Lactate- and immunomagnetic-purified hiPSC–derived cardiomyocytes generate comparable engineered cardiac tissue constructs
Kalina J. Rossler, Willem J. de Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge
Kalina J. Rossler, Willem J. de Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge
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Resource and Technical Advance Cardiology Stem cells

Lactate- and immunomagnetic-purified hiPSC–derived cardiomyocytes generate comparable engineered cardiac tissue constructs

Abstract

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.

Authors

Kalina J. Rossler, Willem J. de Lange, Morgan W. Mann, Timothy J. Aballo, Jake A. Melby, Jianhua Zhang, Gina Kim, Elizabeth F. Bayne, Yanlong Zhu, Emily T. Farrell, Timothy J. Kamp, J. Carter Ralphe, Ying Ge

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Figure 6

Global proteomics analysis of hiPSC-ECTs.

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Global proteomics analysis of hiPSC-ECTs. (A) Unique protein identificat...
(A) Unique protein identifications per hiPSC-ECT group. (B) Pearson correlation of hiPSC-ECT replicates with unbiased dendrogram clustering. (C) Heatmap of overall protein expression with expression of cardiac troponin I (cTnI), phospholamban (PLN), cardiac sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a), and vimentin (VIM) shown for each replicate. (D) Pie chart for visual representation of differentially expressed proteins (Padj ≤ 0.05 and |log2 fold change| ≥ 0.6 required to be considered significant). (E) Log2 fold intensity values plotted for cTnI, PLN, SERCA2a, and VIM, as proteins of interest (cTnI, P = 0.15; PLN, P = 0.73; SERCA2a, P = 0.44; VIM, P = 0.61). All tests were performed with biological replicates as lactate (n = 7) and MACS (n = 5). All statistical analyses are 2-tailed Student’s t tests with α = 0.055. Plots show whiskers from 0 to 25th percentile, box from 25th to 75th percentile (mean indicated with line), and whiskers from 75th to 100th percentile. Proteomics assays were performed with hiPSC-ECTs from days 26 to 29, with day 0 being the generation of hiPSC-ECTs.