Organoid protocol | WNT activators/growth factors | Differentiation/Maintenance period | Cell population markers | Embryonic morphology | Cardiac maturation features | References |
---|---|---|---|---|---|---|
Cardioids | 8–30 ng/mL FGF2, 5 µM LY294002 (PI3K inhibitor), 50 ng/mL activin A, 10 ng/mL BMP4, 10 µg/mL insulin, 4–9 µM CHIR99021 and 5 µM IWP2 | 7.5 days/3 months | FHF markers (HAND1, TBX5, NKX2-5), ventricular (IRX4, MYL2), atrial (NR2F2, HEY1); Intermediate WNT inhibition + VEGF resulted in 41% atrial (MYL7) and 53% endothelial (CDH5) cell fractions and EC-derived fibroblasts (SOX9, MSX1/2, COL1A1, and COL3A1) | Concentric layers of epicardium, myocardium and endothelium lined cavity | β-adrenergic receptors 1 and 2, higher expression of HERG, KCNH2, TNNI1, TTN, MYH6, RYR2 and ATP2A2 | [16] |
Gastruloids | 3 µM Chi99021, 30 ng/mL bFGF, 5 ng/mL VEGF | 7 days | Mesp1, FHF markers (NKX2-5, TBX5, and HCN4), SHF markers (TBX1, ISL1, and FGF10), Endothelial (FLK1) and Cardiac (cTnT) | Cardiac crescent-like domain, endocardial-like layer, gut-like tube | ACTN2 and RYR2 | [19] |
Heart forming organoids | 7.5 µM CHIR99021, 5 µM IWP2 | 10 days/4.7 months | Cardiac (NKX2-5, TNNT2), Endothelial (CD31, CDH5), Lung (SOX2, SOX17,) Pharyngeal endoderm (ISL1, PAX9, TBX1), Liver (HNF4α, AFP) | Cardiac mesoderm, proepicardium | – | [20] |
Heart organoids | 2–4 µM CHIR99021, 1.25 ng/mL BMP4, 1 ng/mL activin A, 2 µM C59 | 15 days | FHF (HAND1), SHF (HAND2), epicardial marker (WT1, TJP1); 59% cardiac (TNNT2), 16%, 14% endocardial (NFATC1), 12% fibroblast (THY1), 1.6% endothelial (PECAM1). ~ 48% atrial (MYL7), ~ 18% ventricular (MYL2) | Epicardium, endocardium, myocardium and multiple chambers | – | [21] |
Cardiac and gut (multilineage) organoid | 12 µM CHIR99021, 5 µM IWP2,10 µM ROCKi | 30 days/11.8 months | Day 20 Cardiac (cTnT), ventricular (MLC2v), endothelial (PECAM1), epicardial (TBX18); Day 30 hindgut (FOXA2, CDX2); Day 60 smooth muscle (aSMA) 80% atrial/nodal:20% ventricular, Day 70–100 paneth, goblet cells and > 90% atrial/nodal cells | Gut endoderm, subepicardium, and cardiac mesoderm | High amplitude and stroke velocity at 1 Hz with response to stimulation at 8 Hz after 80 days, > 70% cells with aligned sarcomeres with ~ 4 aspect ratio | [23] |