The University of Minnesota Human Subjects Research Institutional Review Board approved all protocols related to cell line establishment in this study. The hiPSCs used in this study were generated from cardiac fibroblasts as previously reported (Zhang et al., 2018) and maintained on Geltrex-coated (Gibco) 6-well plates in mTesR Plus medium (STEMCell Technologies, Canada) with daily medium changes until 90–100% confluent and then prepared for the differentiation protocol over 7 days (i.e., beginning on day-7). The cells were washed once with Dulbecco’s Phosphate-Buffered Saline (DPBS) and incubated with 0.5 mL Gentle Cell Dissociation Reagent (GCDR; STEMCell Technologies) for 6 min at 37°C; then, the GCDR was aspirated, and 1 mL TeSR E8 3D medium (STEMCell Technologies) supplemented with 10 μM Y-27632 (BD Biosciences Cat# 562822, RRID:AB_2869435) was gently pipetted into each well to dislodge the cells and disaggregate them into small clumps. Cells from all 6 wells per plate were collected, suspended in 40 mL of TeSR E8 3D Seed medium (STEMCell Technologies), placed in a 125-mL shaker flask (Fisher Scientific), and cultured on a Belly Dancer Shaker (IBI Scientific) at 70 rpm with 5% CO₂ at 37°C. On days-6 and 5, 1.2 mL TeSR E8 3D Feed medium (STEMCell Technologies) was added to the culture flasks, and the cells were passaged on day-4. Passaging was performed by disaggregating the cells into smaller clumps and transferring them into a final volume of 80 mL TeSR E8 3D Seed medium; then, the cells were cultured at 50 rpm with daily additions of 2.4 mL TeSR E8 3D Feed medium until day-1, when half of the culture medium was replaced with fresh TeSR E8 3D Seed medium.

Differentiation was initiated on Day 0, and the protocol was optimized by varying the initial cell density (0.26, 0.67, 1.1, 1.6, 2.1, and 5.1 10⁶ cell/mL), CHIR99021 concentration (4, 5, 6, 7, 8, and 9 μM), and shaking speed (0, 20, 55, and 75 rpm). Briefly, 1 mL of the hiPSC-spheroid suspension was collected; then, the cells were dissociated with GCDR and counted to calculate the cell density. The remaining spheroids were washed through a 500 μm filter and collected on a reversible 40 μm filter (pluriSelect) to establish a homogenous population prior to differentiation. The spheroids were washed out with RPMI 1640 supplemented with 1 B27 without insulin (RPMI/B27–) and CHIR99021 with a final volume of 2.5 mL in low attachment 6 well plates (Corning) for optimization and 30 mL in 125 mL flasks (Thermo Fisher Scientific) for subsequent experiments. Twenty-four hours later (i.e., on day 1) the medium was replaced with fresh RPMI/B27–, and the culture volume was increased by 20% and maintained at 1.2 the initial volume for all subsequent medium changes. On day 3, half the medium was replaced with RPMI/B27– containing 10 μM IWR-1, and on day 5, the medium was completely refreshed with RPMI/B27–. On day 7, the medium was completely changed to RPMI 1640 supplemented with B27 with insulin (RPMI/B27+), and the cells were cultured for two more days until day 9, when the differentiated hiPSC-CMs cells were purified. Purification was performed via metabolic selection: the medium was completely changed to glucose-free RPMI 1640 supplemented with B27 with insulin and 0.12% sodium DL-lactate, and the cells were cultured for 72 h until day 12, when the media was changed back to RPMI/B27+. The purified hiPSC-CMs were maintained in RPMI/B27+ with partial medium changes every 3 days.

Cells were dissociated into single cells via treatment with cardiomyocyte dissociation media (CMDM, STEMCell Technologies) for 10–20 min at 37°C, resuspended in cardiomyocyte support media (STEMCell Technologies), counted, centrifuged at 300 g for 3 min, washed with DPBS, fixed in 4% paraformaldehyde (PFA) for 20 min, permeabilized with 0.1% Triton-X, blocked with 4% bovine serum albumin (BSA) in 4% fetal bovine serum (FBS), stained with Zenon conjugated anti-Troponin T antibody or primary antibody (Supplementary Table 1), incubated for 60 min, and washed with DPBS. Around 5 × 10⁵ cells were used per sample with spheroids from individual batches analyzed as a single sample. Analysis on an Attune NxT Flow Cytometer (Thermo Fisher) used lasers FSC, SSC, and BL1 with voltages of 80, 310, and 260, respectively. FlowJo (FlowJo, RRID:SCR_008520) was used to gate the single cell population and a threshold was set at 1.3 × 10³ volts for cardiac troponin T positivity with consistent gating used across samples. Each marker was examined in at least 4 independent batches of cells.

Cells were lysed with TRIZOL (Thermo Fisher), and lysates were homogenized by repeatedly drawing/expelling them into/from a pipette; then, the RNA was purified in Direct-zol^TM RNA MiniPrep Plus columns (Zymo Research) and treated with DNAase I. Reverse transcription was performed with SuperScript^TM IV VILO^TM Master Mix (Thermo Fisher) as directed by the manufacturer’s protocol, and samples (5 ng with 500 nM primers; Supplementary Table 2) were analyzed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) with PowerUp SYBR Green Master Mix (Applied Biosystems). Measurements were quantified via normalization to measurements of glyceraldehyde phosphate dehydrogenase (GAPDH) RNA abundance in the same sample. Each marker was examined in at least 4 independent batches of cells with GAPDH replicates used to account for measurement error.

Protein lysates were collected by treating cells with RIPA buffer (Thermo Fisher Scientific) supplemented with HALT Proteinase Inhibitor (Thermo Fisher Scientific) and homogenized via pipetting. Total protein concentrations were calculated via BCA assay (Fisher Scientific); then, 6 μg of each sample was loaded onto a 4–20% Mini-PROTEAN^® TGX^TM Precast Protein Gel (Biorad) and run at 100 V for 1 h. Samples were transferred to a nitrocellulose membrane by using the Trans-Blot Turbo System (Biorad), blocked in 5% milk, and then incubated with primary antibody (Supplementary Table 1) overnight at 4°C and with secondary antibody for 1 h at room temperature. ECL Chemiluminescent Reagent (GE Healthcare Amersham) was applied to the membrane for 5 min, and then the membrane was exposed on the ChemiDoc Touch Imaging System (Biorad). Each marker was examined in at least 4 independent batches of cells.

Samples were fixed in 4% formaldehyde (Pierce, Thermo Fisher Scientific, # 28906) for 1 h and then embedded in either optimal cutting temperature (OCT) compound (Fisher Health Care, United States) or paraffin for histological analysis.

Sections (10-μm) were deparaffinized, rehydrated, stained in hematoxylin (Mayer’s, Merck, 3 min) and eosin Y (2 min) solution, dehydrated, mounted in Permount, and imaged with a bright field microscope (Olympus IX83 epifluorescent microscope). Histological sections were analyzed by a non-blinded clinical cardiac pathologist with expertise in assessing for morphological irregularities and necrotic tissue who was asked to determine the heterogeneity of the cardiac spheroids as well as examine for apoptotic or necrotic regions. Heterogeneity was defined as structural and size differences between cells in different regions of the spheroid.

OCT-embedded sections (10-μm) and chamber slides containing live cells were fixed for 20 min in 4% PFA; blocked and permeabilized for 30 min in 10% donkey serum, 10% Tween20, 3% BSA, and 0.05% Triton-X; incubated with primary antibodies (Supplementary Table 1) for 1 h at room temperature; washed with PBS (3 washes, 5 min per wash), incubated with fluorescent (4′, 6-diamidino-2-phenylindole [DAPI]) secondary antibodies for 1 h at room temperature; mounted in VECTASHIELD hardset Antifade Mounting Medium; and visualized via confocal laser scanning (Olympus FV3000 confocal microscope). Stains were assessed through visual inspection of at least 4 different spheroids from multiple batches and where used, quantification of TUNEL positive cells was determined via manual counting of all nuclei in a single spheroid cross section.

Spheroid and monolayer CM cells were dissociated, replated on 0.4-μm pore Transwell Polycarbonate Membranes, and cultured for 7 days; then, the membranes were fixed in 2.5% glutaraldehyde solution for 1 h at 4°C and delivered to the UAB High-Resolution Imaging Facility. Sample blocks were sectioned along the width of the transwells with a diamond knife, and samples were mounted and viewed with a Tecnai Spirit T12 Transmission Electron Microscope. Both monolayer and suspension culture groups consisted of 4 transwells each, with at least 3 subsequent samples per transwell sectioned and imaged. Each group (monolayer vs. suspension) consisted of at least 12 images each. Sarcomere lengths were determined using ImageJ with the line measure tool and all sarcomeres in an image were measured.

Data are presented as mean ± SEM, and significance was evaluated via the Student’s t-test or analysis of variance (ANOVA). Analyses were performed with GraphPad Prism8 software (GraphPad Prism, RRID:SCR_002798), and p < 0.05 was considered significant.

hiPSCs were cultured in 6-well plates until 90–100% confluent and then in suspension for 7 days before differentiation was induced by culturing the cells in CHIR99021-containing medium for 24 h beginning on Day 0 and then in IWR1-containing medium for 48 h beginning on day 3 (Figure 1A). The differentiation protocol was conducted in low attachment 6 well plates with rotational shaking, and the protocol was optimized by varying either the cell density (0.26, 0.67, 1.1, 1.6, 2.1, and 5.1 10⁶ cell/mL), CHIR99021 concentration (4, 5, 6, 7, 8, and 9 μM), or shaking speed (0, 20, 55, and 75 rpm) while holding the other 2 parameters constant. Differentiation efficiency was determined on day 9 via flow cytometry measurements of cTnT expression; optimal results for both the purity (Figure 1B) and yield (Figure 1C) of cTNT-positive cells was achieved with an initial cell density of 1.6 10⁶ cells/mL (purity: 92.9 ± 1.8%, yield: 5.08 ± 0.42 10⁶ hiPSC-CMs), 6 μM CHIR99021 (83.1 ± 4.4%, 4.88 ± 0.96 10⁶ hiPSC-CMs), and 55 rpm shaking (92.2 ± 1.2%, 4.36 ± 0.32 10⁶ hiPSC-CMs).

Shaker flask suspension culture of iPSC aggregates results in a wide range of spheroid sizes and culture heterogeneity (Otsuji et al., 2014). Therefore, prior to beginning differentiation spheroids were passed through a 500 μm filter and collected on a 40 μm filter to remove large aggregates and small debris. When differentiated under optimized conditions, spheroid sizes remained largely stable: mean diameter was 242.4 ± 3.9 μm before differentiation was initiated, 231.4 ± 4.5 μm on day 12 after 30 days of purification via glucose starvation, and 255.2 ± 4.2 μm on day 30 (Figure 2A). Compared with monolayer differentiation (1.08 ± 0.22 10⁶ CM/mL), the suspension protocol produced greater yields of cells (1.47 ± 0.18 10⁶ CM/mL), however, the difference did not reach statistical significance (p = 0.203) (Figure 2B). The most conspicuous changes in spheroid morphology were observed on days 1 and 5, after completion of the 24-h CHIR99021 and 48-h IWR1 culture periods, respectively (Figure 2C). Histological (Figure 2D) and immunofluorescent (Figure 2E) analyses conducted on day 12 indicated that the suspension-differentiated spheroids were composed of homogeneous hiPSC-CM populations that were morphologically similar to fetal cardiomyocytes; displayed no evidence of glandular or other cell populations and no large structural irregularities; consistently expressed cardiac troponin T (cTnT) and alpha-actinin; and had moderately aligned, striated fibers with uniformly distributed spherical nuclei.

One key concern for spheroid culture is whether cells located in the interior of the spheroid are adequately exposed to nutrients and differentiation factors present in the media; thus, spheroid sections were stained for the expression of phosphorylated mixed lineage kinase domain-like protein (pMLKL) (Linkermann et al., 2014; Negroni et al., 2017) and via terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to identify necrotic and apoptotic cells, respectively. None of the sections contained pMLKL-positive nuclei (Figure 3A) and although 7.7 ± 4.3% of cells were TUNEL-positive (Figure 3B) they were evenly distributed throughout the sections rather than localized in the core, which suggests that apoptosis was not caused by lack of access to nutrients in the media. Markers associated with calcium handling and maturation (SERCA, Cx43, JPH2, and MLC2v) were also uniformly expressed throughout spheroid sections and in cardiomyocytes from dissociated spheroids (Figures 3C–G) thus, differentiation appeared to be equally efficient throughout the entire volume of the spheroid, including the spheroid interior. Expression of the atrial isoform marker MYL7, however, was noted to appear higher at the edges of the spheroid than in the internal region. Although initially attributed to greater cell density this observation also presents the possibility that subpopulations of more atrial and ventricular cells may be localized to different regions of the spheroid. Further examination using techniques such as single cell sequencing could help elucidate these differences but were outside the scope of this work.

Flow cytometry assessments conducted with cells collected on day 12 indicated that cTnT was expressed by a considerably greater proportion of hiPSC-CMs when the cells were differentiated via the suspension protocol (98.2 ± 0.8%) than in monolayers (89.2 ± 4.8%) (Figure 4A) and while expression of the pluripotency markers SOX2, SSEA4, and Tra-1–60 was measurable in suspension-differentiated hiPSC-CMs (SOX2: 0.76 ± 0.18, SSEA4: 2.01 ± 0.22, Tra-1–60 0.75 ± 0.16), the proportion remained at or below that of monolayer-differentiated cells (SOX2: 0.26 ± 0.16, SSEA4: 2.83 ± 0.98, Tra-1–60 2.47 ± 0.74) (Figure 4B). RT-qPCR assessments of mRNA levels indicated that pluripotency gene expression declined immediately and rapidly after suspension differentiation was initiated but peaked on day 3 in monolayer-differentiated cells, while expression of the mesoderm genes Brachyury and MESP peaked on days 1 and 3 (respectively), and cardiac gene expression (Gata4, Mef2c, Nkx2–5, and α-MHC) peaked on day 7, with both protocols (Figure 4C). Peak levels of mesoderm-gene expression in suspension- and monolayer-differentiated cells were similar, but cells differentiated via the suspension protocol tended to express higher levels of cardiac genes, and the monolayer protocol was associated with greater variability between samples for all lineage markers (i.e., pluripotency, mesodermal, and cardiac). Notably, when hiPSC-CMs were imaged via TEM, measurements of mean sarcomere length were significantly greater in suspension-differentiated (1.660 ± 0.155 μm) than in monolayer-differentiated (1.406 ± 0.125 μm) cells (Figure 4D).

The biological processes associated with cardiomyocyte maturation include changes in the composition of the sarcomere, as well as in the expression of genes involved in cellular metabolism, structural organization, and electrophysiology (e.g., calcium handling). Thus, we evaluated the maturity of suspension- and monolayer-differentiated hiPSC-CMs by conducting RT-qPCR assessments of mRNA abundance for individual genes, or the ratio of mRNA abundance for pairs of genes, that typically increase (Beta-MHC, Beta/Alpha-MHC, MLC-2v, MLC-2/2a, TNNI3, TNNI3/1) or decline (Alpha-MHC, MLC-2a, TNNI1) as cardiomyocytes mature (Guo and Pu, 2020). Assessments conducted in cells collected on day 12 indicated that the ratio of βMHC-to-αMHC expression (Mahdavi et al., 1984; Reiser et al., 2001; Yang et al., 2014; LaBarge et al., 2019), as well as both TNNI3 mRNA levels and the TNNI3-to-TNNI1 ratio (Bedada et al., 2014) were greater in suspension-differentiated hiPSC-CMs than in hiPSC-CMs that were differentiated in monolayers (Figure 5A). Furthermore, the same three parameters, as well as MLC2v mRNA levels, the MCL2v-to-MCL2a ratio, and the abundance of CKMT2, LAMA2, PLN, Cx43, NCX1, and Calsequestrin mRNA (Kubalak et al., 1994; Uosaki et al., 2015), increased substantially from days 12 to 30 in suspension-differentiated hiPSC-CMs. Notably, measurements for genes involved in organizational structure (FN1, Col3A1, and ELN) tended to vary more between samples from monolayer-differentiated than suspension-differentiated cells, and the results from Western-blot assessments of protein levels for a subset of key genes were consistent with mRNA measurements (Figure 5B) day 12 measurements in suspension- and monolayer-differentiated cells were similar, while both MLC2v protein levels and the ratio of MCL2v-to-MLC2a protein abundance increased from days 12 to 30. Collectively, these observations confirm that hiPSC-CMs were no less mature when differentiated in suspension than in monolayers.