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Neuropsychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BPD) remain challenging to diagnose due to the absence of objective biomarkers, with current assessments relying largely on subjective clinical evaluations. In this study, we present a computational analysis pipeline designed to identify disease-specific electrophysiological signatures from multi-electrode array (MEA) recordings of patient-derived cerebral organoids (COs) and two-dimensional cortical interneuron cultures (2DNs). Using a Support Vector Machine classifier optimized for high-dimensional data, we achieved 95.8% classification accuracy in distinguishing SCZ from control samples in 2DNs under both baseline and post-electrical-stimulation (PES) conditions with the extracted electrophysiological signatures. In COs, classification accuracy improved from 83.3% at baseline to 91.6% following PES, enabling robust separation of control, SCZ, and BPD cohorts. Key discriminative features included channel-specific measures of network activity, with PES significantly enhancing classification performance, particularly for BPD. These results underscore the potential of MEA-based functional phenotyping, coupled with machine learning, to uncover reliable, stimulation-sensitive electrophysiological biomarkers, offering a path toward more objective diagnosis and personalized treatment strategies for neuropsychiatric disorders.

Bipolar disorder (BD) is increasingly recognized as a disease with both mitochondrial dysfunction and heightened inflammatory reactivity, yet contribution to neuronal activity remains unclear. To address these gaps, this study utilizes iPSC-derived cerebral organoids (COs) from BD patients and healthy controls to model disease-specific metabolic and inflammatory dysfunction in a more physiologically relevant system. BD COs exhibited mitochondrial impairment, dysregulated metabolic function, and increased nod-leucine rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activation sensitivity. Treatment with MCC950, a selective NLRP3 inhibitor, effectively rescued mitochondrial function and reduced inflammatory activation in both BD and control COs. The effect of a Bioactive Flavonoid Extract (BFE), a potential therapeutic, was also explored and yielded a partial rescue of inflammasome activation. These findings highlight a mitochondria-inflammasome axis in BD pathophysiology and establish a novel platform for studying BD-associated cellular mechanisms, ultimately bridging the gap between molecular dysfunction and therapeutic development.

Although lithium (Li) is a widely used treatment for bipolar disorder, its exact mechanisms of action remain elusive. Research has shown that the two stable Li isotopes, which differ in their mass and nuclear spin, can induce distinct effects in both in vivo and in vitro studies. Since sodium (Na+) channels are the primary pathway for Li+ entry into cells, we examined how Li+ affects the current of Na+ channels using whole-cell patch-clamp techniques on SH-SY5Y neuroblastoma cells and human iPSC-derived cortical neurons. Our findings indicate that mammalian Na+ channels in both neuronal models studied here display no selectivity between Na+ and Li+, unlike previously reported bacterial Na+ channels. We observed differences between the two neuronal models in three measured parameters (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{V}_{\text{h}\text{a}\text{l}\text{f}},\:{G}_{\text{m}\text{a}\text{x}},\:z$$\end{document}). We saw no statistically significant differences between any ions in SH-SY5Y cells, but small differences in the half-maximum activation potential (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:{V}_{\text{h}\text{a}\text{l}\text{f}}$$\end{document}) between Na+ and 6Li+ and between 7Li+ and 6Li+ were found in iPSC-derived cortical neurons. Although Na+ channels are widely expressed and important in neuronal function, the very small differences observed in this work suggest that Li+ regulation through Na+ channels is likely not the primary mechanism underlying Li+ isotope differentiation.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12893-9.

Human iPSC-derived models currently used in eye research usually replicate later events of tissue differentiation rather than early steps involving concurrent development of diverse embryonic cell types. Here we present a multi-timepoint morphological and transcriptomic analysis of a 2D model mirroring the early stages of human whole eye development, self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. SEAM cultures maintained a reproducible growth profile over the standard 28-day differentiation process with quantifiable morphological changes accompanying generation of key ocular cell types over time. Bulk and single-cell RNA-seq analyses at Days 0, 14 and 28 identified dynamic transcriptomic changes indicative of the emerging cell types, including rare stem cell-like populations analogous to those comprising the ciliary marginal zone, transit-amplifying cells, limbal epithelial stem cells, and corneal stromal stem cells. Integrated developmental trajectory analysis highlighted intermediate differentiation states underlying SEAM maturation. Cluster-specific interrogation of eye disease-associated genes demonstrated dynamic temporal patterns and enrichment in relevant developing cell types. These analyses establish a comprehensive baseline of SEAM formation, supporting the potential of the model to facilitate mechanistic studies of genetic variants that may uniquely impact humans, thus improving the success rate in resolving cases presenting with a broad range of developmental eye phenotypes.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06602-9.

This study aims to determine if neurons derived from induced pluripotent stem cells (iPSCsNs) and directly converted neurons (iNs) from the same source cells exhibit changes in mitochondrial properties related to aging. This research addresses the uncertainty around whether aged iPSCsNs retain aging-associated mitochondrial impairments upon transitioning through pluripotency while direct conversion maintains these impairments. We observe that both aged models exhibit characteristics of aging, such as decreased ATP, mitochondrial membrane potential, respiration, NAD+/NADH ratio, and increased radicals and mitochondrial mass. In addition, both neuronal models show a fragmented mitochondrial network. However, aged iPSCsNs do not exhibit a metabolic shift towards glycolysis, unlike aged iNs. Furthermore, mRNA expression differed significantly between aged iPSCsNs and aged iNs. The study concludes that aged iPSCsNs may differ in transcriptomics and the aging-associated glycolytic shift but can be a valuable tool for studying specific feature of mitochondrial neuronal aging in vitro alongside aged iNs. Side-by-side modeling of iPSC-derived neurons (iPSCsNs) and directly converted neurons (iNs) from the same aged donor shows both retain mitochondrial aging traits, though iPSCsNs exhibit partial rejuvenation in transcriptomics and metabolic shift.

BackgroundHemophilia B is an inherited disorder caused by a mutation in the FIX gene, which results in insufficient blood clotting factor IX (FIX) production from hepatocytes. Currently, there are no treatments for hemophilia B patients. The patients should be continuously administrated with clotting factor concentrates 2–3 times a month to prevent bleeding. Therefore, this study aimed to develop an engineered FIX-secreting hepatocyte sheet that can release FIX for an extended period. Within this study, the engineered FIX-secreting hepatocyte sheet was developed by integrating two core technologies, including a gene editing platform to generate FIX-secreting cells and cell sheet technology to improve cell delivery efficacy.MethodsThe human FIX gene was inserted into the APOC3 site of iPSCs by CRISPR/Cas9, which secretes the target protein after differentiation into hepatocytes. FIX-secreting hepatocyte sheets were obtained by temperature-responsive polymer grafted cell culture dishes (TRCD). Immunohistochemical and functional tests were performed for hepatocyte-like cells differentiated from FIX KI-iPSCs and wild-type iPSCs (WT-iPSCs). After validating the functional activity and secretion of FIX protein, the engineered hepatocyte-like cell sheets were transplanted to NOD/SCID mice for the in vivo experiments.ResultsThe insertion of the human FIX gene into the APOC3 site demonstrated a significant increase in FIX secretion in hepatocyte-like cells differentiated from FIX KI-iPSCs compared with those obtained from WT-iPSCs. Among the iPSCs to hepatocyte differentiation stages, the hepatic endoderm stage was most suitable for seeding the cells on TRCD and generating cell sheets by temperature changes from 37oC to room temperature when the hepatocyte-like cells have reached maturity. The engineered FIX-secreting cell sheets showed intensive expression of the FIX proteins without losing hepatocyte morphology for 20 days. Furthermore, an in vivo study showed that engineered FIX-secreting cell sheets retained their FIX secretion functions for two weeks, whereas single-cell injected traditionally were barely detected in the experimental animals.ConclusionsThe engineered FIX-secreting cell sheets fabricated from functionally improved iPSCs with practical cell delivery tools could be a promising tool for clinically treating Hemophilia B.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04195-8.

Tibetan adaptation to high-altitude hypoxia remains a classic example of Darwinian selection in humans. Amongst Tibetan populations, alleles in the EPAS1 gene - whose protein product, HIF-2α, is a central regulator of the hypoxia response - have repeatedly been shown to carry some of the strongest signals of positive selection in humans. However, selective sweep signals alone may only account for some of the phenotypes that differentiate high-altitude adapted populations from closely related lowlanders. Therefore, there is a pressing need to functionally probe adaptive alleles and their impact at both the locus-specific and genome-wide levels and across cell types to uncover the full range of beneficial traits. To this end, we established a library of induced pluripotent stem cells (iPSCs) derived from Tibetan and Han Chinese individuals, a robust model system allowing precise exploration of allelic effects on transcriptional responses, and we differentiated them into vascular endothelium. Using this system, we focus first on a hypoxia-dependent enhancer (ENH5) that contributes to the regulation of EPAS1 to investigate its locus-specific effects in endothelium. Then, to cast a wider net, we harness the same experimental system to compare the transcriptome of Tibetan and Han Chinese cells in hypoxia and find evidence that angiogenesis, energy metabolism and immune pathways differ between these two populations with different histories of long-term residence at high altitude. Coupled with evidence of polygenic adaptations targeting the same pathways, these results suggests that the observed transcriptional differences between the two populations were shaped by natural selection. Author summaryUnderstanding the ways in which humans have adapted to extreme environmental conditions is of great interest to evolutionary biologists. For instance, substantial research has focused on characterizing how Tibetan populations have genetically adapted to conditions of low oxygen (hypoxia) at high elevation. Nevertheless, the inherent challenges of measuring unique traits or sampling relevant cell and tissues in such remote environments, limits our ability to assess Tibetan adaptation. Here we present the development of a model system in which to interrogate molecular differences between Tibetans and Han Chinese individuals – a closely related lowland population – in a cell type of interest. Specifically, we build a panel of Tibetan and Han Chinese induced pluripotent stem cells (iPSCs), from which we differentiate endothelial cells, a significant hypoxia-responsive cell type. We use these cells to test how Tibetan genetic ancestry influences the cellular response to hypoxia and find substantial differences between populations in metabolic and inflammatory responses. These findings not only help to inform our understanding of high-altitude adaption but can provide critical information for designing hypothesis-driven field studies in the future.

Signaling interplay between the histamine 1 receptor (H1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in mediating histaminergic itch has been well-established in mammalian models, but whether this is conserved in humans remains to be confirmed due to the difficulties in obtaining human sensory neurons (SNs) for experimentation. Additionally, previously reported species-specific differences in TRPV1 function indicate that use of human SNs is vital for drug candidate screening to have a higher chance of identifying clinically effective TRPV1 antagonists. In this study, we built a histamine-dependent itch model using peripheral SNs derived from human induced pluripotent stem cells (hiPSC-SNs), which provides an accessible source of human SNs for pre-clinical drug screening. We validated channel functionality using immunostaining, calcium imaging, and multielectrode array (MEA) recordings, and confirmed the interdependence of H1R and TRPV1 signalling in human SNs. We further tested the amenability of our model for pre-clinical studies by screening multiple TRPV1 antagonists in parallel, identifying SB366791 as a potent inhibitor of H1R activation and potential candidate for alleviating histaminergic itch. Notably, some of the results using our model corroborated with efficacy and side effect findings from human clinical trials, underscoring the importance of this species-specific platform. Taken together, our results present a robust in vitro human model for histaminergic itch, which can be used to further interrogate the molecular basis of human SN function as well as screen for TRPV1 activity-modifying compounds for a number of clinical indications.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-82549-7.

Cardiac regenerative therapy has recently progressed by reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and advanced by large-scale differentiation-derived cardiomyocytes (hiPSC-CMs). However, repairing damaged cardiac tissues with hiPSC-CMs remains limited due to immune rejection, cardiac arrhythmias, and concerns over tumor formation after hiPSC-CM transplantation. Despite efforts in profiling epigenomic changes during cardiac differentiation, regulatory mechanisms underlying 5-methylcytosine (m5C) deposition in RNA m5C epitranscriptomic landscape during hiPSC-to-cardiomyocyte differentiation remain unclear. Herein, bisulfite RNA-sequencing analysis was conducted in human pluripotent stem cells (hPSCs) from three independent cellular origins, and their derived cardiomyocytes (hPSC-CM), metabolic-maturation of derived cardiomyocytes (hPSC-CM-lac) and biochemical-enhanced derived cardiomyocytes (hPSC-CM-TDI). Integrated analysis of differentially methylated RNA m5C profiles and transcriptome-wide expression facilitated the identification of m5C sites coupled to the cardiomyocyte differentiation and RNA-dependent regulatory mechanisms of stem cell pluripotency. The RNA m5C profiles in this dataset allow the evaluations of the m5C level and distribution of specific m5C loci and facilitate understanding of the m5C epitranscriptomic landscape in biological functions of hPSC-CM beyond in vivo transplantation barriers.

This study presents a novel biotechnological approach using octadecylamine-based solid lipid nanoparticles (OCTNPs) for the first-time reprogramming of human CCD1072-SK fibroblast cells into induced pluripotent stem cells (iPSCs). OCTNPs, with an average size of 178.9 nm and a positive zeta potential of 22.8 mV, were synthesized, thoroughly characterized, and utilized as a non-viral vector to efficiently deliver reprogramming factors, achieving a remarkable transfection efficiency of 82.0%. iPSCs were characterized through immunofluorescence, flow cytometry, and RT-qPCR, confirming the expression of key pluripotency markers such as OCT4, SOX2, and KLF4, with alkaline phosphatase activity further validating their pluripotent state. Following this comprehensive characterization, the iPSCs were successfully differentiated into cardiomyocyte-like cells using 5-azacytidine. Our research highlights the innovative application of OCTNPs as a safe and effective alternative to viral vectors, addressing key limitations of iPSC reprogramming. The novel application of OCTNPs for efficient gene delivery demonstrates a powerful tool for advancing stem cell technologies, minimizing risks associated with viral vectors. These findings pave the way for further innovations in biotechnological applications, particularly in tissue engineering and personalized medicine.

BackgroundType 1 diabetes, an autoimmune disorder leading to the destruction of pancreatic β-cells, requires lifelong insulin therapy. Islet transplantation offers a promising solution but faces challenges such as limited availability and the need for immunosuppression. Induced pluripotent stem cells (iPSCs) provide a potential alternative source of functional β-cells and have the capability for large-scale production. However, current differentiation protocols, predominantly conducted in hybrid or 2D settings, lack scalability and optimal conditions for suspension culture.MethodsWe examined a range of bioreactor scaleup process parameters and quality target product profiles that might affect the differentiation process. This investigation was conducted using an optimized High Dimensional Design of Experiments (HD-DoE) protocol designed for scalability and implemented in 0.5L (PBS-0.5 Mini) vertical wheel bioreactors.ResultsA three stage suspension manufacturing process is developed, transitioning from adherent to suspension culture, with TB2 media supporting iPSC growth during scaling. Stage-wise optimization approaches and extended differentiation times are used to enhance marker expression and maturation of iPSC-derived islet-like clusters. Continuous bioreactor runs were used to study nutrient and growth limitations and impact on differentiation. The continuous bioreactors were compared to a Control media change bioreactor showing metabolic shifts and a more β-cell-like differentiation profile. Cryopreserved aggregates harvested from the runs were recovered and showed maintenance of viability and insulin secretion capacity post-recovery, indicating their potential for storage and future transplantation therapies.ConclusionThis study demonstrated that stage time increase and limited media replenishing with lactate accumulation can increase the differentiation capacity of insulin producing cells cultured in a large-scale suspension environment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03973-0.

BackgroundLack of insight into factors that determine purity and quality of human iPSC (hiPSC)-derived neo-cartilage precludes applications of this powerful technology toward regenerative solutions in the clinical setting. Here, we set out to generate methylome-wide landscapes of hiPSC-derived neo-cartilages from different tissues-of-origin and integrated transcriptome-wide data to identify dissimilarities in set points of methylation with associated transcription and the respective pathways in which these genes act.MethodsWe applied in vitro chondrogenesis using hiPSCs generated from two different tissue sources: skin fibroblasts and articular cartilage. Upon differentiation toward chondrocytes, these are referred to as hFiCs and hCiC, respectively. Genome-wide DNA methylation and RNA sequencing datasets were generated of the hiPSC-derived neo-cartilages, and the epigenetically regulated transcriptome was compared to that of neo-cartilage deposited by human primary articular cartilage (hPAC).ResultsMethylome-wide landscapes of neo-cartilages of hiPSCs reprogrammed from two different somatic tissues were 85% similar to that of hPACs. By integration of transcriptome-wide data, differences in transcriptionally active CpGs between hCiC relative to hPAC were prioritized. Among the CpG-gene pairs lower expressed in hCiCs relative to hPACs, we identified genes such as MGP, GDF5, and CHAD enriched in closely related pathways and involved in cartilage development that likely mark phenotypic differences in chondrocyte states. Vice versa, among the CpG-gene pairs higher expressed, we identified genes such as KIF1A or NKX2-2 enriched in neurogenic pathways and likely reflecting off target differentiation.ConclusionsWe did not find significant variation between the neo-cartilages derived from hiPSCs of different tissue sources, suggesting that application of a robust differentiation protocol such as we applied here is more important as compared to the epigenetic memory of the cells of origin. Results of our study could be further exploited to improve quality, purity, and maturity of hiPSC-derived neo-cartilage matrix, ultimately to realize introduction of sustainable, hiPSC-derived neo-cartilage implantation into clinical practice.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-024-01759-y.