Investigating pediatric disorders with induced pluripotent stem cells

The study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients

Hypoplastic Left Heart Syndrome Sequencing Reveals a Novel NOTCH1 Mutation in a Family with Single Ventricle Defects

Hypoplastic left heart syndrome (HLHS) has been associated with germline mutations in 12 candidate genes and a recurrent somatic mutation in HAND1 gene. Using targeted and whole exome sequencing (WES) of heart tissue samples from HLHS patients, we sought to estimate the prevalence of somatic and germline mutations associated with HLHS. We performed Sanger sequencing of the HAND1 gene on 14 ventricular (9 LV and 5 RV) samples obtained from HLHS patients, and WES of 4 LV, 2 aortic, and 4 matched PBMC samples, analyzing for sequence discrepancy. We also screened for mutations in the 12 candidate genes implicated in HLHS. We found no somatic mutations in our HLHS cohort. However, we detected a novel germline frameshift/stop-gain mutation in NOTCH1 in a HLHS patient with a family history of both HLHS and hypoplastic right heart syndrome (HRHS). Our study, involving one of the first familial cases of single ventricle defects linked to a specific mutation, strengthens the association of NOTCH1 mutations with HLHS and suggests that the two morphologically distinct single ventricle conditions, HLHS and HRHS, may share a common molecular and cellular etiology. Finally, somatic mutations in the LV are an unlikely contributor to HLHS

Production of Single Contracting Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Matrigel Mattress Technique

This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy

Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.</p