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Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are promising candidates to treat myocardial infarction and other cardiac diseases. Such treatments require pure cardiomyocytes (CMs) in large quantities. Methods : In the present study we describe an improved protocol for production of hiPSC-CMs in which hiPSCs are first converted into mesodermal cells by stimulation of wingless (Wnt) signaling using CHIR99021, which are then further differentiated into CM progenitors by simultaneous inhibition of porcupine and tankyrase pathways using IWP2 and XAV939 under continuous supplementation of ascorbate during the entire differentiation procedure. Results : The protocol resulted in reproducible generation of &gt;90% cardiac troponin T (TNNT2)-positive cells containing highly organized sarcomeres. In 2D monolayer cultures CM yields amounted to 0.5 million cells per cm 2 growth area, and on average 72 million cells per 100 mL bioreactor suspension culture without continuous perfusion. The differentiation efficiency was hardly affected by the initial seeding density of undifferentiated hiPSCs. Furthermore, batch-to-batch variations were reduced by combinatorial use of ascorbate, IWP2, and XAV939. Conclusion : Combined inhibition of porcupine and tankyrase sub-pathways of Wnt signaling and continuous ascorbate supplementation, enable robust and efficient production of hiPSC-CMs.

theranostics

Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations