Single‐Cell Transcriptomics of Engineered Cardiac Tissues From Patient‐Specific Induced Pluripotent Stem Cell–Derived Cardiomyocytes Reveals Abnormal Developmental Trajectory and Intrinsic Contractile Defects in Hypoplastic Right Heart Syndrome

Abstract

Background To understand the intrinsic cardiac developmental and functional abnormalities in pulmonary atresia with intact ventricular septum (PAIVS) free from effects secondary to anatomic defects, we performed and compared single‐cell transcriptomic and phenotypic analyses of patient‐ and healthy subject–derived human‐induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) and engineered tissue models. Methods and Results We derived hiPSC lines from 3 patients with PAIVS and 3 healthy subjects and differentiated them into hiPSC‐CMs, which were then bioengineered into the human cardiac anisotropic sheet and human cardiac tissue strip custom‐designed for electrophysiological and contractile assessments, respectively. Single‐cell RNA sequencing (scRNA‐seq) of hiPSC‐CMs, human cardiac anisotropic sheet, and human cardiac tissue strip was performed to examine the transcriptomic basis for any phenotypic abnormalities using pseudotime and differential expression analyses. Through pseudotime analysis, we demonstrated that bioengineered tissue constructs provide pro‐maturational cues to hiPSC‐CMs, although the maturation and development were attenuated in PAIVS hiPSC‐CMs. Furthermore, reduced contractility and prolonged contractile kinetics were observed with PAIVS human cardiac tissue strips. Consistently, single‐cell RNA sequencing of PAIVS human cardiac tissue strips and hiPSC‐CMs exhibited diminished expression of cardiac contractile apparatus genes. By contrast, electrophysiological aberrancies were absent in PAIVS human cardiac anisotropic sheets. Conclusions Our findings were the first to reveal intrinsic abnormalities of cardiomyocyte development and function in PAIVS free from secondary effects. We conclude that hiPSC‐derived engineered tissues offer a unique method for studying primary cardiac abnormalities and uncovering pathogenic mechanisms that underlie sporadic congenital heart diseases.

Decoding Genetics of Congenital Heart Disease Using Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

“…Single-cell RNA-seq analysis of human and mouse hearts has provided unprecedented resources on the trajectory of cardiac development in vivo at single-cell resolution and revealed a blueprint on how normal cell fate determination is altered under genetic perturbation and pathological conditions such as CHD ( Cui et al, 2019 ; De Soysa et al, 2019 ; Litvinukova et al, 2020 ; Paik et al, 2020 ). Single-cell transcriptional profiling of healthy and diseased iPSCs during cardiac differentiation would decipher how a given genetic variant affects cardiac differentiation and developmental trajectories, and uncover new molecular insights in the pathogenesis of CHD ( Churko et al, 2018 ; Kathiriya et al, 2020 ; Lam et al, 2020 ; Miao et al, 2020 ; Paige et al, 2020 ). As heart development is dependent on interaction among multiple cell types in the embryo, cardiac organoids and 3D bio-printing may serve as another tier of disease modeling using patient iPSCs ( Lee et al, 2019 ; Nugraha et al, 2019 ).…”

Ex uno, plures–From One Tissue to Many Cells: A Review of Single-Cell Transcriptomics in Cardiovascular Biology

“…Furthermore, single-cell transcriptomic data revealed that the expression of Gdf15, a cardiac-produced hormone and marker of cardiac dysfunction, was regulated by distinct cell-type-specific networks. More recently, two studies have used 10x Chromium technologies to investigate different models of congenital heart defects [ 55 , 56 , 57 , 58 ]. The first study [ 55 ] profiled the cardiogenic region of wild-type versus Hand2 null mouse embryos over three phases of cardiac development (E7.75, E8.5, E9.25).…”

“…Further, scRNAseq has been employed to analyze patient-derived hiPSC and dissect the cellular basis of cardiomyopathy associated with Duchenne muscular dystrophy (DMD) [ 71 ] and the defective cardiac development in Hypoplastic Left Heart Syndrome (HLHS) [ 56 ] and a type of Hypoplastic Right Heart Syndrome (HRHS) [ 58 ]. DMD is the most common form of muscular dystrophy, it is caused by mutation in the DMD gene, and it generally leads to dilated cardiomyopathy (DCM).…”

“…HRHS is associated with pulmonary or tricuspid valve atresia, and it is prevalent in the Asian population. To probe a class of HRHS, Pulmonary Atresia with Intact Ventricular Septum syndrome, the authors profiled hiPSC-CM derived from three patients and three healthy controls, cultured in three different conditions: regular cultures, anisotropic organoids, and cardiac tissue strips [ 58 ]. The two bioengineered constructs are devised to measure electrophysiological and contractile responses, respectively, and promote CM maturation.…”

Mending a broken heart: In vitro, in vivo and in silico models of congenital heart disease

“…Mature 3D EHTs have been fabricated from iPSCs in a custom-designed bioreactor that generates electromechanical stimulation ( Ronaldson-Bouchard et al, 2019 ), an attractive system with great potential for translational research, enabling assessment of cardiac contractility, and facilitating investigation of various genetic mutations and molecular mechanisms of CHD-related heart failure. Recently, by using single-cell RNA sequencing (scRNAseq), 2D and 3D cardiac tissues served as powerful tools with which to understand the transcriptomic basis and molecular regulation of iPSC-cardiomyocytes that carry disease-associated genes, further expanding the repertoire of CHD research ( Lam et al, 2020 ; Ni et al, 2020 ).…”