Induced Pluripotent Stem Research Articles

Efficient Light-Based Bioprinting via Rutin Nanoparticle Photoinhibitor for Advanced Biomedical Applications.

Digital light processing (DLP) bioprinting, known for its high resolution and speed, enables the precise spatial arrangement of biomaterials and has become integral to advancing tissue engineering and regenerative medicine. Nevertheless, inherent light scattering presents significant challenges to the fidelity of the manufactured structures. Herein, we introduce a photoinhibition strategy based on Rutin nanoparticles (Rnps), attenuating the scattering effect through concurrent photoabsorption and free radical reaction. Compared to the widely utilized biocompatible photoabsorber tartrazine (Tar), Rnps-infused bioink enhanced printing speed (1.9×), interlayer homogeneity (58% less overexposure), resolution (38.3% improvement), and print tolerance (3× high-precision range) to minimize trial-and-error. The biocompatible and antioxidative Rnps significantly improved cytocompatibility and exhibited resistance to oxidative stress-induced damage in printed constructs, as demonstrated with human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs). The related properties of Rnps facilitate the facile fabrication of multimaterial, heterogeneous, and cell-laden biomimetic constructs with intricate structures. The developed photoinhibitor, with its profound adaptability, promises wide biomedical applications tailored to specific biological requirements.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Generation of two induced pluripotent stem cell lines from patients with Li-Fraumeni Syndrome carrying TP53 mutation

Li-Fraumeni syndrome (LFS) is a rare autosomal dominant inherited genetic disorder that greatly increases the risk of developing several types of cancer, including young children and young adults. LFS is primarily caused by specific mutations in the tumor suppressor gene TP53. In this study, we successfully generated two human induced pluripotent stem cell (iPSC) lines derived from patients diagnosed with LFS, each carrying a distinct heterozygous mutation in the TP53 gene. These LFS patient-derived iPSC lines exhibited robust expression of key pluripotency markers, demonstrated the capacity to differentiate into all three germ layers (endoderm, mesoderm, and ectoderm), and maintained a normal karyotype. The establishment of these iPSC lines provides a valuable tool for modeling LFS in vitro, enabling researchers to investigate the underlying pathological mechanisms associated with the disease across various cell types and tissues.

Abstract Mo091: Analysis of GLA Gene Mutation Using a Female Fabry Disease Patient-derived Induced Pluripotent Stem Cells.

Introduction: Fabry disease is an inborn error of metabolism caused by mutations in the alpha-galactosidase gene ( GLA ) located on the X chromosome, resulting in intracellular accumulation of globotriaosylceramide and systemic tissue injury. In addition to enzyme replacement therapy, chaperone therapy is effective for many missense mutations. One female patient had c.547G>A mutation, which has been reported as p.G183S, and we checked the indication for migalastat, a pharmacological chaperone. However, migalastat reactivity testing using HEK293 cells transfected with mutant cDNA had not been performed for this mutation. Aims: We analyzed the c.547G>A mutation and examined the efficacy of migalastat for this mutation. Methods: Peripheral blood mononuclear cells from patients were transfected with reprogramming factors using episomal vectors to generate induced pluripotent stem (iPS) cells. Cardiomyocytes were induced from the iPS cells. The mutation site was located at the 3' end of exon 3, and splicing mutation was suspected. Thus, PCR amplification was performed between exon 2 and exon 5 of the cDNA, and the PCR product was sequenced to confirm the mRNA sequence generated in the patient's cells. Real-time PCR was performed using Taqman probe to amplify sites unaffected by the mutation, and expression levels of normal and mutant GLA were compared. iPS cells were treated with migalastat and the effect on α-galactosidase activity was examined. Results: Since the patient was female, five iPS cell lines expressing only normal GLA and four iPS cell lines expressing only mutant GLA were established due to X-chromosome inactivation. Mutant cDNA was found to have a deletion of 36 bases, part of exon 3, resulting in a deletion of 12 amino acids. Real-time PCR result showed that the GLA expression level of mutant GLA-expressing cells was about half that of normal GLA-expressing cells, which was comparable to the GLA expression level of the cells from a healthy subject. Mutant GLA-expressing iPS cells had no α-galactosidase activity, which was not increased by treatment with migalastat. Conclusions: In patient-derived iPS cells and iPS cell-derived cardiomyocytes, c.547G>A produced p.C172_G183del, not p.G183S, and migalastat was ineffective. Further therapeutic development is needed for patients with this mutation.

Abstract We031: Optimizing Immunosuppressive Strategies for Enhanced Survival of Allogeneic Pluripotent Stem Cell-Derived Cardiomyocytes in Non-Human Primate Transplantation Model

Introduction: Accumulating evidence suggests that the transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) regenerates injured hearts in animal studies. Currently, several clinical studies are underway, transplanting allogeneic human PSC-CMs. In these clinical studies, patients have been treated with the same combination of immunosuppressants used after heart transplantation, including steroids, calcineurin inhibitors (CNIs), and mycophenolate mofetil (MMF). However, the optimal treatment following PSC-CM transplantation has yet to be established. The purpose of this study is to determine the optimal treatment for grafted PSC-CM survival by regulating the immune response using an allogeneic non-human primate transplantation model. Methods and Results: We prepared 9 male cynomolgus monkeys as recipient animals. Donor cardiomyocytes were generated from induced pluripotent stem (iPS) cells derived from another monkey, with MHC class I and class II types differing from those in the recipients. The recipient animals underwent ischemia/reperfusion injury of the left anterior descending artery two weeks before iPSC-CMs transplantation. Initially, animals were treated with the same combination of immunosuppressants (steroid/CNIs/MMF: N=3), and grafted PSC-CMs survived without apparent immune rejection. Subsequently, we conducted experiments aiming to reduce immunosuppressants. The animals were treated with the same combination, but steroids were terminated at 4 weeks post-transplantation (N=4). Histological analysis at 8 or 12 weeks post-transplantation revealed that grafted cardiomyocytes were rejected, with a significant infiltration of CD3-positive lymphocytes. When recipients were treated with another combination of immunosuppressants (steroid/abatacept/MMF), and steroids were terminated at 4 weeks post-transplantation, grafted cardiomyocytes survived without apparent infiltration of CD3-positive lymphocytes at 8 weeks post-transplantation (N=2). Conclusion: A different combination of immunosuppressants from heart transplantation allows transplanted allogeneic PSC-CMs to survive without the long-term use of steroids.

Abstract We103: Integration of deep learning assisted high-content screening and deep tissue-phenotyping to identify cardioprotective compounds in dilated cardiomyopathy

With a prevalence of 4:10,000, dilated cardiomyopathy (DCM) is the most common hereditary heart disease, characterized by dilatation of one or both ventricles and impaired systolic and diastolic function. Numerous mutations have been identified to cause DCM. TITIN-truncating variants (TTNtv) are commonly observed in 20% of the genetic DCM cases. Progress in drug discovery for DCM has been hampered by the lack of robust high-throughput/high-content analysis pipelines of disease-specific cell and tissue platforms for deep structural and functional phenotyping. To address this challenge, we have developed a two-tiered human induced pluripotent stem cell (iPSC)-based screening approach, comprised of high-content cardiomyocyte imaging with artificial intelligence (AI)-driven image and data analyses as well as deep tissue phenotyping in engineered heart muscle (EHM) models. As a DCM use case, we have developed several isogenic human iPSC-lines with and without a previously reported and pathologically relevant A-band-TTNtv mutation (c.70692_70693insAT; p.T23565SfsX5; Gerull et al. 11788824, Gramlich et al. 25759365, Fomin et al. 34731013) Using fixed weights from a convolutional deep neural network trained on ImageNet, we generated unbiased deep embeddings from each 2D cell model and applied these to train machine learning (ML) models to detect morphological disease phenotypes. Our ML model is able to confidently separate healthy controls from TTNtv cells with high accuracy (>93%), specificity (WT >79%) across different batches/plate layouts and generate concentration-response curves, demonstrating platform robustness and sensitivity. Building upon this work, we will present our latest efforts applying the platform technology for a chemogenomic screening campaign to identify novel cardioprotective drugs using a library of 7,100 bioactive compounds and validation assays, including measuring functional changes in EHMs. The phenotypic profiling platform, as presented here, can be readily adapted to other disease-relevant cell types and pathologies. Our results demonstrate the power of combining iPSC-CMs with cytological profiling and deep learning as well as tissue-level phenotyping to accelerate drug discovery for patients with DCM.

Generation of a human induced pluripotent stem cell (iPSC, DVSi001-A) with a heterozygous mutation in KRAS (A209T)

Human varicose veins are commonly claimed to be responsible for lower limb symptoms. Mutation in KRAS gene has been implicated in various diseases, including cancers and vascular diseases. While little known about the novel mutation in KRAS gene and its contribution to the development of varicose veins. Here, we have generated human induced pluripotent stem cell (iPSC) line, which harboured a novel mutation in KRAS (c.209A>T) gene. This cell line provided a novel tool for understanding the mechanism of KRAS mutation in the pathogenesis of varicose veins.

Abstract We015: Modeling Cardiac Arrhythmogenicity of hiPSC-CMs and Cardiac Fibroblasts Nanopatterned Coculture and Machine Learning

Background: Sudden cardiac death is one of the most threatening heart conditions in the U.S. Most individuals have an underlying structural cardiac disease associated with cardiac fibrosis and ventricular arrhythmia. The relationship between fibrosis and arrhythmias is found to be related to cardiovascular cell couplings, especially cardiomyocytes (CMs) and cardiac fibroblasts (CFs). While animal models have been applied to model cardiac arrhythmias, the disparities between animal models and humans limit the accuracy of pro-arrhythmic predictions. The use of human induced pluripotent stem cells (hiPSCs)-derived CMs has permitted highly modular testing conditions, specifically the ratio of CFs to CMs in an engineered coculture system. Moreover, machine learning (ML) has become an emerging tool in cardiology, such as cardiac toxicity and function evaluation and classification, with its unique advantages of high accuracy and efficiency with less human bias and intuition. Hypothesis: We hypothesize that cardiac fibrosis-associated arrhythmia will be modeled by nanopatterned coculture of hiPSC-CMs and CFs at varied ratios, and the arrhythmic samples will be accurately and efficiently classified by ML algorithms. Approach: The hiPSC-CMs were cocultured and nanopatterned with CFs at ratios of 0 to 30% for 7 days. Upon the electrical pacing at 1Hz, the action potential was recorded by live-cell optical mapping with FluoVolt. The recorded membrane potential and electrical propagation were analyzed by Matlab-based Electomap software. Results: The cocultured hiPSC-CMs and CFs were aligned with aligned sarcomeres and uni-directional contraction ( Fig. 1a ). The cardiac arrhythmia was observed in 15% CFs with spiral wave ( Fig. 1b ) in comparison to the hiPSC-CMs with 0% CF. Beat-to-beat and depolarization durations were found significant with top ranks in Feature Importance to facilitate the distinction between arrhythmic and non-arrhythmic samples by the unsupervised K-means Clustering ( Figs. 1c and d ). Conclusions: We have successfully established a nanopatterned coculturing system of hiPSC-CMs and CFs for modeling cardiac fibrosis-induced arrhythmia, which was further analyzed and classified by ML algorithms. This system reveals great potential for better understanding the relationship between cardiac fibrosis-associated arrhythmia and sudden cardiac death and also drug evaluation for anti-cardiac arrhythmia.

CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a fabry disease vasculopathy model derived from human induced pluripotent stem cells

Background and aimsThe objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs). MethodsWe differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing. ResultsGLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated while SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs. ConclusionsCRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.

Abstract We044: MATURITY OF HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES PROMOTED BY BRACHYURY PRIMING

Introduction: Cardiac differentiation protocols enable derivation of cardiomyocytes (CM). from human induced pluripotent stem cell (iPS). However, limited tools are available to ensure enhanced iPS-CM maturation without the need for prolonged and complex culture. Research Question: This study assessed whether the mesodermal and cardiopoiesis inductor, Brachyury (T), complements standard cardiac differentiation and augments structural and functional maturation of iPS-CM. Aims: The impact of T mRNA introduction prior to initiation of cardiac differentiation was characterized on: i) cardiac transcription factor expression; ii) structural and functional hallmarks of maturity; and iii) cardiotoxicity. Methods: RT-qPCR was employed to monitor the expression of cardiac-specific transcription factors. Transmission electron microscopy enabled the assessment of cellular ultrastructure. Seahorse measurements were used to determine basal respiration. Fluo4 staining was used to measure calcium transients with external field stimulation. Cardiotoxicity was measured as the impact of doxorubicin on calcium handling and cell survival. Results: Transfection with T prompted early and sustained upregulation of the cardiac transcription factors Nkx2.5 (10-fold; FDR<0.001) and MEF2C (7.2-fold; FDR<0.001). Mitochondrial area was increased with T transfection (P<0.001) and T-transfected iPS-CM exhibited greater basal respiration (FDR<0.05), greater calcium handling resilience in response to field stimulation, and a more mature transient morphology. Following doxorubicin exposure T transfected iPS-CM exhibited a greater compromise in calcium handling (FDR<0.001), and exaggerated cell death. Conclusion: Induction of human iPS with T prior to initiation of cardiac differentiation enhanced signatures of structural and functional maturity, and provided more robust readouts of drug toxicity.

Abstract Mo097: Inhibiting the Sarcomere Improves Diastolic Dysfunction in Patient-Derived hiPSC-CM Models of Pediatric Restrictive Cardiomyopathy

Introduction: Pediatric patients with Restrictive Cardiomyopathy (RCM) develop heart failure due to stiffening of the wall of the cardiac ventricle leading to diastolic dysfunction but preserved cardiac contractility. Treatment options are extremely limited, and the evaluation of new therapeutic strategies is hindered due to a lack of models that recapitulate this ventricular stiffening. Here, we evaluate two novel patient-derived human induced pluripotent stem cell (hiPSC) models of restrictive cardiomyopathy due to mutations in the thin filament, and evaluate separate sarcomere-targeted therapeutic strategies to improve diastolic function. Hypothesis: Directly decreasing the thin filament’s response to calcium will improve diastolic function in RCM models with less effect on systolic function than myosin inhibition. Methods: hiPSC-cardiomyocytes (hiPSC-CMs) were differentiated from hiPSC lines generated from two patients with pediatric RCM, as well as their isogenic controls. After replating on polyacrylamide gels and staining with fluorescent calcium indicators, diastolic tension, generated tension, and calcium sensitivity were measured at baseline and upon treatment with either a troponin calcium desensitizer (W7) or a myosin inhibitor (mavacamten). Results: hiPSC-CMs from both patients showed increased diastolic tension, generated tension, and calcium sensitivity compared to their isogenic controls. Treatment with Mavacamten improved diastolic function but severely compromised systolic function, while treatment with W7 improved diastolic function with less effect on systolic function. Conclusions: Directly inhibiting the sarcomere in models of RCM improved measures of diastolic dysfunction, but directly targeting the thin filament in these lines showed less effect on systolic function than myosin inhibition, suggesting cardiac troponin calcium desensitizers may be more beneficial in this severe diastolic disease.

Abstract We007: High Throughput 3D-Printed Human Engineered Heart Tissues for Cardiac Disease Modeling

While cell models have been used to study cardiac diseases, there is currently no standardization or fully mature in vitro system. Obtaining a mature cardiomyocyte (CM) phenotype is necessary to create a physiologically relevant environment and accurately model and study cardiac diseases. Increased success in maturation of human induced pluripotent stem cell derived CMs (hiPS-CMs) has been achieved in 3D in vitro models in the form of engineered heart tissues (EHTs). Digital light processing (DLP) offers a high throughput and efficient method to fabricate hydrogels that recapitulate properties of native tissues. In this study, we use DLP-printed EHTs to seed and mature differentiated hiPS-CMs and test their potential for modeling pathological cardiac hypertrophy. To further encourage maturity of CMs, we utilized a low glucose maturation medium supplemented with fatty acids. DLP-printed EHTs displayed high reproducibility and increased CM maturity. To determine degree of maturity of our system, we performed RT-qPCR, western blot analysis, and immunofluorescence (IF) microscopy. We found increased expression of fatty-acid transport and beta oxidation genes and improved sarcomere alignment compared to 2D hiPS-CMs. To model pathological cardiac hypertrophy, we used two different approaches: 1. acute adrenergic agonism with isoproterenol and phenylephrine; 2. chronic culturing of EHTs in pathologically stiff conditions. To assess pathology, we conducted RT-qPCR, western blot, ELISA, IF microscopy, and functional analyses. We found that agonist-treated EHTs displayed increased pathology-associated gene expression compared to standard 2D hiPS-CMs. Both agonist-treated and stiff EHTs had increased secreted B-Type Natriuretic Peptide (BNP) and phosphorylation of proteins involved in pathological remodeling, including ERK and P38. Additionally, we observed increased nuclear area, increased calcium handling, and increased contractile force in the treatment groups. In conclusion, we have established a high throughput, reproducible, mature EHT platform that can be used to model pathological cardiac hypertrophy. This platform can be easily adopted by other laboratories to study a wide array of cardiac diseases.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.

Abstract Mo062: Cpne5 is Necessary for Normal Sinoatrial Node Function

Background: Cpne5 (Copine 5) is a Ca2+-dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing (scRNA-seq) of the developing mouse heart, we found Cpne5 gene expression to be highly enriched throughout the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesize that Cpne5 is necessary for normal CCS function. Objective: Determine whether Cpne5 is necessary for the normal development and/or function of the CCS. Methods: To assess the necessity of Cpne5, homozygous systemic knockout (KO) mice were generated using CRISPR/Cas9. Cpne5KO and littermate wild-type (WT) control mice were characterized using surface electrocardiogram (ECG), continuous telemetry and echocardiogram. Protein expression of Cpne5 and CCS-related ion channels were assessed by immunofluorescence of tissue sections, isolated adult murine SAN cells and whole mount immunostaining with 3D volumetric imaging (iDISCO+). Further, CRISPR-mediated deletion of CPNE5 was performed in human induced pluripotent stem cells (hiPSC). Differentiated SAN-like cells were phenotyped via calcium transient (CaT) dynamics and microelectrode array. Results: Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, sinus pauses, and increased heart rate variability compared to WT controls on long term telemetry. Additionally, CPNE5KO hiPSC-derived SAN-like cells exhibited irregular CaT as well as significant beat period variability compared to isogenic WT controls. Conclusion: Systemic loss-of-function of Cpne5 in mice resulted in sinus node dysfunction, consistent with an hiPSC-derived SAN model and human GWAS data, implicating a novel, cell-intrinsic role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies are ongoing to further elucidate the underlying mechanism.

Abstract Tu031: Immunosuppression Drug Modulation of Endothelial Cell Function

Heart transplantation is a life-saving therapy for patients with end-stage heart failure with over 4,000 transplants occurring per year. The use of immunosuppression drugs is necessary in post transplantation to prevent graft rejection. Proliferation signal inhibitors have previously been shown to effectively reduce the incidence of cardiac allograft vasculopathy (CAV) compared to calcineurin inhibitors but the differential mechanisms that drive this benefit and differences in vascular profile of immunosuppression drugs remain incompletely understood. In this study, we examined the direct effect of immunosuppressive agents on endothelial function to define cellular mechanisms that may contribute to differential vascular phenotypes. We used endothelial cells derived from human induced pluripotent stem cells (iPSCs) and treated them with calcineurin inhibitor (tacrolimus), mTOR inhibitor (sirolimus), or vehicle for 5 days with daily media changes. We examined endothelial function using scratch wound assay, tube formation assay, and LDL uptake. We also examined metabolic changes using Seahorse assay owing to metabolic changes induced by immunosuppression drugs in other organs. Our results indicate mTOR inhibitor (sirolimus) resulted in a reduction in endothelial cell proliferation and migration using the scratch wound assay and tube formation assay compared to vehicle or calcineurin inhibitor (tacrolimus)-treated cells. Sirolimus also reduced LDL uptake in iPSC-derived endothelial cells compared to vehicle or tacrolimus-treated cells. Tacrolimus was associated with a trend towards altered endothelial energetics. Tacrolimus and sirolimus exert differential endothelial cell functional profiles that do not fully explain the clinical outcomes observed suggesting a need to explore the drug endothelial profiles in the context of vascular injury and inflammation.

Abstract We027: Gut Microbiota Modulation by Immunosuppression and Cardiac Cell Therapy in a Nonhuman Primate Ischemia/Reperfusion Model of Cardiac Regeneration

End-stage ischemic heart disease necessitates heart transplantation, and emerging cell therapy presents a promising solution to address donor scarcity. Disruption of gut microbiota significantly influences various diseases and treatments, including transplantation. However, the impact of immunosuppression and cardiac cell therapy on gut microbiota remains largely unexplored. To elucidate this relationship, we investigated gut microbiota dynamics in response to immunosuppression and cell therapy in a nonhuman primate (NHP) cardiac ischemia/reperfusion (IR) model, with controlled genetic, dietary, and environmental factors. Immunosuppression enriched anaerobes (Faecalibacterium, Streptococcus, Anaerovibrio, Dialister), increasing gut microbiota diversity. These changes correlated with metabolic shifts towards amino acid metabolism and nucleosides/nucleotides biosynthesis. Combined treatment of human induced pluripotent stem cell (iPSC)-derived endothelial cells (EC) and cardiomyocytes (CM) also increased gut microbiota diversity, with specific genera alterations. The EC/CM co-treatment group displayed gut microbiota resembling the pre-injury group, with host metabolism shifting towards amino acid and fatty acid/lipid biosynthesis post-cell therapy. These observed microbiota changes and metabolic shifts could serve as biomarkers for monitoring cell therapy and immunosuppression outcomes, offering potential therapeutic targets to enhance efficacy.

Abstract Mo084: Stem Cell-Based Functional Genomics Unravel A Novel Vascular Smooth Muscle Cell State Induced By The 9p21 Coronary Artery Disease Risk Locus

Background: Genome-wide association studies have identified common genetic variants (Single Nucleotide Polymorphisms – SNPs) at ~300 human genomic loci linked to coronary artery disease (CAD) susceptibility. Among these genomic regions, the most impactful is the 9p21.3 CAD risk locus, which spans a 60 kb gene desert and encompasses ~80 SNPs in high linkage disequilibrium. We have previously generated isogenic induced Pluripotent Stem Cells (iPSCs) lines from risk and non-risk donors at 9p21.3 and performed a complete deletion of the locus. By using iPSC-derived vascular smooth muscle cells (iPSC-VSMCs) we demonstrated that the risk haplotype at 9p21.3 causes altered expression of numerous genes essential for muscle function, including contraction and adhesion. Notably, deletion of the risk haplotype restores the non-risk phenotype, suggesting a gain of function effect. Here, we aimed to identify the transcriptomic signature and state trajectories of VSMCs induced by the 9p21.3 risk haplotype and validate them through functional assays and ex-vivo analysis of human coronary arteries. Methods: We have used iPSCs from individuals carrying the risk and non-risk haplotype at 9p21.3 and isogenic knockout lines. After VSMCs differentiation induction, mature VSMCs were used for single cell RNA sequencing using 10X Chromium platform and functional assays. Results: Our analysis revealed that the 9p21.3 risk haplotype prompts VSMCs to acquire a novel cellular state showing reduced plasticity and divergent from other VSMC states previously linked to CAD. We found markers of alternative lineages, including osteogenic and neuronal, coupled with altered expression of genes within other CAD loci, and functional defects. We identified a set of new molecular markers crucial to define risk-associated VSMCs and uncovered their upstream regulators. Conclusions: Our study provides insights into CAD pathogenesis driven by the 9p21.3 risk locus and identified new gene regulatory networks essential for maintaining the normal functionality of the muscle layer of the arteries. Leveraging the power of iPSCs we present novel concepts about the biology of VSMCs and shed light on the impact of the strongest CAD genetic risk factor in preserving a healthy cellular state within the vasculature.

Early nuclear phenotypes and reactive transformation in human iPSC-derived astrocytes from ALS patients with SOD1 mutations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons (MNs). Glial cells play roles in MN degeneration in ALS. More specifically, astrocytes with mutations in the ALS-associated gene Cu/Zn superoxide dismutase 1 (SOD1) promote MN death. The mechanisms by which SOD1-mutated astrocytes reduce MN survival are incompletely understood. To characterize the impact of SOD1 mutations on astrocyte physiology, we generated astrocytes from human induced pluripotent stem cell (iPSC) derived from ALS patients carrying SOD1 mutations, together with control isogenic iPSCs. We report that astrocytes harboring SOD1(A4V) and SOD1(D90A) mutations exhibit molecular and morphological changes indicative of reactive astrogliosis when compared to isogenic astrocytes. We show further that a number of nuclear phenotypes precede, or coincide with, reactive transformation. These include increased nuclear oxidative stress and DNA damage, and accumulation of the SOD1 protein in the nucleus. These findings reveal early cell-autonomous phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in alterations of astrocyte-MN communication in ALS.

Abstract Tu092: Role of Z-disc as a Driver of Hypertrophy and Sarcomere Assembly in Hypertrophic Cardiomyopathy

The Z-disc is an electron-dense structure demarcating the lateral boundaries of the sarcomere and postulated to play a key role in both cell signaling and sarcomere assembly. We performed EM on both HCM patient septal myectomies and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with 3 different HCM MYH7 mutations, finding marked Z-disc widening along with a hallmark feature of HCM, myofibrillar disarray. Mutations in MYH7 that cause HCM do so by increasing force at the sarcomere level, leading to the proposal that the Z-disc may act as a mechanosensor that converts myosin-generated force into the intracellular signaling that drives hypertrophy. We focused on understanding the mechanisms of Z-disc mechanotransduction of both hypertrophy and sarcomere disarray. RNA-seq from HCM myectomies demonstrated 52 Z-disc differentially expressed genes (DEGs). scRNA-seq from HCM hiPSC-CMs identified 24 Z-disc DEGs. 14 Z-disc DEGs were shared across both datasets, most notably 5 upstream regulators of calcineurin signalling. To correlate changes in gene expression with altered force we transfected hiPSC-CMs with a FRET-based tension sensor in the Z-disc protein vinculin allowing the direct measurement of force experienced by the Z-disc. Live-cell confocal imaging of hiPSC-CMs expressing EGFP-α-actinin-2 showed that indistinct α-actinin-2 foci coalesced into sarcomeres over the course of 24-48 h after replating. Imaging at nm scale using cryo-electron tomography revealed marked disorder in individual thick filaments in HCM hiPSC-CMs, distinct from the classical disarray at the level of whole myofibrils and which would not be visible on conventional EM. Our data suggest that early alterations in sarcomeric micro-structure and signaling induced by altered myosin force may precede hypertrophy and could occur decades before clinical signs (hypertrophy on ECG or echo) are present, with potential implications for early preventive intervention.

Abstract We035: PTMA-MBD3 axis is a core heart regenerative driver in mammals

Introduction: The adult mammalian heart has an extremely limited ability for regeneration after injury, whereas the fetal heart can steadily regenerate. Uncovering the molecular underpinnings of proliferative cardiomyocytes and manipulating the core genes activate cardiomyocyte proliferation may provide new insight into adult heart regrowth and functional repair post-injury. Methods and Results: We performed single-cell RNA-sequencing of mouse embryonic hearts from various developmental stages (E8.5-E17.5), and identified a cardiomyocyte population in proliferative phase with 120 highly expressed feature genes. Combining with our bulk RNA-seq data on isolated cardiomyocytes from different heart developmental periods (E14.5, P1-P56), we screened and demonstrated that Ptma is the core conservative factor in promoting mouse, rat and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation. Cardiac specifically knocking out Ptma blocked cardiomyocyte proliferation and neonatal mouse heart regeneration after apical resection injury. AAV9-delivered Ptma overexpression extended neonatal heart regenerative time window and showed therapeutic benefit in driving adult cardiomyocyte proliferation and cardiac repair. As PTMA is a polypeptide, we further validated that PTMA derived conservative peptide Tα1 displayed more effective in treating adult cardiac injury compared to Decapeptide. PTMA is an unstructured, intrinsically disordered nuclear protein that functions by binding to target proteins. Using Co-IP, IP and Native PAGE, we found PTMA promoted cardiomycyte proliferaton by binding to MBD3, which decreases the formation of MBD3-HDAC1 NuRD complex . We further decreased MBD3-HDAC1 NuRD complex formation by inhibiting MBD3, boosting both rat and mouse cardiomyocyte proliferation. Conclusions: We identified the PTMA-MBD3 axis as an effective target for promoting mammalian heart regeneration and provided a new insight into cardiomyocyte proliferation through blocking MBD3-HDAC1 NuRD complex.