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Table 1 A comparison of embryonic heart organoid protocols and features

From: An evidence appraisal of heart organoids in a dish and commensurability to human heart development in vivo

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]