Human Induced Pluripotent Stem Cells Research Articles

Generation of human iPSC-derived 3D bile duct within liver organoid by incorporating human iPSC-derived blood vessel

In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs. After 3 weeks, liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms. Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFβ and Notch pathways. Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation, highlighting BVLO potential as a model for Alagille syndrome, a congenital biliary disease. Overall, we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction.

Blood-tumor barrier in focus - investigation of glioblastoma-induced effects on the blood-brain barrier.

Glioblastoma (GBM) is the most prevalent, malignant, primary brain tumor in adults, characterized by limited treatment options, frequent relapse, and short survival after diagnosis. Until now, none of the existing therapy and treatment approaches have proven to be an effective cure. The availability of predictive human blood-tumor barrier (BTB) test systems that can mimic in-vivo pathophysiology of GBM would be of great interest in preclinical research. Here, we present the establishment of a new BTB in-vitro test system combining GBM spheroids and BBB models derived from human induced pluripotent stem cells (hiPSCs). We co-cultured hiPSC-derived brain capillary endothelial-like cells (iBCECs) with GBM spheroids derived from U87-MG and U373-MG cell lines in a cell culture insert-based format. Spheroids were monitored over 168hours (h) of culture, characterized for GBM-specific marker expression and treated with standard chemotherapeutics to distinguish inhibitory effects between 2D mono-culture and 3D spheroids. GBM-induced changes on iBCECs barrier integrity were verified via measurement of transendothelial electrical resistance (TEER), immunocytochemical staining of tight junction (TJ) proteins claudin-5 and occludin as well as the glucose transporter-1 (Glut-1). GBM-induced secretion of vascular endothelial growth factor (VEGF) was additionally quantified. Our hypothesis was validated by reduced expression of TJ proteins, occludin and claudin-5 together with significant barrier breakdown in iBCECs after only 24h of co-culture, demonstrated by reduction in TEER from 1313 ± 265 Ω*cm2 to 712 ± 299 Ω*cm2 (iBCECs + U87-MG) and 762 ± 316 Ω*cm2 (iBCECs + U373-MG). Furthermore, 3D spheroids show more resistance to standard GBM chemotherapeutics in-vitro compared to 2D cultures. We demonstrate the establishment of a simplified, robust in-vitro BTB test system, with potential application in preclinical therapeutic screening and in studying GBM-induced pathological changes at the BBB.

Robust Differentiation of Human Pluripotent Stem Cells into Lymphatic Endothelial Cells Using Transcription Factors

Introduction: Generating new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, protocols to differentiate human induced pluripotent stem cells (hiPSCs) into lymphatic endothelial cells (LECs) are still lacking. Methods: Transcription factors, ETS2 and ETV2 are key regulators of embryonic vascular development, including lymphatic specification. While ETV2 has been shown to efficiently generate blood endothelial cells, little is known about ETS2 and its role in lymphatic differentiation. Here, we describe a method for rapid and efficient generation of LECs using transcription factors, ETS2 and ETV2. Results: This approach reproducibly differentiates four diverse hiPSCs into LECs with exceedingly high efficiency. Timely activation of ETS2 was critical, to enable its interaction with Prox1, a master lymphatic regulator. Differentiated LECs express key lymphatic markers, VEGFR3, LYVE-1, and Podoplanin, in comparable levels to mature LECs. The differentiated LECs are able to assemble into stable lymphatic vascular networks in vitro, and secrete key lymphangiocrine, reelin. Conclusion: Overall, our protocol has broad applications for basic study of lymphatic biology, as well as toward various approaches in lymphatic regeneration and personalized medicine.

Human induced pluripotent stem cell based hepatic-modeling of lipid metabolism associated TM6SF2 E167K variant.

TM6SF2 rs58542926 (E167K) is related to increased prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD). Conflicting mouse study results highlight the need for a human model to understand this mutation's impact. This study aims to create and characterize a reliable human in vitro model to mimic the effects of the TM6SF2-E167K mutation for future studies. We used gene editing on human human-induced pluripotent stem cells (iPSC) from a healthy individual to create cells with the TM6SF2-E167K mutation. After hepatocyte directed differentiation, we observed decreased TM6SF2 protein expression, increased intracellular lipid droplets and total cholesterol in addition to reduced VLDL secretion. Transcriptomics revealed upregulation of genes involved in lipid, fatty acid, and cholesterol transport, flux, and oxidation. Global lipidomics showed increased lipid classes associated with ER stress, mitochondrial dysfunction, apoptosis, and lipid metabolism. Additionally, the TM6SF2-E167K mutation conferred a pro-inflammatory phenotype with signs of mitochondria and ER stress. Importantly, by facilitating protein folding within the ER of hepatocytes carrying TM6SF2-E167K mutation, VLDL secretion and ER stress markers improved. Our findings indicate that induced hepatocytes generated from iPSCs carrying the TM6SF2-E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to identify potential clinical targets and highlights the therapeutic potential of targeting protein misfolding to alleviate ER stress and mitigate the detrimental effects of the TM6SF2-E167K mutation on hepatic lipid metabolism.

A deep learning method that identifies cellular heterogeneity using nanoscale nuclear features

Cellular phenotypic heterogeneity is an important hallmark of many biological processes and understanding its origins remains a substantial challenge. This heterogeneity often reflects variations in the chromatin structure, influenced by factors such as viral infections and cancer, which dramatically reshape the cellular landscape. To address the challenge of identifying distinct cell states, we developed artificial intelligence of the nucleus (AINU), a deep learning method that can identify specific nuclear signatures at the nanoscale resolution. AINU can distinguish different cell states based on the spatial arrangement of core histone H3, RNA polymerase II or DNA from super-resolution microscopy images. With only a small number of images as the training data, AINU correctly identifies human somatic cells, human-induced pluripotent stem cells, very early stage infected cells transduced with DNA herpes simplex virus type 1 and even cancer cells after appropriate retraining. Finally, using AI interpretability methods, we find that the RNA polymerase II localizations in the nucleoli aid in distinguishing human-induced pluripotent stem cells from their somatic cells. Overall, AINU coupled with super-resolution microscopy of nuclear structures provides a robust tool for the precise detection of cellular heterogeneity, with considerable potential for advancing diagnostics and therapies in regenerative medicine, virology and cancer biology.

Generation of human induced pluripotent stem cells carrying albumin-sfGFP reporter

Current methodologies for hepatocyte induction from human induced pluripotent stem cells (hiPSCs) have limited efficacy due to lack of a functional hepatocyte reporter. To address this, we developed an endogenous albumin (ALB)-sfGFP reporter system in hiPSCs using homologous directed recombination (HDR)-mediated knock-in. The hiPSCs maintained the characteristic morphology, pluripotency, and normal karyotype while demonstrating successful differentiation into all three germ layers both in vitro and in vivo. Co-expression of EGFP and ALB was observed in the derived hepatocyte-like cells (HLCs). This reporter system holds promise for functional hepatocyte induction.

KCNJ16-depleted kidney organoids recapitulate tubulopathy and lipid recovery upon statins treatment

BackgroundThe KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid–base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistic links between Kir5.1 and tubulopathy, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the tubulopathy and potential therapeutic interventions.MethodsWe used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/− and KCNJ16−/−) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air–liquid interface.ResultsKCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16−/− organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16−/− kidney organoids.ConclusionsMature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.

Clinical-grade human induced pluripotent stem cell models for understanding mitochondrial structural abnormalities, bioenergetics and differentiation potential in mitochondrial diseases

Clinical-grade human induced pluripotent stem cell models for understanding mitochondrial structural abnormalities, bioenergetics and differentiation potential in mitochondrial diseases

Gradient conducting polymer surfaces with netrin-1-conjugation promote axon guidance and neuron transmission of human iPSC-derived retinal ganglion cells

Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies.

Stimulus effects of extremely low-frequency electric field exposure on calcium oscillations in a human cortical spheroid.

High-intensity, low-frequency (1 Hz to 100 kHz) electric and magnetic fields (EF and MF) cause electrical excitation of the nervous system via an induced EF (iEF) in living tissue. However, the biological properties and thresholds of stimulus effects on synchronized activity in a three-dimensional (3D) neuronal network remain uncertain. In this study, we evaluated changes in neuronal network activity during extremely low-frequency EF (ELF-EF) exposure by measuring intracellular calcium ([Ca2+]i) oscillations, which reflect neuronal network activity. For ELF-EF exposure experiments, we used a human cortical spheroid (hCS), a 3D-cultured neuronal network generated from human induced pluripotent stem cell (hiPSC)-derived cortical neurons. A 50 Hz sinusoidal ELF-EF exposure modulated [Ca2+]i oscillations with dependencies on exposure intensity and duration. Based on the experimental setup and results, the iEF distribution inside the hCS was estimated using high-resolution numerical dosimetry. The numerical estimation revealed threshold values ranging between 255-510 V/m (peak) and 131-261 V/m (average). This indicates that thresholds of neuronal excitation in the hCS were equivalent to those of a thin nerve fiber.

Simultaneous electro-dynamic stimulation accelerates maturation of engineered cardiac tissues generated by human iPS cells

Electrical and dynamic stimulation are commonly employed to enhance the maturation of engineered cardiac tissue (ECT) derived from human induced pluripotent stem cells (iPSCs), reflecting the physiological environment of the heart. While electrical stimulation mimics natural bioelectrical signals and dynamic stimulation replicates mechanical forces, the combined effects of these stimuli on ECT maturation have not been thoroughly explored. We hypothesized that simultaneous electro-dynamic stimulation would enhance ECT maturation and function more effectively than either stimulus alone. Human iPSC-derived cardiovascular cells were co-cultured with Collagen I and Matrigel for 2 weeks, followed by a comparative analysis of four groups: no stimulation, dynamic stimulation, electrical stimulation, and simultaneous electro-dynamic stimulation. The functionality of ECTs was assessed by measuring contractile capacity and calcium indicators, and histological assessments examined structural maturation. Our results demonstrated that simultaneous electro-dynamic stimulation significantly increased the CM component, elevated TNNT2 mRNA expression levels, and enhanced calcium transient capacity. Additionally, ECTs subjected to simultaneous stimulation exhibited a positive force-frequency relationship in contractility and an elevation in peak calcium flux, indicative of advanced tissue maturation. Moreover, simultaneous stimulation promoted vascular network formation within the ECTs, suggesting improved structural organization. These findings underscore the importance of simultaneous stimulation for developing effective cardiac tissue engineering strategies.

Dynamic convergence of autism disorder risk genes across neurodevelopment.

Over a hundred risk genes underlie risk for autism spectrum disorder (ASD) but the extent to which they converge on shared downstream targets to increase ASD risk is unknown. To test the hypothesis that cellular context impacts the nature of convergence, here we apply a pooled CRISPR approach to target 29 ASD loss-of-function genes in human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Two distinct approaches (gene-level and network-level analyses) demonstrate that convergence is greatest in mature glutamatergic neurons. Convergent effects are dynamic, varying in strength, composition, and biological role between cell types, increasing with functional similarity of the ASD genes examined, and driven by cell-type-specific gene co-expression patterns. Stratification of ASD genes yield targeted drug predictions capable of reversing gene-specific convergent signatures in human cells and ASD-related behaviors in zebrafish. Altogether, convergent networks downstream of ASD risk genes represent novel points of individualized therapeutic intervention.

Genotype-First Approach Identifies an Association between rs28374544/FOG2S657G and Liver Disease through Alterations in mTORC1 Signaling.

Metabolic dysfunction-associated Fatty Liver Disease (MAFLD) has emerged as one of the leading cardiometabolic diseases. Friend of GATA2 (FOG2) is a transcriptional co-regulator that has been shown to regulate hepatic lipid metabolism and accumulation. Using meta-analysis from several different biobank datasets, we identified a coding variant of FOG2 (rs28374544, A1969G, S657G) predominantly found in individuals of African ancestry (minor allele frequency~20%), which is associated with liver failure/cirrhosis phenotype and liver injury. To gain insight into potential pathways associated with this variant, we interrogated a previously published genomics dataset of 38 human induced pluripotent stem cell (iPSCs) lines differentiated into hepatocytes (iHeps). Using Differential Gene Expression Analysis and Gene Set Enrichment Analysis, we identified the mTORC1 pathway as differentially regulated between iHeps from individuals with and without the variant. Transient lipid-based transfections were performed on the human hepatoma cell line (Huh7) using wild-type FOG2 and FOG2S657G and demonstrated that FOG2S657G increased mTORC1 signaling, de novo lipogenesis, and cellular triglyceride synthesis and mass. In addition, we observed a significant downregulation of oxidative phosphorylation in FOG2S657G cells in fatty acid-loaded cells but not untreated cells, suggesting that FOG2S657G may also reduce fatty acid to promote lipid accumulation. Taken together, our multi-pronged approach suggests a model whereby the FOG2S657G may promote MAFLD through mTORC1 activation, increased de novo lipogenesis, and lipid accumulation. Our results provide insights into the molecular mechanisms by which FOG2S657G may affect the complex molecular landscape underlying MAFLD.

Generation of human induced pluripotent stem cell lines from patients with a RYR2 gene variant c.14201A>G (p.Y4734C): Implications for idiopathic ventricular fibrillation and catecholaminergic polymorphic ventricular tachycardia

Human induced pluripotent stem cell (iPSC) lines were generated from peripheral blood mononuclear cells (PBMCs) isolated from two related patients diagnosed with either idiopathic ventricular fibrillation or catecholaminergic polymorphic ventricular tachycardia, carrying an unknown variant in the RYR2 gene, c.14201A>G (p.Y4734C) and one healthy related individual. Reprogramming was done using a commercially available Epi5 Reprogramming Kit. The pluripotency of the iPSC lines was verified by the expression of pluripotency markers and by their capacity to differentiate into all three embryonic germ layers in vitro. These iPSC lines are available for functional analysis and in vitro studies of RYR2 channelopathy.

Evaluating the embryotoxicity of benzophenone-based photoinitiators in stem cells and zebrafish embryos

Benzophenones (BPs) are widely used as photoinitiators (PIs) or printing inks in food packaging, which may migrate into foods. However, the toxicity information of some BP analogues, such as 4,4′-bis(diethylamino)-benzophenone (DEAB), 4-phenylbenzophenone (4-PBP), 4 (hydroxymethyl)benzophenone (4-HMBP), those are used as PIs is lacking. Developmental toxicity is a health concern associated with PIs exposure. Recently, alternative non-in vivo methods have been proposed to evaluate the concerned chemicals or better understand the modes of action of certain toxicological endpoints. In this study, using in silico methods, we predicted that BP, DEAB, 4-PBP and 4-HMBP might exhibit developmental toxicity. However, we found that only DEAB is strong embryotoxic and disturbs the early differentiation of mouse embryonic stem cells into three germ layers and cardiomyocytes. DEAB treatment also prevented cardiomyocyte differentiation in human induced pluripotent stem cells (hiPSCs) on day 10. However, BP, 4-PBP and 4-HMBP had no similar effects on cardiomyocyte differentiation on day 10. Transcriptomic analysis revealed that treatment with DEAB significantly decreased the mRNA levels of differentiation-related transcription factors SOX17 and FOXA1, in hiPSCs on day 4. Furthermore, DEAB treatment caused tail malformations and yolk sac edema in zebrafish embryos. To conclude, DEAB may be embryotoxic because it disturbs the early differentiation of stem cells. Further studies are warranted to better understand the health effects of DEAB exposure.

Exosomes from Human iPSC-Derived Retinal Organoids Enhance Corneal Epithelial Wound Healing.

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2'-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing.

Generation of four distinct isogenic cell lines with truncating variants in I-band or A-band titin

Truncating variants in TTN (TTNtv) are present in 15–25 % of patients with idiopathic dilated cardiomyopathy. Interestingly, the pathogenicity of TTNtv seems to be linked to their location within the gene. More proximal I-band TTNtv (TTNtvI) harbour less pathogenic potential than distant A-band TTNtv (TTNtvA). We created isogenic human induced pluripotent stem cell lines (hiPSC) with TTNtvI and TTNtvA using CRISPR/Cas9, for the investigation of the pathomechanism in hiPSC-derived cardiomyocytes (hiPSC-CMs). Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted.

Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Duchenne muscular dystrophy skeletal muscle cells derived from human induced pluripotent stem cells recapitulate various calcium dysregulation pathways

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in premature death. As a major secondary event, an abnormal elevation of the intracellular calcium concentration in the dystrophin-deficient muscle contributes to disease progression in DMD. In this study, we investigated the specific functional features of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) generated from DMD patients to regulate intracellular calcium concentration. As compared to healthy hiPSC-skMCs, DMD hiPSC-skMCs displayed specific spontaneous calcium signatures with high levels of intracellular calcium concentration. Furthermore, stimulations with electrical field or with acetylcholine perfusion induced higher calcium response in DMD hiPSC-skMCs as compared to healthy cells. Finally, Mn2+ quenching experiments demonstrated high levels of constitutive calcium entries in DMD hiPSC-skMCs as compared to healthy cells. Our findings converge on the fact that DMD hiPSC-skMCs display intracellular calcium dysregulation as demonstrated in several other models. Observed calcium disorders associated with RNAseq analysis on these DMD cells highlighted some mechanisms, such as spontaneous and activated sarcoplasmic reticulum (SR) releases or constitutive calcium entries, known to be disturbed in other dystrophin-deficient models. However, store operated calcium entries (SOCEs) were not found to be dysregulated in our DMD hiPSC-skMCs model. These results suggest that all the mechanisms of calcium impairment observed in other animal models may not be as pronounced in humans and could point to a preference for certain mechanisms that could correspond to major molecular targets for DMD therapies.

PITX2 deficiency leads to atrial mitochondrial dysfunction.

Reduced left atrial PITX2 is associated with atrial cardiomyopathy and atrial fibrillation. PITX2 is restricted to left atrial cardiomyocytes in the adult heart. The links between PITX2 deficiency, atrial cardiomyopathy and atrial fibrillation are not fully understood. To identify mechanisms linking PITX2 deficiency to atrial fibrillation, we generated and characterized PITX2-deficient human atrial cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) and their controls. PITX2-deficient hiPSC-derived atrial cardiomyocytes showed shorter and disorganised sarcomeres and increased mononucleation. Electron microscopy found an increased number of smaller mitochondria compared to the control. Mitochondrial protein expression was altered in PITX2-deficient hiPSC-derived atrial cardiomyocytes. Single-nuclear RNA-sequencing found differences in cellular respiration pathways and differentially expressed mitochondrial and ion channel genes in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2 repression in hiPSC-derived atrial cardiomyocytes replicated dysregulation of cellular respiration. Mitochondrial respiration was shifted to increased glycolysis in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2-deficient human hiPSC-derived atrial cardiomyocytes showed higher spontaneous beating rates. Action potential duration was more variable with an overall prolongation of early repolarization, consistent with metabolic defects. Gene expression analyses confirmed changes in mitochondrial genes in left atria from 42 patients with atrial fibrillation compared to 43 patients in sinus rhythm. Dysregulation of left atrial mitochondrial (COX7C) and metabolic (FOXO1) genes was associated with PITX2 expression in human left atria. In summary, PITX2 deficiency causes mitochondrial dysfunction and a metabolic shift to glycolysis in human atrial cardiomyocytes. PITX2-dependent metabolic changes can contribute to the structural and functional defects found in PITX2-deficient atria.