Generation of Human Cardiac Organoids from Embryonic Stem Cells via Stepwise Mesoderm Induction and 3D Self-organization

Bingwei Wang; Chunxiang Zhang

doi:10.3791/68738

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Method Article

Generation of Human Cardiac Organoids from Embryonic Stem Cells via Stepwise Mesoderm Induction and 3D Self-organization

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DOI:

10.3791/68738

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October 7th, 2025

Bingwei Wang¹^,² , Chunxiang Zhang¹^,²^,³^,⁴^,⁵

¹Basic Medicine Research Innovation Center for Cardiometabolic Diseases, Ministry of Education, Southwest Medical University, ²Nucleic Acid Medicine, Key Laboratory of Luzhou, Southwest Medical University, ³Department of Cardiology, The Affiliated Hospital of Southwest Medical University, Southwest Medical University, ⁴Key Laboratory of Medical Electrophysiology, Ministry of Education, Southwest Medical University, ⁵Institute of Cardiovascular Research, Southwest Medical University

Summary

This protocol describes a step-by-step method for generating functional human cardiac organoids from human embryonic stem cells (hESCs). It includes cell thawing, differentiation, and maturation steps, providing a robust platform for cardiac development and disease modeling.

Abstract

Human embryonic stem cell (hESC)-derived cardiac organoids are multicellular three-dimensional (3D) structures that recapitulate key aspects of early human heart development and function. These self-organizing organoids exhibit spontaneous contractility, cardiomyocyte marker expression, and tissue-like architecture reminiscent of native myocardium. Here, we present a robust and reproducible protocol to generate cardiac organoids from the H9 hESC line via stepwise lineage differentiation. Mesoderm induction is initiated by treating spheroids for 36-40 h with Activin A (50 ng/mL), bone morphogenetic protein 4 (BMP4, 10 ng/mL), fibroblast growth factor 2 (FGF2, 30 ng/mL), laduviglusib (CHIR99021, 3 µM), and a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002, 5 µM). Cardiac lineage specification is subsequently directed by daily exposure for four days to BMP4 (10 ng/mL), FGF2 (10 ng/mL), a Wnt pathway inhibitor (XAV-939, 5 µM), and retinoic acid (0.5 µM). Cardiomyocyte differentiation and maturation are further promoted from Day 5.5 onward using BMP4 (10 ng/mL), FGF2 (10 ng/mL), and insulin (10 µg/mL). Functional validation is achieved through time-lapse imaging and immunofluorescence analysis, confirming the generation of contractile cardiomyocytes marked by cardiac troponin T (cTnT) expression. Additionally, 3D immunostaining reveals the presence of α-SMA and CDH5, indicating the emergence of smooth muscle and endothelial-like cell populations. These cardiac organoids consistently demonstrate rhythmic contractions; however, direct electrophysiological validation of electromechanical coupling was not performed. Known limitations include the inability to passage organoids and potential central necrosis during extended cultures. In summary, this model provides a scalable and physiologically relevant platform for studying human cardiogenesis, drug responses, and congenital heart diseases.

Introduction

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide, underscoring the urgent need for reliable in vitro models that recapitulate human cardiac development and function¹^,². Traditional two-dimensional (2D) monolayer cultures of cardiomyocytes, while valuable, fail to reproduce the spatial complexity, multicellular interactions, and electromechanical integration of the native human heart³^,⁴. 3D cardiac organoids derived from pluripotent stem cells have emerged as powerful platforms that address these limitations, providi....