Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells.

Harshal Nemade,Aviseka Acharya,Erastus Nembo,Hinrich Abken,Umesh Chaudhari,Nicole Riet,Jürgen Hescheler,Agapios Sachinidis,Symeon Papadopoulos,Filomain Nguemo

doi:10.3390/cells9030554

Abstract

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.

Highlights

Generation of functional cardiomyocytes (CMs) from human pluripotent stem cells including, human embryonic stem cells and human-induced pluripotent stem cells, offer a relevant tool to study the cardiovascular development, and for disease modelling, toxicity studies, and drug screening [1]
First we investigated the effects of Sulindac on the cardiomyogenesis in four human pluripotent stem cells (hPSCs) lines and second, to ascertain the role of COX-1 and COX-2 in cardiogenesis, we knocked down COX-1 and COX-2 expression in hPSCs either by introducing siRNAs targeted towards COX-1 and/or COX-2 or by treatment with different non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., Piroxicam: COX-1 inhibitor, Nimesulide: COX-2 inhibitor and Diclofenac: Non-selective COX-1 and COX-2 inhibitor)
Further fluorescence analysis showed that inhibition of COX-1/2 results in reduced TCF-LEF promoter activity suggesting reduced Wnt signaling. These findings demonstrate for the first time that (1) Sulindac and other NSAIDs can efficiently differentiate hPSCs into functional

Summary

Introduction

Generation of functional cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) including, human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), offer a relevant tool to study the cardiovascular development, and for disease modelling, toxicity studies, and drug screening [1]. For an in vitro cardiotoxicity screening, it is controversially discussed whether both hESCs and hiPSC-derived CMs are suitable [2,3,4,5,6,7]. Recent advancements in differentiation techniques allowed the generation of functional CMs from hPSCs, demonstrated by their ability to express CM-specific markers that are considered as reliable and renewable cellular source for cardiac regeneration [1,8,9].

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Results

Conclusion