisely coordinated lineage decisions to establish the trophectoderm (TE), inner cell mass (ICM), epiblast (EPI), and primitive endoderm (PrE). Glucose metabolism and epigenetic regulation are increasingly recognised as key determinants of lineage specification during preimplantation development. However, how glucose-dependent metabolic cues interface with epigenetic mechanisms to regulate embryonic cell fate remains poorly understood. Here, we investigated the role of glucose in regulating protein methylation by protein arginine methyltransferases (PRMT) in bovine preimplantation development. PRMT1 and its associated histone mark H4R3me2a were detected throughout bovine oocyte maturation and embryo development. Pharmacological inhibition of Type I PRMTs using two structurally distinct inhibitors, GSK3368715 and MS023, markedly reduced global protein asymmetric dimethylarginine (ADMA) and H4R3me2a levels. PRMT inhibition impaired blastocyst cell proliferation, reduced total cell number, and disrupted both first and second lineage decisions, as demonstrated by decreased CDX2- and SOX2-positive TE and ICM cells and reduced NANOG- and GATA6-positive EPI and PrE cell allocation. Mechanistically, Type I PRMT inhibition downregulated key components of the Hippo-associated TE programme, including YAP, TEAD4, and TFAP2C. Consistent effects were observed in mouse embryos, where MS023 treatment reduced ADMA, CDX2, YAP, and TFAP2C expression and impaired TE and ICM allocation. Collectively, our findings identify Type I PRMT-mediated ADMA as an essential epigenetic regulator of early mammalian lineage specification and support a conserved ADMA-Hippo regulatory axis linking arginine methylation to embryonic cell fate decisions. In briefType I protein arginine methyltransferase (PRMT)-mediated asymmetric dimethylarginine (ADMA) is required for proper lineage specification during mammalian preimplantation development. ADMA depletion disrupts Hippo signalling, cell proliferation, and trophectoderm and inner cell mass allocation in bovine and mouse embryos.

Insulin production is a cardinal feature of pancreatic {beta} cells. Studies in rodents show that {beta} cells can switch between low and high insulin gene activity states and that elevated insulin production makes {beta} cells more vulnerable to stresses associated with diabetes. In people, genetically elevated insulin production increases the risk of type 1 diabetes. Via effects on obesity, hyperinsulinemia contributes to the pathogenesis of type 2 diabetes. Here, we characterize {beta} cells in low and high INS gene activity states sorted from primary human islets transduced with INS-GFP adenovirus and differentiated INS-EGFP knock-in embryonic stem cells (SC{beta} cells). We profile {beta} cell function, protein synthesis, resilience to diabetes associated stress, single {beta} cell transcriptomes and their co-activity networks, and purified {beta} cell proteomes. We show that human {beta} cells transition between distinct states. High INS cells have elevated maturity marker mRNAs and proteins, increased protein translation, are larger, but also more susceptible to cell death when exposed to diabetes-relevant stresses. We also catalogue thousands of differences in proteins in high INS stem cell-derived {beta} cells compared directly with high INS primary {beta} cells. Our study improves our understanding of the delicate balance between insulin production and {beta} cell resilience and guides the engineering of better {beta} cells. Blurbtranscriptional, proteomic, and functional analyses of insulin gene expression states in human {beta} cells from donor islets and stem cells Key findingsO_LIWe identify high and low INS gene activity states in human insulin-producing cells from donor islets and embryonic stem cell differentiations. C_LIO_LIWe characterize the relationship between insulin production and fragility, demonstrating that increased insulin production comes at a cost of reduced resilience to multiple stresses. C_LIO_LIFunctional, transcriptomic, and proteomic analyses identify similarities and differences between how primary and stem cell-derived {beta} cells manage stress and insulin production. C_LIO_LIWe report a comprehensive side-by-side proteomic analysis of purified primary and stem cell- derived {beta} cells in the high INS state and identify differences in protein production and secretion machinery, providing a roadmap for making better {beta} cells. C_LI

Background/Objectives: Cancer survivors frequently experience long-term neurocognitive impairments following chemotherapy and cranial irradiation, yet experimental models that enable mechanistic investigation of therapy-induced neurotoxicity at the transcriptional level remain limited. This study aimed to develop a human three-dimensional (3D) neural tissue model derived from L-Myc immortalized neural stem cells (LMNSCs) and use transcriptomic profiling to identify molecular pathways underlying chemotherapy- and radiation-induced neural injury and extracellular vesicle (EV)-mediated recovery. Methods: LMNSCs were differentiated in a 3D, methylcellulose-based culture to generate neural tissue containing neurons, astrocytes, and oligodendrocytes. Cultures were exposed to methotrexate (MTX) or ionizing radiation to induce neural injury and subsequently treated with LMNSC-derived EVs. Neural injury and repair mechanisms were evaluated by immunocytochemistry and bulk transcriptomics. Results: MTX and irradiation induced dose-dependent injury, exhibited by loss of neuronal complexity and reduced glial populations. LMNSC-EV treatment promoted recovery of neuronal and glial populations following MTX- and irradiation-induced injury. Transcriptomic analysis of irradiated cultures revealed activation of inflammation, DNA damage, and stress-response pathways, which were attenuated after treatment with LMNSC-EVs. Conclusions: LMNSC-based 3D neural tissue provides a human-relevant platform for modeling cancer therapy-induced neurotoxicity. Furthermore, LMNSC-EVs represent a promising cell-free regenerative therapeutic that restores injury-associated inflammatory, stress, and metabol-ic transcriptional programs after radiation-induced neural injury.