Table 2.

Efforts to promote hiPSC-CM maturation by 3D tissue engineering

Reference		Huang et al.²⁶	Ronaldson-Bouchard et al.^{4, 27}	Shadrin et al.²⁸	Mills et al.²⁹	Ruan et al.³⁰	Hirt et al., Mannhardt et al., Lemoine et al.^31–33	Nunes et al.³⁴
Engineered tissue size and treatments		0.5 mm × 0.2 mm, T3 + Dex + IGF1 for 1 wk	6 mm × 2 mm, early ramped field stim. 2–6 Hz	7 mm × 7 mm, RPMI + B27 + insulin for 1wk, 5% FBS for 2 wks	1 mm × 0.5 mm, low glucose, high palmitate, no insulin	20 mm × 0.3 mm, static stress for 2 wks + electrical stim. for 1 wk	8 mm × 0.2–1.3 mm, ± pacing	~600 μm wide gel on inelastic silk core; ramped field stim. 1–6 Hz
Myofibril assembly	isoform switching	↑MYH6, ↑MLC2v; ↓MYH7, ↓MLC2a, ↓ TNNI1	↑MYH7, ↑TNNI3	↑MLC2V, ↑TNNI3, ↑MYH7; ↓MLC2a	↑MLC2v; ↑TTN N2B, ↑MYH7/6, ↑TNNI3/1	not described	MLC2v detectable	↓MYH6
	Sarcomere organization	Orderly register of A-bands, I-bands, H-zone and Z-lines; no M-lines.	Orderly register of A-bands, I-bands, Z-lines and M-lines.	Orderly register of A-bands, I-bands, H-zone and Z-lines; no M-lines.	Clear Z-lines, I-bands and A-bands; no M-line	Improved; lack detailed analysis of TEM	Regular Z-lines; inconsistent I- and A-bands; no M-line	Regular Z-lines; I-band and H-zone detectable; no M-line
	sarcomere length	2 μm	2.2 μm	2.1 μm	2.3 μm	not described	1.6 μm	not described
Electrophysiology and Ca²⁺ handling	expression of channels & regulators	↑KCNJ2, ↑RYR2, ↑SERCA, ↑NCX1	↑RYR2, ↑SERCA; ↓HCN4	↑CASQ2, ↑S100A1	not described	↑SERCA,↑RYR2	no detectable changes	↑KCNJ2
	T-tubule	adjacent to sarcomeres; unclear alignment	well developed and aligned	not detectable	adjacent to sarcomeres; unclear alignment	not detectable	not detectable	not detectable
	resting Vm	not quantified	–70 mV	–71 mV	–60 mV	nd	–73.5 mV	–80 mV
	Max dV/dt	not quantified	23 V/s	38 V/s	148 V/s	nd	219 V/s	125 V/s
	APD	APD80 1000 ms at 0.5 Hz pacing	APD90 500 ms	ADP80 450 ms	APD90 110 ms, APD50 60 ms	nd	nd	APD90 120 ms
	AP “notch”	not detectable	Yes	not described	Yes	nd	Yes	nd
	Ca²⁺ transient	enhanced	enhanced	visible	enhanced	nd	nd	nd
	Ca²⁺ storage & SR release	nd	enhanced	nd	nd	nd	nd	enhanced
metabolism	metabolic gene expr.	↑PPARA, ↑PGC1a	↑TFAM, ↑PGC1a	↑COX6A2, ↑CKMT2, ↑CKM	↑redox and FAO genes	nd	nd	nd
	mitochondria amount	increase by TEM	increase by TEM	increase by TEM	mtDNA increase	nd	nd	nd
	mitochondria alignment	close to sarcomeres	close to sarcomeres	nd	close to sarcomeres	nd	nd	close to sarcomeres
	mitochondria cristae	nd	well developed	well developed	nd	nd	immature	nd
	mitochondria functions	nd	OCR and ECAR increased	nd	Incr. maximal OCR and OCR reserve	nd	nd	nd
Proliferation & hypertrophy	cell-cycle gene expr.	nd	nd	nd	cell cycle gene downregulation	nd	nd	nd
	proliferation rate	nd	nd	decrease	decrease	nd	nd	decrease
	CM size	Incr. to 735 μm²	Incr. to 1500 μm²	nd	nd	Incr. to 795 μm²	nd	Incr. to 917 μm²
Tissue integration and physiology	ICD	ICD on TEM, Cx43 at cell poles	ICD on TEM, Cx43 at cell poles	NCad at cell poles; Cx43 mislocalized	ICD on TEM; Cx43 and NCad mislocalized	Primitive ICD on TEM	Cx43 mislocalized,	nascent ICD
	contractility	2.1–4.4 mN/mm²	3 mN/mm²	23 mN/mm²	0.3 mN	1.3 mN/mm²	up to 0.15 mN	nd
	Frank-Starling relationship	nd	nd	detectable	nd	detectable	detectable	nd
	Force-freq. relationship	flat	positive	flat or slightly negative	nd	positive	flat	nd
	Response to β-agonists	Incr. contraction rate & amplitude	Incr. contraction rate & amplitude	nd	Incr. contraction rate & amplitude	Incr. contraction rate but not force amplitude	Incr. force amplitude; rate not described	Incr. rate, force not described
	Post-pause potentiation	nd	present	nd	nd	nd	present	nd
	Conduction vel. (cm/s)	up to 40	25	25.1	nd	2.76	nd	15
	inotropic response to extracellular Ca²⁺ (EC₅₀)	nd	~0.4 mM	nd	1 mM	nd	0.6 mM	nd

Abbreviations: Dex, dexamethasone; stim, stimulation; Vm, membrane voltage, APD, action potential duration; AP, action potential; nd, not described; ICD, intercalated disc; TEM, transmission electron microscopy; NCad, N-cadherin