Biology subjects

Mital, S.

Publications and source records attributed to Mital, S..

6 recordsLinked to original sources

bioRxiv2023

Myosin inhibitor reverses hypertrophic cardiomyopathy in pediatric iPSC-cardiomyocytes to mirror variant correction

Hypertrophic cardiomyopathy (HCM) is mainly caused by sarcomere gene variants in MYH7 and MYBPC3. Targeted drugs like myosin ATPase inhibitors have shown efficacy in adult HCM but have not been evaluated in children. We generated iPSC-cardiomyocytes (CMs) from four children with HCM harboring variants in MYH7 (V606M; R453C) or MYBPC3 (G148R; P955fs and TNNI3_A157P), variant-corrected controls, and a healthy individual. All CMs showed hypertrophy and sarcomere disorganization. All 3 single variant CMs showed higher contractility, slower relaxation, higher calcium transients and higher ATPase activity. Only MYH7 variant CMs showed stronger myosin-actinin binding. Targeted myosin ATPase inhibitor showed complete rescue of the phenotype in affected CMs and in cardiac Biowires to mirror isogenic controls. The response was stronger compared to verapamil or metoprolol, highlighting the need for clinical trials of myosin targeted therapy in pediatric HCM patients. The phenotype and response to drug therapy are influenced by the underlying genotype.

genetics↗

bioRxiv2022

Advanced physiological maturation of iPSC-derived human cardiomyocytes using an algorithm-directed optimization of defined media components

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold tremendous promise for in vitro modeling to assess native myocardial function and disease mechanisms as well as testing drug safety and efficacy. However, current iPSC- CMs are functionally immature, resembling in vivo CMs of fetal or neonatal developmental states. The use of targeted culture media and organoid formats have been identified as potential high-yield contributors to improve CM maturation. This study presents a novel iPSC-CM maturation medium formulation, designed using a differential evolutionary approach targeting metabolic functionality for iterative optimization. Relative to gold-standard reference formulations, our medium significantly matured morphology, Ca2+ handling, electrophysiology, and metabolism, which was further validated by multiomic screening, for cells in either pure or co-cultured microtissue formats. Together, these findings not only provide a reliable workflow for highly functional iPSC-CMs for downstream use, but also demonstrate the power of high-dimensional optimization processes in evoking advanced biological function in vitro.

bioengineering↗

bioRxiv2021

Abnormal Differentiation and Proliferation of Coronary Arterial Endothelium in Hypoplastic Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdevelopment of the left ventricle. Early serial postmortem examinations revealed high rate of coronary artery abnormalities in HLHS fetal hearts, such as thickened wall, kinking arteries and ventriculo-coronary arterial connection. However, it is unclear if there is an intrinsic defect in the HLHS coronary vessels and what the underlying molecular mechanism is. Here, we profiled both human fetal heart with an underdeveloped left ventricle (ULV) and ECs differentiated from induced pluripotent stem cells (iPSCs) derived from HLHS patients at single cell resolution. CD144+/NPR3-vascular ECs were selected and further classified as venous, arterial and late arterial subclusters. To study the arterial EC phenotype, we specifically generated iPSC-arterial ECs (AECs, CD34+CDH5+CXCR4+NT5E-/low) derived from three HLHS patients and three age-matched healthy controls. Gene ontology analysis revealed that ULV late arterial EC subcluster showed specific defects in endothelial development, proliferation, and Notch signaling compared to control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation shown as the reduced CXCR4+NT5E-/low AEC progenitor population. Mature HLHS iPSC-AECs also exhibited increased G0/G1 cell cycle arrest with decreased expression of cell cycle related genes (e.g., Ki67, CCND1/2). Additionally, NOTCH targeted genes (e.g., DLL4, HEY1, GJA5) were found suppressed in both ULV AECs and HLHS iPSC-AECs compared to control. We also found the HLHS de novo mutation gene KMT2D directly regulated the transcription of NOTCH targeted genes participating in arterial differentiation and cell proliferation, contributing to the HLHS AEC dysfunctionalities. Intriguingly, the treatment of NOTCH ligand JAG1 improved cell proliferation of HLHS AECs and upregulated G1/S transition genes downstream of NOTCH pathway. In summary, our results revealed that KMT2D directly regulated transcription activity of NOTCH signaling, contributing to the poor differentiation and low proliferation of HLHS coronary AECs.

developmental biology↗

bioRxiv2019

Single-Cell RNA-Seq Reveals Endocardial Defect in Hypoplastic Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is one of the most challenging forms of congenital heart diseases. Previous studies were mainly focused on intrinsic defects in myocardium. However, this does not sufficiently explain the abnormal development of the cardiac valve, septum, and vasculature, known to originate from the endocardium. Here, using single-cell RNA profiling, induced pluripotent stem cells, and human fetal heart tissue with an underdeveloped left ventricle, we identified a developmentally impaired endocardial population in HLHS. The intrinsic endocardial deficits contributed to abnormal endothelial to mesenchymal transition, NOTCH signaling, and extracellular matrix organization, all of which are key factors in valve formation. Consequently, endocardial abnormalities conferred reduced proliferation and maturation of cardiomyocytes through a disrupted fibronectin-integrin interaction. Several recently described HLHS de novo mutations were associated with abnormal endocardial gene and FN1 regulation and expression. Our studies provide a rationale for considering endocardial function in future regenerative strategies for HLHS.

developmental biology↗

bioRxiv2019

Control iPSC lines with clinically annotated genetic variants for versatile multi-lineage differentiation

Induced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSCs, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-lymphocytes and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole genome sequencing (WGS) based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modeling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.

developmental biology↗

bioRxiv2019

Quality of Whole Genome Sequencing from Blood versus Saliva Derived DNA in Cardiac Patients

Whole-genome sequencing (WGS) is becoming an increasingly important tool for detecting genomic variation. Blood derived DNA is the current standard for WGS for research or clinical purposes. We compared the level of microbial contamination, sequencing coverage, as well as yield and concordance of single-nucleotide polymorphism (SNP) and copy number variant (CNV) calls in WGS from paired blood and saliva samples from 5 pediatric heart disease patients. We found that although saliva samples contained a higher proportion of sequence reads that map to the human oral microbiome, these reads were readily excluded by mapping the reads to the human reference genome. Sequencing coverage was low only in 1 of 5 saliva samples. Over 95% SNPs (including rare SNPs) but <80% CNVs called in blood genomes were detected in paired saliva genomes. These findings suggest that most good quality saliva samples can serve as an alternative to blood samples for detection of sequence variants from WGS in cardiovascular disease patients.

genomics↗