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.

Keywords: Cardiology; Cardiovascular disease; Stem cells; iPS cells.

Figure 1. Purification of 2D hiPSC-CMs for 3D hiPSC-ECT generation.

(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.

Figure 2. Global proteomics analysis of hiPSC-CMs after purification.

(A) Unique protein identifications per hiPSC-ECT group. (B) Principal component analysis (PCA) of sample log₂ protein abundances illustrates clear separation between purification techniques. (C) Volcano plot illustrating differential fold-change in protein expression between lactate and MACS purification. The number of significantly upregulated proteins is shown in the top outside corners of the plot (P_adj ≤ 0.05 and |log₂ fold change| ≥ 0.6 required to be considered significant). Some significant proteins are indicated by name on the plot. (D) Pie chart for visual representation of differentially expressed proteins. (E) Hierarchal unbiased dendrogram clustering and heatmap depicting normalized intensities of Z scores for the top 50 significantly differently expressed proteins between lactate and MACS purification. (F) UniProt/Reactome pathways with pathways including keywords “muscle”, “cardiomyopathy”, “reactive oxygen species”, “glycolysis”, “beta-oxidation”, “microtubule”, “hypoxia”, “senescence”, “TGFβ”, “adrenaline”, “trafficking”, “hypertrophy,” and “mitochondrial”. Points indicate individual proteins identified in the pathway, and the box plots indicate average expression change of those proteins. Significantly differentially expressed proteins and pathways are indicated by log₂ fold change greater than 0.6 or less than –0.6 (indicated by dashed gray line). All tests were performed with biological replicates as lactate (n = 5) and MACS (n = 5). Analysis was performed on hiPSC-CM cultures at day 24, with day 0 as the start of hiPSC differentiation. Lactate purification occurred from hiPSC-CM day 17 to day 24. MACS purification occurred at day 24 the day of proteomics analysis.

Figure 3. Sarcomere lengths of hiPSC-ECTs.

(A) Three-dimensional representations of hiPSC-ECTs from lactate and MACS purification methods. Three-dimensional hiPSC-ECT images taken at day 58 prior to assessment with 50× Olympus microscope. (B) α-Actinin and DAPI immunofluorescence labeling on representative lactate and MACS hiPSC-ECTs. (C) Sarcomere length comparison (100 total measurements per condition, P = 0.45) using manual annotation of Z disc length via α-actinin staining. All tests were performed with biological replicates as lactate (n = 3) and MACS (n = 3), unless otherwise stated. All statistical analyses are 2-tailed Student’s t tests with α = 0.05. 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. IHC was performed with hiPSC-ECTs at day 28, with day 0 being the generation of hiPSC-ECTs.

Figure 4. Twitch force assessment of hiPSC-ECTs.

(A and B) Raw twitch force (TF) (A) and normalized averaged TF (NForce) (B) curves for lactate (red) and MACS (blue) hiPSC-ECTs. SEM at 25%, 50%, and 75% are shown for each curve. (C–H) TF parameters for lactate (red) and MACS (blue) hiPSC-ECTs as follows: cross-sectional area (CSA, P = 0.59) (C); automaticity (lactate, n = 11 versus MACS, n = 5;P = 0.68) (D); TF amplitude (P = 0.33) (E); time from pacing stimulus to TF peak (CT₁₀₀, P = 0.91) (F); time from TF peak to 50% twitch force decay (RT₅₀, P = 0.94) (G); and time from 50% to 90% TF decay (RT_50–90; P = 0.97) (H). All tests were performed with biological replicates as lactate (n = 15) and MACS (n = 9), unless otherwise stated. All statistical analyses are 2-tailed Student’s t test with α = 0.05. 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. Functional assays were performed with hiPSC-ECTs from days 26 to 31, with day 0 being the generation of hiPSC-ECTs.

Figure 5. Calcium transients assessment of hiPSC-ECTs.

(A) Normalized averaged calcium transients (NCa²⁺TR) curves for lactate (red) and MACS (blue) hiPSC-ECTs. (B–E) Ca²⁺TR parameters for lactate (red) and MACS (blue) hiPSC-ECTs as follows: Ca²⁺TR peak (dR, P = 0.20) (B); time to Ca²⁺TR peak (CaT₁₀₀, P = 1.0) (C); time from Ca²⁺TR peak to 50% Ca²⁺TR decay (CaDT₅₀, P = 0.66) (D); and time from 50% Ca²⁺TR decay to 75% Ca²⁺TR decay (CaDT_50–75, P = 0.40) (E). All tests were performed with biological replicates as lactate (n = 8) and MACS (n = 6). All statistical analyses are 2-tailed Student’s t tests with α = 0.05. 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. Functional assays were performed with hiPSC-ECTs from days 26 to 31, with day 0 being the generation of hiPSC-ECTs.

Figure 6. Global proteomics analysis of hiPSC-ECTs.

(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 Ca^2+-ATPase2a (SERCA2a), and vimentin (VIM) shown for each replicate. (D) Pie chart for visual representation of differentially expressed proteins (P_adj ≤ 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.

Figure 7. Top-down proteomics of the hiPSC-ECT cardiac sarcomere.

(A) Selected base peak chromatograms for lactate and MACS replicates with identified proteins based on retention time (RT). (B–I) Spectra and isotopic resolution given for cardiac troponin T (cTnT) (B), slow skeletal troponin I (ssTnI) (C), troponin C (TnC) (D), α tropomyosin (α-Tpm) (E), myosin light chain 1v (MLC-1v) (F), myosin light chain 1a (MLC-1a) (G), MLC-1 isoforms (H), and myosin light chain 2v (MLC-2v) (I) with RT shown with extraction ion chromatograms (EICs). (J) Differential quantitation of myofilament proteins (cTnT, P = 0.94; ssTnI, P = 0.63; MLC-1v, P = 0.22; α-Tpm, P = 0.97; MLC-1a, P = 0.46; MLC-2v, P = 0.13; cα-actin, P = 0.62; TnC, P = 0.32). (K) Relative phosphorylation of myofilament proteins (pcTnT, P = 0.60; pα-Tpm, P = 0.10; pMLC-2v, P = 0.86). All tests were performed with biological replicates as lactate (n = 9) and MACS (n = 6), unless otherwise stated. All statistical analyses are 2-tailed Student’s t tests with α = 0.05. 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. Proteomic assays were performed with hiPSC-ECTs from days 20 to 28, with day 0 being the generation of hiPSC-ECTs.