Maturation of human pluripotent stem cell-derived cardiomyocytes in three dimensional tissue constructs

Abstract

Cardiomyopathy is the leading cause of morbidity and mortality. Due to variability in disease mechanisms, pharmaceutical responses may vary among patients. Human pluripotent stem cells (hPSC) derived ventricular cardiomyocytes (VCMs) provide a platform for disease modeling, cardiotoxicity screening, drug discovery and cell-based therapies. However, demonstrated by our group previously, hPSC-VCMs display immature phenotypes. Although studies have separately shown that thyroid hormone, (T3), and electro-mechanical conditioning act as developmental cues for the maturation of metabolic, electrophysiological and calcium handling properties, there is no investigation on the effect of the combinatorial treatment. In this dissertation, fabricated into three-dimensional tissue constructs, hPSC-VCMs were treated with both T3 and electro-mechanical conditioning. We found that upon treatment, hPSC-VCMs elicited stronger contractile force, indicating that the combinatorial treatment improved maturation of hPSC-VCMs. For morphological and molecular maturation in hPSC-VCMs, the cellular shape and organization were both studied by using confocal and transmission electron microscopy. The results showed that combinatorial treatment induced cell elongation and enhanced sarcomeric arrangement. Qualitative analysis also illustrated that treated hPSC-VCMs possessed more developed mitochondrial structure and anchoring junctions, which improved the conduction velocity.

The aforementioned improvement in conduction velocity can also be attributed to the maturation in 〖Ca〗^(2+) handling properties. Combinatorial treatment led to up-regulation of the gene expression of crucial 〖Ca〗^(2+) handling proteins, which resulted in better mechanistic for Ca2+ release and Ca2+ homeostasis. Consequently, using optical mapping and calcium sensitive dye, we observed shortened upstroke time in the calcium transient data. Together with the functional data, this indicated that treated hPSC-VCMs had improved excitation-contraction coupling. Furthermore, quantitative reverse transcription polymerase chain reaction analysis showed that the combinatorial approach up-regulated the gene expression of β1-adrenergic receptor, which increased the sensitivity to catecholamine. Testing the dose response curve of hPSC-VCMs to isoproterenol, indeed, treated hPSC-VCMs revealed stronger positive inotropic effect. Pretreating hPSC-VCMs with milrinone, a phosphodiesterase inhibitor, increased number of cyclic adenosine monophosphate amplified the effect of adrenergic agonist. To sum it up, combinatorial treatment 1) improved the contractility of hPSC-VCMs, 2) enhanced the morphological and molecular maturation, 3) developed the 〖Ca〗^(2+) handling properties, and 4) increased the sensitivity of hPSC-VCMs to adrenergic stimulation, which may facilitate clinical and other applications of hPSC-VCMs.