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 1

Purification of 2D hiPSC-CMs for 3D hiPSC-ECT generation.

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Purification of 2D hiPSC-CMs for 3D hiPSC-ECT generation. (A) Timeline f...
(A) Timeline for the generation of hiPSC-CMs and hiPSC-ECTs. (B) Efficiency of purification methods are given by percentage of pure hiPSC-CMs recovered from successful differentiation batches. Viable differentiation batches were visually evaluated as viable with observance of greater than 80% of cells contracting in the well. Percentage recovery was calculated as follows: Percentage recovery = (Count of cTnT+ cells after purification process/Count of total cells before purification process) × 100. Lactate differentiation was significantly more successful in pure hiPSC-CM recovery in comparison with MACS (lactate = 82.2% ± 2.86 %, versus MACS = 60.9% ± 8.73 %; P = 0.0035). All tests were performed with 4 separate differentiation batches split to purification process. Lactate purification occurred from hiPSC-CM day 17 to day 24, with day 0 as the start of hiPSC differentiation. MACS purification occurred at day 30 before generation of hiPSC-ECTs. Statistical analysis involved 2-tailed Student’s t test with α = 0.05. (C) Flow cytometry with cTnT labeling demonstrates effective hiPSC-CM enrichment using each purification method. Representative experiment performed once. (D) Two-dimensional representations of hiPSC-CMs from lactate and MACS purification methods. Two-dimensional hiPSC-CM images were taken on the day of hiPSC-ECT generation with 400× Olympus microscope. Representative experiment performed once.