biostudies-literatureRossler KJNational Science Foundation Graduate Research Fellowship ProgramNHLBI Division of Intramural ResearchNIH Office of the DirectorNHLBI NIH HHSDepartment of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-MadisonNCI NIH HHSNIGMS NIH HHSNIH HHSe172168https://www.ebi.ac.uk/biostudies/studies/S-EPMC10906451biostudies-literatureUnknown9(1)Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared with magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs affects the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed that lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for 4 weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transient measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates that lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and it suggests that lactate purification does not result in an irreversible change in a hiPSC-CM phenotype.JCI insightLactate- and immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs.PMC10906451R01 HL109810S10OD018475R01 GM125085. DGE-1747503S10 OD018475P30 CA014520S10 OD023526U01 HL134764R01 HL096971Kim GZhang JZhu YAballo TJFarrell ETRalphe JCMann MWBayne EFKamp TJRossler KJGe Yde Lange WJMelby JAfalseLactate- and immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs.Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared with magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs affects the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed that lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for 4 weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transient measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates that lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and it suggests that lactate purification does not result in an irreversible change in a hiPSC-CM phenotype.2024-01-01T00:00:00Z2024 Jan2025-04-04T20:40:53.7Z2025-04-04T20:40:53.7ZS-EPMC109064513798817010.1172/jci.insight.172168