Pluripotent Stem Cell Models of Human Heart Disease

Stem cells & developmental biolog

SUBTY PE-SPECIFIC PROMOTER-DRIVEN ACTION POTENTIAL IMAGING FOR PRECISE DISEASE MODELING AND DRUG TESTING IN HIPSC-DERIVED CARDIOMYOCYTES

Stem cells & developmental biolog

Comparison of delay times between symptom onset of an acute ST-elevation myocardial infarction and hospital arrival in men and women <65 years versus ≥65 years of age.: Findings from the Multicenter Munich Examination of Delay in Patients Experienci

Early administration of reperfusion therapy in acute ST-elevation myocardial infarctions (STEMI) is crucial to reduce mortality. Although female sex and old age are key factors contributing to an inadequate long prehospital delay time, little is known whether women ≥65 years are a particular risk population. Hence, we studied the interaction of sex and age (<65 years or ≥65 years) and the contribution of chest pain to delay time during STEMI. Bedside interview data were collected in 619 STEMI patients from the Munich Examination of Delay in Patients Experiencing Acute Myocardial Infarction (MEDEA) study. Sex and age group stratification disclosed an excess delay risk for women ≥65 years, accounting for a 2.39 (95% confidence interval (CI) 1.39 to 4.10)-fold higher odds to delay longer than 2 hours compared with all other patient groups including younger women (p ≤0.002). Median delay time was 266 minutes in women ≥65 years and 148 minutes in younger women (p <0.001). Chest pain during STEMI had the lowest frequency both in women (81%) and men ≥65 years (83%) and the highest frequency (95%) in younger women. Experiencing non-chest pain was 2.32-fold (95% CI, 1.20 to 4.46, p <0.05) higher in women ≥65 years than in all other patients. Mediation analysis disclosed that the effect accounted for only 9% of the variance. Age specific educational strategies targeting women ≥65 years at risk are urgently needed. To tailor adequate strategies, more research is required to understand age- and sex driven barriers to timely identification of ischemic symptoms

Isogenic Sets of Human Pluripotent Stem Cells as Model of LQT2 Syndrome

Stem cells & developmental biolog

Generation of two human iPSC lines, HMGUi003-A and MRIi028-A, carrying pathogenic biallelic variants in the PPCS gene.

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency

Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells

Direct Nkx2-5 transcriptional repression of Isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1+ precursors. Embryos deficient for Nkx2-5 in the Isl1+ lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity

Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes

Stem cells & developmental biolog

Cell cycle defects underlie childhood-onset cardiomyopathy associated with Noonan syndrome.

Childhood-onset myocardial hypertrophy and cardiomyopathic changes are associated with significant morbidity and mortality in early life, particularly in patients with Noonan syndrome, a multisystemic genetic disorder caused by autosomal dominant mutations in genes of the Ras-MAPK pathway. Although the cardiomyopathy associated with Noonan syndrome (NS-CM) shares certain cardiac features with the hypertrophic cardiomyopathy caused by mutations in sarcomeric proteins (HCM), such as pathological myocardial remodeling, ventricular dysfunction, and increased risk for malignant arrhythmias, the clinical course of NS-CM significantly differs from HCM. This suggests a distinct pathophysiology that remains to be elucidated. Here, through analysis of sarcomeric myosin conformational states, histopathology, and gene expression in left ventricular myocardial tissue from NS-CM, HCM, and normal hearts complemented with disease modeling in cardiomyocytes differentiated from patient-derived PTPN11N308S/+ induced pluripotent stem cells, we demonstrate distinct disease phenotypes between NS-CM and HCM and uncover cell cycle defects as a potential driver of NS-CM