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

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 function^1,2. 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 heart^3,4. 3D cardiac organoids derived from pluripotent stem cells have emerged as powerful platforms that address these limitations, providing self-organizing, multicellular structures that mimic early cardiac morphogenesis and functional maturation in vitro⁵.

hESCs, such as the H9 line, possess the intrinsic ability to differentiate into all somatic cell types, including cardiomyocytes, endothelial cells, and cardiac fibroblasts⁶. Under controlled culture conditions, hESCs can be guided through the sequential stages of mesoderm induction, cardiac mesoderm specification, and cardiomyocyte differentiation^5,7. This stepwise process can be regulated using specific signaling molecules and inhibitors, including Activin A, BMP4, FGF2, PI3K inhibitors, CHIR99021, Wnt inhibitors, and retinoic acid^5,8. By closely mimicking the in vivo signaling environment of embryonic cardiac development, these cues promote efficient and reproducible cardiogenesis in vitro^5,7.

Unlike many organoid systems that require embedding in extracellular matrix (ECM) components such as Matrigel, cardiac organoids can self-assemble in low-adhesion conditions without matrix support^5,9. Aggregation of differentiating hESCs in suspension yields spherical 3D structures that exhibit spontaneous beating within the first week of differentiation¹⁰. These organoids contain functionally relevant cardiac cell populations and demonstrate characteristic features, such as sarcomeric organization, calcium transients, and rhythmic contractions^11,12, offering a physiologically relevant model of early human heart tissue.

The ability to generate hESC-derived cardiac organoids offers exciting opportunities for developmental biology, disease modeling, drug screening, and cardiotoxicity testing^13,14. Moreover, integration with genome editing technologies and patient-specific induced pluripotent stem cells (iPSCs) enables personalized disease modeling and therapeutic exploration for inherited cardiac disorders.

In this article, we present a detailed, step-by-step protocol for the generation of cardiac organoids from human embryonic stem cells, highlighting key stages of differentiation, culture conditions, and validation techniques. This platform provides a robust, scalable system for recapitulating human cardiac development and establishing a foundation for future translational research.

Protocol

All procedures involving human embryonic stem cells (H9) were conducted in accordance with institutional guidelines and approved by Biomedical Ethics Committee of Southwest Medical University, approval 20241023-031. All tissue culture work detailed below should be done in a Class II laminar flow hood. Always ensure that media and reagents are at room temperature, equilibrated naturally before use. Do not use a water bath to warm media.

1. Thawing and routine culture of HESCs (H9)

1. Vitronectin coating of culture plates
   1. Thaw Vitronectin stock on ice and dilute to a working concentration of 10 µg/mL using cold E8 medium.
   2. Add 1 mL per well of the diluted Vitronectin solution to a 6-well plate. Incubate the plate in a 37 °C, 5% CO₂ incubator for 1-4 h before use.
   3. Prior to seeding cells, aspirate excess Vitronectin and do not rinse.
      NOTE: Incubation in the cell culture incubator improves coating uniformity and enhances cell attachment, especially important for sensitive hESCs. Do not allow the coating solution to dry. This protocol uses vitronectin-coated plates only during the hESC thawing and expansion stages to provide a defined, surface-bound ECM substitute. Cardiac organoids are subsequently formed through self-aggregation in low-adhesion suspension conditions, without the need for ECM.
2. Thawing cryopreserved HESCs (H9)
   1. At least 30-60 min in advance, allow all required media and reagents (e.g., E8 medium, E8 + ROCK inhibitor, PBS) to equilibrate naturally to room temperature.
   2. Retrieve one cryovial of H9 human embryonic stem cells from liquid nitrogen storage using appropriate personal protective equipment. Immediately immerse the cryovial in a 37 °C water bath. Gently swirl the vial and monitor closely.
   3. Once a small ice crystal remains (~90% thawed, ~1-2 min), promptly transfer the vial into a biosafety cabinet. Disinfect the outside of the cryovial with 75% ethanol before opening.
   4. Slowly transfer the contents of the cryovial dropwise into a 15 mL conical tube containing 5 mL of room temperature E8 medium supplemented with 10 µM Y-27632 (ROCK inhibitor). Mix gently by pipetting up and down with a wide-bore pipette tip.
   5. Centrifuge at 200 × g for 5 min at room temperature. Carefully aspirate the supernatant and resuspend the cell pellet in 2 mL of E8 + Y-27632 medium.
   6. Plate cells onto a Vitronectin-coated 6-well plate (prepared in advance) at the desired seeding density (typically 150,000-200,000 cells/well). Gently rock the plate to distribute the cells evenly, and transfer to a 37 °C, 5% CO₂ incubator.
      NOTE: The use of ROCK inhibitor (Y-27632) is critical during the first 24 h post thaw to prevent anoikis and support survival.
3. Routine maintenance and passaging
   1. Change medium daily using 2 mL of fresh E8 medium per well.
   2. Monitor daily under a phase-contrast microscope; passage cells when they reach ~70-80% confluency, typically every 3-4 days.
      1. To passage: Aspirate medium and rinse gently with 1 mL of DPBS.
      2. Add 0.5 mL of stem cell dissociation reagent and incubate at room temperature for 3-5 min. Tap the plate gently to dislodge colonies.
      3. Collect the cell suspension and replate at a 1:3-1:4 split ratio onto freshly Vitronectin-coated wells in E8 medium containing 10 µM Y-27632. After 24 h, replace the medium with E8 without ROCKi.
         NOTE: For optimal recovery and subsequent differentiation efficiency, HESCs should be passaged as small clusters of 2-10 cells.
         Use of wide-bore tips is recommended to minimize shear stress. A 5 mL pipette tip or a 10 mL serological pipette can be used for mixing.

2. Differentiation of HESCs (H9) into cardiac lineage

1. Induction of mesodermal differentiation (Day 0-1.5)
   NOTE: Avoid directly differentiating freshly thawed HESCs (H9) into cardiac organoids. It is crucial that the cells are first passaged and maintained for at least one passage before initiating differentiation to ensure optimal cell viability and healthy differentiation. Differentiating cells immediately post thawing can result in poor outcomes due to compromised cell health.
   1. Preinduction seeding: When hPSCs reach approximately 70% confluency, gently dissociate the cells into small clusters using an appropriate dissociation reagent. Resuspend the clusters in 2 mL of E8 medium supplemented with 10 µM ROCK inhibitor (Y-27632).
   2. Aggregation: Seed 200 µL of the cell suspension (typically 12,000-15,000 cells) into each well of a 96-well ultra-low attachment plate and then centrifuge the plate at 200 × g for 3 min at room temperature to facilitate cell aggregation at the bottom of each well.
   3. Incubation: Place the plate in a 37 °C, 5% CO₂ incubator and culture for 24 h to allow formation of compact and uniform embryoid bodies (EBs).
   4. Prepare Basal Culture Medium (BC-M) by combining 50% IMDM, 50% F12 nutrient mixture, 15 µg/mL of Insulin-Transferrin-Selenium (ITS-G), 450 µM of monothioglycerol, and 5 mg/mL bovine serum albumin fraction V.
   5. Prepare MD-M by supplementing BC-M with Activin A (50 ng/mL), BMP4 (10 ng/mL), CHIR99021 (3 µM), FGF2 (30 ng/mL), and LY294002 (5 µM).
   6. Mesodermal Induction: After 24 h of aggregation, induce EBs for 36 h with MD-M.
      NOTE: To ensure the induction efficiency and consistency of cardiac organoids, we strictly limited the use of H9 hESCs to within three passages after thawing. This strategy is critical for maintaining high differentiation efficiency and overall experimental reliability.
      Gentle handling during medium exchange is critical to preserve the integrity of the formed aggregates. Avoid disrupting the spherical morphology of the EBs.
2. Induction of cardiac mesodermal differentiation (Days 1.5-5.5)
   NOTE: The Wnt signaling inhibitor (XAV-939) is critical to suppress posterior mesodermal signals and promote cardiac lineage specification. Retinoic acid plays a dose-sensitive role in anteroposterior patterning; excessive RA may lead to foregut specification.
   1. Supplement BC-M (composition detailed in Step 2.1) with BMP4 (10 ng/mL), FGF2 (10 ng/mL), Retinoic Acid (0.5 µM), and XAV-939 (5 µM) to prepare Cardiac Mesodermal Differentiation Medium (CMD-M).
   2. After 36-40 h of mesodermal induction, carefully aspirate the MD-M and replace with CMD-M for 4 days with medium change every day.
      ​NOTE: Handle aggregates gently during medium changes to avoid mechanical disruption or stress-induced apoptosis.
      ​During this period, spheroids become more compact and organized. Early signs of cardiac lineage commitment can be observed under brightfield microscopy by Day 5.
3. Cardiac organoid specification (Days 5.5-7.5)
   1. Supplement BC-M (composition detailed in Step 2.1) with BMP4 (10 ng/mL), FGF2 (10 ng/mL), and insulin (10 mg/mL) to prepare cardiac organoid specification medium (COS-M).
   2. On Day 5.5, gently aspirate CMD-M and replace with COS-M for 2 days with medium change every day.
      NOTE: BMP4 and FGF2 synergistically promote cardiac progenitor expansion and suppress non-cardiac fates at this stage. Insulin supports metabolic activity and structural development of differentiating cardiomyocytes.
4. Cardiac organoid maturation
   NOTE: Avoid full dissociation or pipetting of spheroids during transfer. Cardiomyocytes are highly sensitive to shear stress.
   1. Supplement BC-M (composition detailed in Step 2.1) with insulin (10 mg/mL) to prepare Cardiac Organoid Maturation Medium (COM-M).
   2. From Day 7.5 onwards, transition to COM-M. Perform medium changes daily to maintain nutrient levels and remove metabolic waste while minimizing stress.
   3. From Day 8 to Day 12, look for spontaneous beating of cardiac organoids, which typically becomes evident under a light microscope.
   4. By Day 12+, confirm that contractile rhythm is regular and sustained.
      NOTE: Beat rate and morphology can be quantified using timelapse microscopy and image analysis tools.
      Electrophysiological analysis (e.g., calcium imaging or MEA) can be performed from Day 12 onwards to assess functional maturity.

3. Preparation for cryopreservation

1. Carefully transfer cardiac organoids from the culture plate using a wide-bore pipette to avoid mechanical stress. Place them in a 15 mL conical tube and allow the organoids to settle naturally by gravity for 3-5 min without centrifugation.
2. Prepare Cryopreservation Medium (Cryo-CM) with the following components (final concentrations): 80% CM-M, 10% FBS, 10% DMSO.
3. Gently resuspend cardiac organoids in 500 µL of Cryo-CM per cryovial.
4. Place cryovials into an isopropanol-based freezing container. Store at -80 °C for 12-24 h to ensure gradual cooling at ~1 °C/min.
5. After overnight freezing, transfer vials into liquid nitrogen storage for long-term preservation.
   NOTE: We cryopreserved the cardiac organoids at a density of approximately 20-30 mature organoids per cryovial, suspended in 1 mL of cryopreservation medium.

4. Cardiac organoid recovery and thawing

1. To recover organoids, quickly thaw vials in a water bath, transfer the cardiac organoids to CM-M medium, gently centrifuge to remove DMSO, and resuspend the organoids in fresh CM-M..
   NOTE: Postthaw beating typically resumes within 24-48 h, depending on the maturity of organoids and handling quality.

Results

The successful differentiation of H9 hESCs into cardiac organoids begins with the robust revival and maintenance of pluripotent stem cells. Upon thawing, hESCs demonstrate high viability and adherence when plated on vitronectin-coated plates in E8 medium supplemented with a ROCK inhibitor. Within 24-48 h, compact colonies with defined edges and high nucleus-to-cytoplasm ratios can be observed, indicating recovery of typical undifferentiated morphology (Figure 1A). After thawing, hESCs rapidl...

Discussion

The successful generation of cardiac organoids from H9 hESCs relies on precise temporal and spatial regulation of key signaling pathways to mimic early cardiac development. This protocol recapitulates the critical stages of mesoderm induction, cardiac lineage specification, and self-organization into 3D contractile structures in a chemically defined system. The method provides a robust and reproducible platform for modeling human heart development, congenital heart disease, and cardiotoxicity screening.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.02% EDTA	Beyotime	C0198
6 well  plate	CORNING	3516
7.5% bovine serum albumin	ACMEC	AC11954
96-well ultra-low attachment plate	CORNING	7007
Alexa Fluor 488 Donkey anti-Mouse IgG	Yeasen	34106ES60
Alexa Fluor 555 Goat anti-rabbit	Invitrogen	A-21428
CHIR-99021 monohydrochloride	Yeasen	52965ES10
DMSO	Solarbio	D8370
Dulbecco Phosphate-Buffered Saline (D-PBS)	Yeasen	60152ES76
FBS	Gibco	10099141C
FGF2 Protein, Human, Recombinant	TargetMol	TMPY-00749-50 μg
Ham's F-12 Nutrient Mix	life technologies	11765054
hPSC-CDM (Essential 8 medium)	Cauliscell	400105
Human BMP-4 Recombinant Protein	Gibco	PHC9534
IMDM, GlutaMAX Supplement	life technologies	31980030
Insulin-Transferrin-Selenium (ITS -G)	Gibco	41400045
LY294002	Selleck	s1105-50mg
Monothioglycerol	Sigma-Aldrich	M6145
Mouse anti-αSMA	Abmart	MN50104
ProLong Diamond Antifade Mountant with DAPI	Invitrogen	P36966
Rabbit anti-CDH5	Abmart	TA6265
Rabbit anti-cTNT	Invitrogen	701620
Recombinant Human BMP-4 Protein	Yeasen	92053ES20
Recombinant Human Insulin	Yeasen	40112ES25
Recombinant Human/Mouse/Rat Activin A Protein	Yeasen	91702ES10
ReLeSR	Stemcell	100-0483
Retinoic Acid	Sigma-Aldrich	R2625
Vitronectin	Gibco	A14700
XAV-939	Selleck	S1180
Y-27632 2HCl (ROCK inhibitor)	Selleck	S1049