Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs

Abstract

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.

CRISPR/Cas9-mediated introduction of the sodium/iodide symporter gene enables noninvasive in vivo tracking of induced pluripotent stem cell-derived cardiomyocytes

“…Rhesus CD34 + hematopoietic stem and progenitor cells or skin fibroblasts were isolated and reprogrammed as previously described 6,11,12 . RhiPSCs were cultured either on mouse embryonic fibroblast (GlobalStem) feeders or growth factor‐reduced Matrigel (BD Biosciences)‐coated plates.…”

“…Cardiac differentiation of RhiPSCs was performed as previously described 12 . Briefly, RhiPSCs were dissociated and cultured with Activin A (10 ng/mL, R&D), bone morphogenic protein 4 (BMP4; 10 ng/mL, R&D), and fibroblast growth factor 2 (FGF2; 10 ng/mL, Peprotech) for 3 days.…”

“…RhiPSC‐CMs were dissociated and plated onto glass coverslips as single cells as previously described 12 . For action potential (AP) measurements, cells were inserted into a small‐volume chamber and bathed in Tyrode's solution (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 10 mM HEPES, 10 mM glucose, pH 7.4) and maintained at 33°C to 35°C.…”

Combining Optical Approaches with Human Inducible Pluripotent Stem Cells in G Protein-Coupled Receptor Drug Screening and Development

“…Genetically-encoded biosensors offer an attractive alternative, and a toolkit of genetically-encoded calcium and voltage indicators are now available [85]. Rhesus monkey-derived iPSCs stably expressing the calcium indicator GCaMP6 were generated by introducing the calcium sensor into the AAVS1 safe harbor site [86]. These cells were differentiated into cardiomyocytes whereupon stimulation with the βAR agonist isoproterenol increased the frequency of calcium transients which was reduced by a calcium channel blocker, verapamil [86].…”

“…Rhesus monkey-derived iPSCs stably expressing the calcium indicator GCaMP6 were generated by introducing the calcium sensor into the AAVS1 safe harbor site [86]. These cells were differentiated into cardiomyocytes whereupon stimulation with the βAR agonist isoproterenol increased the frequency of calcium transients which was reduced by a calcium channel blocker, verapamil [86]. Evaluating the intracellular signaling properties of iPSC-derived cells has so far not received the same attention but can be addressed using similar techniques.…”