Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes

N Chirico, E L Kessler, RGC Maas, J Fang, J Qin, I Dokter, S Ciccone, T Saric, JW Buikema, Z Lei, P Doevendans, JPG Sluijter, A Van Mil

Published: 27 December 2022


Funding Acknowledgements

Type of funding sources: Other. Main funding source(s): Gravitation Program “Materials Driven Regeneration” by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013) and the Marie Skłodowska-Curie Actions (Grant agreement RESCUE #801540). The EU-funded project BRAV3 (H2020, ID:874827)


Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature human primary cardiomyocytes: the ability to use fatty acids as an energy source, high mitochondrial mass, increased nuclei polyploidism, synchronized electrical conduction, and forceful contractions. Instead, their phenotype is similar to immature cardiomyocytes in the late fetal stage. This immaturity represents a bottleneck to their application in 1) disease modeling – as most cardiac (genetic) diseases have a middle-age onset – and 2) clinical use, where integration and functional coupling are key. So far, the mainly used methods to enhance iPSC-CM maturation include prolonged time-in-culture, 3D culture, cyclic mechanical stretch, and electrical stimulation with specialized media. However, these protocols are laborious, costly, and not easily scalable.


In this study, we developed a simple, low cost, and rapid protocol using two peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A/PGC-1α) activating small molecules: Asiatic Acid (AA) and GW501516 (GW) to promote cardiomyocyte maturity by inducing a metabolic switch to fatty acid utilization and increased mitochondrial biogenesis.


Monolayers of iPSC-CMs were incubated with AA and GW every other day for 10 days resulting in increased expression of fatty acid-metabolism-related genes (5 and 10-fold increase in CPT1B gene expression, respectively), mitochondria biogenesis (protein expression of ATP5A) and fusion (50 and 100-fold increase in OPA1 gene expression, respectively). In addition, AA treated iPSC-CMs responded in the seahorse mitochondria stress test more rapidly to an artificial increase in mitochondrial activity and showed a higher flexibility in substrate utilization in the seahorse stress test. A more mature electrophysiological functionality was shown by increased ion channel gene expression (KCNA4, SCN5A, GJA1, CACNA1C, and SCN1B) and enhanced synchronous contraction in treated samples. Moreover, maturation was further shown by increased sarcomeric gene expression (5 and 7-fold increase in TNNI3 in AA and GW respectively) and nuclear polyploidism (>4N fold 2.16 and 1.48-fold increase in AA and GW respectively).


Collectively, these findings show that AA and GW trigger a metabolic switch and induce extensive maturation of iPSC-CMs, providing a rapid and cost-effective method to obtain iPSC-CMs that more closely resemble their adult counterparts.

Full Access Link: Stem cell research & therapy