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Modeling early human heart development using an iPSC-based 3D bioprinted model of embryonic heart tube

ABSTRACT: Human heart development is a precisely coordinated process involving dynamic transcriptional and morphological changes. Due to limited access to the developing human heart in utero and lack of accurate experimental models, the cell-microenvironmental interactions underlying human heart development remain elusive. Advances in 3D bioprinting and stem cell technologies have enabled the fabrication of in vitro analogues of cardiac tissues with complex structures, while incorporating cell intrinsic (genetic) and microenvironment factors. Here we present a perfusable 3D bioprinted model of human embryonic heart tube (eHT), composed of distinct layers of myocardium, cardiac jelly, and endocardium. Human induced pluripotent stem cell-derived cardiomyocytes and endothelial cells were cocultured in the engineered eHTs under dynamic flow. This system demonstrated robust cell viability and cardiac function, including full lumen endothelialization, progressive myocardial compaction, and global tissue contraction. A promoting effect of dynamic flow on cardiac maturation and cell lineage commitment was revealed by single-cell RNA sequencing. The established eHT model offers a novel research-enabling platform to study human heart development, various (congenital) heart disease, and therapeutic interventions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE315240 | GEO | 2026/02/18

REPOSITORIES: GEO

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Project description:Rationale Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and is associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with findings that microtubule detyrosination (dTyr-MTs) is markedly elevated in heart failure. Acute reduction of dTyr-MTs by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, presenting a new perspective for HCM treatment. Objective In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model (Mybpc3-knock-in; KI), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents, in 3-week-old KI mice, and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show: i) TTL for 6 weeks dose-dependently reduced dTyr-MTs and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes. ii) TTL for 12 weeks reduced the abundance of dTyr-MTs in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in KI mice. iii) TTL for 10 days normalized cell area in HCM hiPSC-cardiomyocytes. iv) TTL overexpression activates transcription of several tubulin isoforms, and omics GO analysis revealed enrichment of components of the cytoskeleton, sarcomere Z-disc, mitochondria, and intercalated disc in KI mice. v) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.

Project description:Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart and high concentrations of RA have been shown to steer differentiation towards atrial CMs, whereas lower concentrations of RA promote a more ventricular-like CM profile. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of retinoic acid (RA) during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. The maturation and growth of EHTs were induced in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. The function of CMs and EHTs was analyzed under different conditions. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPRC and immunofluorescence staining analysis. RNA sequencing was conducted on EHTs to examine how RA affects both the function and structure of EHT. Results: In the HRA group, hiPSC-CMs exhibited the first onset of beating and showed the highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are the marker genes for left ventricular CMs, was also the highest in the HRA group. The transcription factor MEF2C associated with RA and ventricular development genes, NPPA and MYH7, were highly expressed in the HRA group, while GATA4 was less expressed in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was more functionally similar to the left ventricle. The expression of TBX5 and NKX2.5 was found to be the highest expression in the HRA group of EHT, while expression of TBX20 and ISL1 was found to be the highest expression in control group. When the electrical stimulation frequency of EHT in the HRA group was raised, it correspondingly increased its contractile force. The heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paved the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine.

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Modeling early human heart development using an iPSC-based 3D bioprinted model of embryonic heart tube

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