Culture in Glucose-Depleted Medium Supplemented with Fatty Acid and 3,3′,5-Triiodo-l-Thyronine Facilitates Purification and Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

With recent advances in stem cell technology, it is becoming efficient to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, which can subsequently be used for myriad purposes, ranging from interrogating mechanisms of cardiovascular disease, developing novel cellular therapeutic approaches, as well as assessing the cardiac safety profile of compounds. However, the relative inability to acquire abundant pure and mature cardiomyocytes still hinders these applications. Recently, it was reported that glucose-depleted culture medium supplemented with lactate can facilitate purification of hPSC-derived cardiomyocytes. Here, we report that fatty acid as a lactate replacement has not only a similar purification effect but also improves the electrophysiological characteristics of hPSC-derived cardiomyocytes. Glucose-depleted culture medium supplemented with fatty acid and 3,3′,5-Triiodo-l-thyronine (T3) was used during enrichment of hPSC-derived cardiomyocytes. Compared to untreated control cells, the treated cardiomyocytes exhibited enhanced action potential (AP) maximum upstroke velocity (as shown by a significant increase in dV/dtmax), action potential amplitude, as well as AP duration at 50% (APD50) and 90% (APD90) of repolarization. The treated cardiomyocytes displayed higher sensitivity to isoproterenol, more organized sarcomeric structures, and lower proliferative activity. Expression profiling showed that various ion channel and cardiac-specific genes were elevated as well. Our results suggest that the use of fatty acid and T3 can facilitate purification and maturation of hPSC-derived cardiomyocytes.

A Comparative Assessment of Human and Chimpanzee iPSC-derived Cardiomyocytes with Primary Heart Tissues

“…All of the cardiomyocyte cultures started beating 6–8 days post induction of differentiation. On days 20–27, a subset of cultures was treated with T3 (methods), which was shown to aid in the maturation of iPSC-derived cardiomyocytes 20 , 24 , 26 . We assessed the purity of the iPSC-derived cardiomyocytes using flow cytometry (Methods) for cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2).…”

ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

“…To assess whether ETV1 has a conserved function in human HPS specification, we investigated whether ectopic ETV1 induction in hiPSC-CMs would convert these cells toward a HPS lineage. Using a protocol previously validated for the generation of purified and mature hiPSC-CMs 71 , we infected hiPSC-CM monolayers with Ad-Etv1-EGFP or Ad-EGFP. Two weeks post-transduction, cells were collected for either RNA or cellular electrophysiological analysis (Fig.…”

“…NCRM1 iPSC line from Codex BioSolutions Inc. (MD, USA) were plated on Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco, A1413202) coated plates and cultured with Essential 8 Medium (Gibco, A1517001). The differentiation and maturation protocol has been previously published 71 . Briefly, day one hiPSC were treated with small molecule CHIR99021 (Tocris, 4423, final concentration 10 μM) in the RPMI-BSA medium [RPMI 1640 Medium (HyClone, SH30027.01) supplemented with 213 μg/ml AA2P (l-ascorbic acid 2-phosphate magnesium) (A8960, Sigma) and 0.1% bovine serum albumin (BSA) (A1470, Sigma)] for 24 h followed by RPMI-BSA medium replacement.…”

Metabolic Regulation of Cardiac Differentiation and Maturation in Pluripotent Stem Cells: A Lesson from Heart Development

“…Furthermore, metabolically selected PSC-derived cardiomyocytes enhance the ratio of OCR/ECAR by 1.8-fold compared to non-purified cardiomyocytes (61) , suggesting that this method forces the metabolic maturation from embryonic to neonatal cardiomyocytes. These metabolic shifts of cardiomyocytes result into more mature action potentials and calcium handling with increased expression of cardiomyocyte-related ion channels and cardiac contraction genes (62) . Researchers reported that activated glycolysis inhibited cardiac maturation through pentose phosphate pathway, which supplies NADPH, nucleotides, and lipids for proliferation.…”