Differentation and maturation of pluripotent stem cell-derived cardiomyocytes

Abstract

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) may serve as tools in pharmacology for drug screening and safety evaluations and in future cardiac regenerative therapies. However, the use of hPSC-CMs is hampered by their immature foetal-like structural and functional characteristics. This project aimed to evaluate two pro-maturation stimuli to enhance the maturation of hPSC-CMs: administration of triiodothyronine (T3), a hypertrophic stimulus known to be crucial for cardiac development, and overexpression of Bin1(-EGFP), which may be implicated in the formation of t-tubules (TTs). T3-signalling may be suboptimal during in vitro culture and TTs, a hallmark of mature ventricular cardiomyocytes, are absent in hPSC-CMs. To investigate the effect of the pro-maturation stimuli initially cardiomyocytes were derived from the human embryonic stem cell line Shef3 via spontaneous or directed differentiation methods but due to low differentiation efficiency all maturation studies were conducted on iCell-CMs, a commercially produced line of human induced pluripotent stem cell-derived cardiomyocytes. To assess their initial state of maturity and subsequently to evaluate the impact of the pro-maturation stimuli on iCell-CMs the molecular, structural and electrophysiological properties of the cells were extensively characterised using qRT-PCR, immunostaining, bright field and transmission electron microscopy, micro electrode array and the whole-cell patch-clamping technique. Characterisation of iCell-CMs revealed an overall immature phenotype: iCell-CMs were smaller than adult cardiomyocytes; exhibited relatively low expression of key ion channels (Kir2.1 and Kv4.3), major Ca2+ handling components (e.g. RyR2, SERCA2a and CSQ2) and TT-associated proteins (e.g. Bin1 and caveolin 3); showed poor ultrastructural organisation and lacked TTs. However, the sarcoplasmic reticulum (SR) Ca2+ contributed to cell contraction similarly to adult cardiomyocytes. Administration of T3 increased the expression of the hypertrophic marker ANP and enhanced the maturation of iCell-CMs as measured by a shift in the expression levels of SERCA2a, Kv4.3 and Cav3.1 closer to the levels of adult heart controls. Overexpression of Bin1(-EGFP) increased the expression of CSQ2, Kir2.1 and Kv4.3 and induced formation of TT-like sarcolemmal invaginations. However, the tubular structures were not aligned to myofibrils and may have been connected to the SR at only few foci. Localisation of L-type Ca2+ channels on the induced tubules could not be definitely confirmed but the increased Ca2+ current density in transfected cells suggested that the channels may have been recruited to the tubules. Also, overall the expression levels of most analysed genes in T3-treated or Bin1-EGFP-transfected cells remained markedly below adult heart controls including RyR2, CSQ2, Kir.2.1, Kv4.3 and caveolin 3. In conclusion, both T3-treatment and overexpression of Bin1(-EGFP) promoted the maturation of iCell-CMs to some measure but did not induce a fully adult-like phenotype. Thus the ultimate maturation strategy for hPSC-CMs still remains to be found.