Advanced physiological maturation of human iPSC-derived cardiomyocytes using an algorithm-directed optimization of defined media components

Domain: biology
Published: 2026-03-31
Editorial outcome: minor_revision

Abstract
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold tremendous promise for in vitro modeling to assess native myocardial function and disease mechanisms as well as testing drug safety and efficacy. However, current hiPSC-CMs are functionally immature, resembling in vivo CMs of fetal or neonatal developmental states. The use of targeted culture media and organoid formats have been identified as potential high-yield contributors to improve CM maturation. This study presents an hiPSC-CM maturation medium formulation, designed using a differential evolutionary approach targeting metabolic functionality for iterative optimization. Relative to existing high-performing reference formulations, our medium significantly matured morphology, Ca2+ handling, electrophysiology, and metabolism, which was further validated by multi-omic screening, for cells in either pure or co-cultured microtissue formats. Together, these findings not only provide a reliable workflow for highly functional hiPSC-CMs for downstream use, but also demonstrate the power of high-dimensional optimization processes in evoking advanced biological function in vitro.

Introduction
Cardiovascular diseases (CVDs) including heart failure, myocardial infarction, and arrhythmias often have multifactorial etiologies, are progressive and prone to sequelae, and cannot usually be curatively treated; for this reason, they form the single most common cause of death in developed countries 1. For decades, understanding of CVDs has been advanced using animal-derived models of function and disease, but the need for robust and accurate in vitro models to inform human-specific pathophysiology has become apparent 2. Similarly, the incidence of cardiotoxicity, which has remained unpredicted by preclinical in vivo screening with murine models, is a leading reason for failure of new drug trials or post-marketing drug recalls 3,4. hiPSC-CMs provide a promising avenue for cardiac pharmaceutical efficacy screening, both for preclinical screening and personalized efficacy testing 5–7.Both disease phenotypes and drug responses are emergent composites of interactions across multiple aspects of physiology 8,9, necessitating higher-fidelity in vitro models of myocardium. Important advances have been made in evoking physiological maturation in both hPSC-CMs monolayers and organoids in translational and preclinical applications 10–12, including transcriptomic or proteomic profiles 13,14,23–30,15–22, morphometrics 13,17,28–31,18,19,21–24,26,27, metabolic profile 13,14,28–31,15,18–24, Ca2+ handling 15,16,29–31,17–19,21,22,24,27,28, electrophysiology 15,16,18,19,21–24,28,31, and disease modeling 13,16,18,19,21,22,24,32,33. However, despite the advancements of many physiological parameters of current hiPSC-CMs to neonatal levels or beyond, they remain phenotypically immature in most aspects of CM-specific physiology; they do not recapitulate the contractile force, morphology, electrophysiology, Ca2+ handling characteristics, or metabolic profile associated with adult myocardium 34.

Culture medium optimization has been explored as a high-yield avenue for further hiPSC-CMs maturation. Notable advances have been made using one- or two-factor experiments including metabolic substrates such as fatty acids or galactose 15,16,30, and hormones such as triiodothyronine (T3) 17,35–39, insulin 40 or insulin-like growth factor 36,41, glucocorticoids 17,36,40, or the B27 supplement comprising a mixture of hormones and small molecules optimized for functional neuronal culture 15,19,20,29,35,37,42. However, there has been comparatively little focus to date on supplementation of endogenous cofactors or otherwise functional small molecules. The energetic demands of a fully mature CM to fuel electrophysiological and contractile function (e.g., via oxidative phosphorylation) may preclude secondary metabolic pathways that would produce such molecules from more basal media. Furthermore, pairwise and higher-order interactions have been found to be ubiquitous across biological systems such as cell fate, comprising differentiation or maturation and their associated homeostatic changes 43,44. As a result, small stepwise optimization experiments are unlikely to converge, even in aggregate, on high-efficacy maturation medium formulations.

In this study, we sought to mature hiPSC-CMs using a suite of soluble factors comprising metabolic substrates, hormones, cofactors, and other small molecules based on the potential for widescale interactions between factors. Our general strategy in choosing factors was to mirror the exogenous availability of soluble signals to which the myocardium is exposed during late cardiac development, specifically during the perinatal window and early childhood 45, developmental phases that coincide with a switch to a predominantly oxidative metabolism 46. This metabolic specialization prioritizes oxidative catabolic flux and outsources peripheral metabolic processes (e.g,. anaerobic catabolism, glycogen-related processes, cofactor synthesis, etc.) to other tissues. This transition to a highly efficient energetic phenotype may drive maturation in other functional metrics by increasing the accessible short-term energetic capacity within the cell 47. To maximize factor synergies and high-order interactions, a high dimensional, differential evolution (HD-DE) algorithm was employed to functionally interrogate a large factor space, even in the presence of factor interactions or nonlinear responses 48. By combining a set of 17 independent soluble factors each at 5 dosage levels, a traditional full factorial screen would require c.a. 763 billion runs to cover the solution space; using the HD-DE methodology, we developed a high-performing custom formulation after 4 iterative generations comprising 169 unique formulations. Candidate formulations were scored based on their oxidative uncoupled:control ratio using cell respirometry as a self-normalizing correlate of overall functional maturation. This approach identified a medium formulation that best enhances hiPSC-CMs maturity to maximize multiple functional performance metrics compared to existing high-performing commercial and published formulations. Recent high-performing hPSC-CM maturation efforts 13,25,49 have resulted in cell phenotypes that eclipse the specificity of single expressional markers of CM maturity 50, and the field has accordingly embraced more comprehensive physiological profiling both for the purpose of demonstrating application and for more precisely stratifying maturation efficacy. Accordingly, our formulation demonstrated an unmatched degree of advancement of many specific functional metrics of maturity including morphology, contractility, electrophysiology, Ca2+ handling, metabolism, and gene and protein expression profiles.

Results
Differential evolution and selection of an hiPSC-CM maturation medium
To efficiently optimize hiPSC-CM maturation medium without full mechanistic understanding of the processes involved, we implemented an HD-DE algorithm-driven process (Figure 1a), using oxidative uncoupled:control ratio (UCR; the ratio of pharmacologically electron transport chain-uncoupled to steady-state oxygen consumption rates) as an objective metric to score successive generations of candidate formulations relative to a commercially available control medium (STEMCELL Cardiomyocyte Maintenance Medium) (Figure 1b). A total of 17 soluble factors (Supplementary Table 1, Figure 1c) were chosen for iterative optimization; 6 of these factors as well as M199 basal medium had not previously been tested, nor appreciable analogues, for their use in CM maturation. Factors were surveyed at any of 5 discrete doses (including null; Supplementary Table 1) within a given candidate formulation. In all, 169 unique formulations were tested over 204 runs across 4 iterative generations each consisting of 3 weeks of treatment with candidate formulations (culture timelines in Supplementary Figure 1a-b). Furthermore, the first generation of formulations was defined using a low-discrepancy sequence so that the entire solution space was evenly queried at the widest possible coverage. This generation included at least one formulation that did not include each soluble factor, to ensure their relative contributions to the utility of the medium as a whole. By principal component analysis (PCA), the top 10 performing formulations formed 2 main clusters of composition, with 2 outliers (Figure 1d-e); metabolic substrates and cofactors were correlated most strongly in PC1, while hormones and small molecules primarily associated with signaling were aligned within PC2 (Supplementary Table 2). A final formulation for further characterization was manually defined by including specific dose-factor combinations present in the top-right cluster of PCA but absent from the bottom-left quadrant, resulting in a composite of high-performing candidate formulations and containing 15 of the 17 queried factor additives in addition to the M199 base. This 16-component hiPSC-CM maturation formulation (“C16”) was confirmed to be a viable and high-performing candidate against high-performing control formulations from STEMCELL, iCell, and the homemade RPMI + B27 cocktail, using both morphological and contractile comparisons after 3 weeks of culture. Compared to treatment with both STEMCELL and a leading high-efficacy maturation formulation by Feyen et al. 21, hiPSC-CM monolayers treated with C16 medium exhibited increased spontaneous alignment (circular variance p < 0.0001), cell elongation (eccentricity p = 0.0019), and intracellular organization (α-actinin:connexin-43 (Cx43) colocalization coefficient p < 0.0001; Figure 1f), while confocal microscopy demonstrated that C16 medium enhanced myofibrillar density, α-actinin striation, and Cx43 junctional expression (Figure 1g). C16-treated cells demonstrated clear sarcomeric striations under phase-contrast in C16-treated monolayers (Supplementary Figure 1d-e) with increased sarcomere length (p = 0.0003; Figure 1h), as well as increased hypertrophy (projected area p < 0.0001; perimeter p < 0.0001) and anisotropy (form factor p < 0.0001), but not α-actinin integrated density (p = 0.91) (Supplementary Figure 1f). Furthermore, C16 treatment induced striated expression of hallmark CM functional proteins DHPR, SERCA (Figure 1i), and RyR2 (Figure 1j). Transmission electron microscopy demonstrated increased C16-specific prevalence of t-tubules (Figure 1k) and mitochondria (Figure 1l). Optical contractile traces, obtained using particle image velocimetry, demonstrated accelerated full contractile cycles in C16-cultured monolayers derived from two hiPSC-CM lines from healthy donors 51 (PGPC17 and PGPC14), as well as pathologically slowed and biphasic contraction in a MYBPC3-KO CRISPR-generated PGPC17 mutant hiPSC line (Supplementary Figure 1g).

Electrophysiological characterization and identification in maturing hiPSC-CMs
The presence of stable resting (diastolic) membrane potentials and the absence of spontaneous beating are hallmarks of mature working myocardium. Importantly, in addition to the maturation metrics already listed, hiPSC-CM monolayers treated with C16 medium were not spontaneously contractile, unlike RPMI + B27-treated cells, suggesting marked electrophysiological differences between treatments. To assess the electrical properties of our cultures, we measured intracellular voltages in randomly-selected cells after 6 weeks of treatment with either C16 or RPMI + B27 (Figure 2a). These electrical differences were associated with classical differences in the resting diastolic membrane potentials. Specifically, RPMI + B27 cells underwent spontaneous depolarization after reaching minimum diastolic membrane potentials (MDPs) of -56.1 ± 6.8 mV (Figure 2b), a pattern that is characteristic of immature cardiomyocytes, while C16-treated cells displayed fixed resting diastolic membrane potentials (-81.1 ± 7.7 mV). RPMI + B27-treated cell APs were accompanied simultaneously by contraction and Ca2+ transients (Figure 2c) at a rate of ~ 0.2 Hz. By contrast, C16-treated cells did not display spontaneous action potentials (APs), beating or Ca2+ transients in the absence of electrical stimulation. Previous studies have established that the absence of spontaneous APs in mature tissues is linked to the presence of background inward rectifier K+ channels (IK1), which are able to "clamp" the resting membrane potential at values close to the equilibrium potential for K+ ions (EK) 52. To assess whether IK1 underlies the differences in spontaneous APs and beating between C16 and RPMI + B27, we applied extracellular Ba2+ at a dose (400 µmol L-1) that potentially blocks IK1 channels. While Ba2+ addition had minimal effect on either MDPs (49.2 ± 5.3 mV) or the beating rates of RPMI + B27 cells, it caused C16-treated cells to display spontaneous beating and APs accompanied by spontaneous depolarizations from MDPs of -53.7 ± 1.8 mV. Our findings suggest that C16-treated cells express high number of IK1 channels compared to RPMI + B27-treated cells. Indeed, in voltage-clamp studies, C16-treated cells demonstrated robust background K+ currents with characteristic kinetic behaviour, classical inward rectification properties (Figure 2d-e) and reversal properties (i.e., ~EK) which are hallmark features of IK1. Moreover, these background currents in the C16 treatment were eliminated by 400 µmol L-1 Ba2+ (Figure 2e). RPMI + B27 cells also displayed Ba2+-sensitive background K+ currents but these were ~4-fold smaller than in C16-treated cells (i.e., Ba2+-sensitive densities measured at -115 mV were -21.9 ± 8.2 pA pF-1 in C16 versus -5.5 ± 3.9 pA pF-1 in the RPMI + B27 treatment, Figure 2f) which consistent with the observed effects of Ba2+ on APs and beating of C16- vs. RPMI + B27-treated cells. Measurement of IK1 density slope between -115 and -95 mV further demonstrated significant differences between C16-treated cells to those treated with RPMI + B27 or the high-performing medium of Feyen et al. 21 (Figure 2g).

Physiological characterization of maturing hiPSC-CMs
hiPSC-CM monolayers treated with C16 medium demonstrated differential Ca2+ handling and metabolic flux compared high-performing control formulations after 6 weeks of culture. Measurement of steady-state Ca2+ handling at 1 Hz pacing using line-scanning microscopy demonstrated accelerated onset (Figure 3a-b) and decay kinetics (Figure 3c-e) in randomly-selected C16-treated cells vs. controls, which may reflect a greater dependence of Ca2+ cycling on the sarcoplasmic reticulum (SR) seen in mature versus immature myocardium 34. More direct evidence of enhanced SR function in the C16 culture conditions was uncovered by perfusing cells with high levels of caffeine to cause complete release of the SR's Ca2+ into the cytosol. Specifically, when cells were pre-treated with verapamil to prevent Ca2+ entry into the cytosol via L-type channels, a bolus of caffeine generated a rise in intracellular Ca2+ within C16 cells that was ~2-fold higher than for cells cultured in STEMCELL medium (p < 0.0001) (Figure 3f-h). Also consistent with increased SR contributions, administration of the β-adrenergic receptor agonist, isoproterenol, further accelerated Ca2+ transient onset in C16-treated cells only (Figure 3i).

C16-treated hiPSC-CMs also diverged from high-performing control formulations in metabolic profiling using Seahorse XFe96 respirometry (Figure 3j-k). Specifically, the per-cell basal oxygen consumption rate outpaced control treatments (Figure 3l), while the FCCP-enabled UCR of C16-cultured cells was higher than those of the iCell (p = 0.035), RPMI + B27 (p = 0.023), and STEMCELL (p = 0.013) control treatments (Figure 3m). These findings were consistent with targeted metabolomic analyses of hiPSC-CM monolayers at the same timepoint, treated with either the C16 formulation, or the formulation of Feyen et al.21, or RPMI + B27 as a control. C16-treated monolayers demonstrated greater incorporation (i.e., lowered m+0 proportion) of labeled glucose and lactate into aerobic pathways (Figure 3n-q), with reduced anaerobic metabolism (Figure 3r-t) while maintaining pentose phosphate flux (Figure 3u).

Transcriptomic profiling of maturing hiPSC-CMs
RNA sequencing was used to evaluate the transcriptomic profiles of hiPSC-CMs treated with C16 or g high-performing control maintenance media to help identify targets for further physiological analysis. PCA was used to compare the overall transcriptional profiles of each medium relative to immature day 20 (d20) controls, demonstrating that C16-treated cells were highly divergent from both controls and other media (Figure 4a). Many of the GO terms significantly upregulated in C16 treatments were associated with general cardiomyocyte functions, including the cardiac action potential, contraction, regulation of contractility, and oxidative metabolism, while GO terms heavily downregulated in C16 treatments included differentiation, motility, and anaerobic metabolism (Figures 4b-j). Similarly, gene set enrichment analysis (GSEA) comparing C16 to high-performing control treatments revealed GO terms pertaining to heart function, lipid metabolism and oxidative phosphorylation, cell-cell adhesion, and transcriptional regulation (Supplementary Figures 2, 3).

Meta-analysis of (hiPSC-)CM maturation
RNA-seq datasets from several studies of CM maturation were pooled into a single meta-analysis to better understand transcriptional trajectories, or the gene expressional landscape around the physiological maturation process. To enable interpretation, external samples were divided into one of 7 broad categories. In vivo myocardial or CM samples were assigned to the ‘Adult’, ‘Infant’ or ‘Fetal’ category based on developmental timepoint at isolation. Matured in vitro hiPSC-CM samples were assigned to the “2D”, “3D” or “EB” categories, depending on the culture methodology and type of maturation approach employed (see Supplementary Table 3 for detailed descriptions). ‘Control’ samples were immature hiPSC-CMs, collected at ~d18-20 of differentiation, after the onset of spontaneous beating but prior to starting any type of maturation protocol.

To adjust for batch effects introduced by the inclusion of multiple datasets, the count matrix was adjusted to minimize variation among samples from different studies, while preserving variation among the designated sample categories (Supplementary Figure 4). Following batch correction, tSNE and PCA analyses demonstrated that the external samples could agnostically be re-separated into distinct clusters according to their pre-assigned categories (Figure 4k-l). Both GSEA of principal components (Supplementary Figure 5) and differential expression analysis (Supplementary Figure 6) revealed similar trends in functional gene expression between unique clusters; the PC1 positive direction (2D cluster) was enriched for electrophysiological function and electrical conduction, PC2 positive (3D cluster) enriched for contractile function and Ca2+ handling, and PC1 negative (EB cluster) enriched for cardiac developmental and morphogenic processes. DEA further identified specific functions enriched in each cluster that were not identified according to the PCA loadings, such as GPCR signaling and lipid handling in the “3D” cluster, and Ca2+ handling in the “2D” cluster.

Proteomic profiling of maturing hiPSC-CMs
Transcriptomic analysis suggested marked changes to transcriptional regulation, protein synthesis and turnover, and protein trafficking. To directly examine protein changes in the 2D system, global proteomic analysis of urea-soluble fractions was conducted on hiPSC-CMs after 6 weeks treatment in C16, iCell, RPMI + B27, and STEMCELL media, as well as the formulation designed by Feyen et al. 21 (Figure 5). Principal component analysis (PCA), conducted on a set of 1869 proteins that had valid (greater than 0 LFQ intensity) values in all three samples from at least one experimental treatment, demonstrated that PC1 largely separated the STEMCELL and C16 conditions, while PC2 separated the high-performing Feyen medium from the other four conditions (Figure 5a). Both PC loadings included metabolic and mitochondrial-specific proteins; the positive direction of PC1 also heavily featured sarcoplasmic synthesis and maintenance ontologies, ionoregulatory proteins, and membrane trafficking components, while the positive direction of PC2 heavily featured cytoskeletal proteins associated with contractility. Enriched GO terms associated with maturation conditions included membrane functionalization, oxidative metabolism, and mRNA processing, while downregulated terms centred on apoptotic induction and protein localization (Figure 5b-c). Of particular note, MYH7 protein expression was not significantly downregulated in C16-treated samples, in contrast with its transcriptional downregulation in identical samples. This may suggest that regulation beyond the level of mRNA transcription may contribute to the functional expression of maturity-associated protein products.

Maturation of hiPSC-CM-containing engineered cardiac microtissues
Marked differences in the level of attainable hiPSC-CM function relative to monolayers have previously been found in the context of engineered and co-cultured micro tissues, leveraging physiological mechanical and electrical cues to drive maturation. We used the Biowire II platform32 to examine further the impact of our maturation medium on parameters of CM function (Figure 6). These studies involved the creation of compacted multicellular muscle strips 32, followed by 3 weeks of culture in either modified C16 (see Methods) or RPMI + B27, and with or without continual electrical field pacing (1 Hz). As in monolayer culture, Biowire tissues in C16 media exhibited lower rates of spontaneous contractility than in RPMI + B27 (Figure 6a; Supplemental Movies 1-4) after 3 weeks in of treatment. Contractile forces normalized to cross-sectional area (i.e., twitch/systolic stress) was ~5-fold higher with C16 versus RPMI + B27 treatment after 1, 2, or 3 weeks in culture (Figure 6b), with or without electrical stimulation during the culture period. After 3 weeks in culture with electrical stimulation, the twitch stress was 1.69 ± 1.42 mN mm-2 with C16 treatment vs. 0.35 ± 0.37 mN mm-2 with RPMI + B27 treatment. By 3-way ANOVA, C16 treatment (p = 0.0002) and culture time (p < 0.0001) both independently enhanced twitch stress, and these parameters positively interacted (p < 0.0001), establishing greater benefit of culture time in C16 medium versus the high-performing control. Neither tissue morphology nor diastolic stresses were significantly affected by treatments (Supplementary Figure 7).

Furthermore, positive force-frequency responses after 3 weeks in culture were enhanced when measured at either 2 Hz or 3 Hz stimulation (relative to tissue-matched baseline stresses at 1 Hz) by both culture time (p < 0.0001) and C16 treatment (p < 0.0001) (Figure 6c); again, there was a positive interaction (p < 0.0001) between these factors. Tissues treated for 3 weeks with the C16 formulation also exhibited higher-density SERCA2a and DHPR staining than those treated with RPMI + B27, similarly to in monolayers (Figure 6d). However, DHPR expression was variable between cells, suggesting stratification of phenotypes within the tissue. Compared to RPMI + B27 treatment, C16-treated tissues under transmission electron microscopy demonstrated increased myofibrillar bundling, sarcomeric striation and zonation, mitochondrial density and myofibrillar association, electron-dense intercalated discs, and nuclear invaginations by the SR 53 (Figure 6e-h). Consistent with both the functional and morphological differences observed in C16-treated tissues, the two formulations were highly stratified by PCA of bulk RNA sequencing primarily by medium treatment and not electrical field stimulation (Figure 6i). Highly enriched GO terms in C16-treated cultures pertained most strongly to oxidative metabolic and contractile ontologies, while RPMI + B27-treated culture segregation was driven by developmental ontologies (Figure 6j-l; Supplementary Figure 8).

Methods
No statistical methods were used to predetermine sample size. Assignment of experimental wells was randomized, but blinding of conditions could not be used during experiments or outcome assessment due to physical differences (e.g. medium colour) between the treatments used and the morphological differences in the resulting cells.

Design of an iterative algorithmic search for optimized maturation medium
The C16 maturation medium was non-prescriptively optimized in a large solution space (~763 billion discrete formulations for an analogous 517 full factorial experimental design), with generations of formulations iteratively scored and evaluated according to the respirometric uncoupled:control ratio (UCR) as measured by Seahorse XFe96 analysis as an objective metric. This design philosophy allowed for agnostic testing and the evolution of emergent physiological interactions between multiple defined soluble factors within a formulation; several of these factors had not been surveyed previously for efficacy toward PSC-CM maturation. Additionally, we used exclusively M199-based formulations for two reasons: firstly, the ionic profile of M199 is closer to that of human plasma than DMEM- or RPMI 1640-based media, especially in [Ca2+], for which homeostasis is vital to developing CMs; and secondly, that M199 contains a much more diverse source of cofactors and vitamins than either basal DMEM or RPMI 1640 media. For the latter consideration, the use of a defined formulation negates the possibility of serum providing these essential components, especially in a highly-specialized cell such as a maturing CM, which may outsource many non-core biosynthetic and secondary metabolic pathways to other tissues of the body. Soluble factors were chosen to be included in the screen based on inclusion in other published PSC-CM maturation media (e.g., lactate, galactose, lipid supplements, albumin), inclusion in adult primary cell culture media (e.g., creatine, carnitine, taurine, insulin, transferrin, selenium), hormones implicated in myocardial development (e.g., LIF, IGF-1, triiodothyronine, hydrocortisone, neuregulin), secondary metabolites implicated in membrane stability (e.g., putrescine, ethanolamine), and exogenous compounds with impacts on central carbon metabolism and mitochondrial activity (e.g., 2-deoxyglucose, metformin).

The formulation search was performed using an existing iterative High-Dimensional, Differential Evolution (HD-DE) decision tree algorithm programmed in MATLAB 48; the published script was modified for R2018a version compatibility, for the use of 17 variable factors, and for duplicate runs. Briefly, the algorithm generates formulation candidate vectors within a defined number of runs, factors, and levels of said factors. Maximal coverage during the initial generation is generated using a Sobol quasi-random distribution (e.g., minimizing discrepancy). After a generation is produced, objective metric scores are reported, theoretically forming a rough Pareto distribution. Formulations within 10% of the top scorer are chosen to continue in the next generation, while poor-scoring formulations are either replaced with randomly-generated vectors, or with offspring vectors from the intergenerational memory of top scorers. The process is formally terminated when the median candidate of the Pareto-ranked generation does not improve by at least 10% after 3 consecutive generations. Via formulation screening by the UCR objective metric, the top 10 scoring formulations were subjected to PCA to establish trends and differences in high-performing formulations, as well as possible mechanistic explanations thereof. From analysis of the top 11 HD-DE-generated formulations, a manual adjustment was made from the clusters formed to generate the C16 medium. The C16 formulation maintained most of the characteristics from its parent cluster with 3 key changes: 1) lactate, which was deemed to be redundant in the presence of multiple other carbon sources, was removed to reduce manufacturing burden, 2) ethanolamine, which was otherwise ubiquitously present in other HD-DE lineages, was added at a low dose, and 3) growth hormone, which was variably present or excluded from lineages, was removed to reduce cost and manufacturing burden.

Materials
For iterative formulation candidate screening and characterization of the C16 formulation, soluble components used were 2-deoxyglucose (DXG498; BTioShop), sodium L-lactate (L7022; Sigma), Chemically Defined Lipid (11905031; Gibco), D-galactose (GAL500; BioShop), creatine monohydrate (CREE200; BioShop), L-carnitine hydrochloride (C0283; Sigma), taurine (TAU303; BioShop), insulin-transferrin-selenium (41400045; Gibco), bovine serum albumin fraction V (10735078001; Roche), β-mercaptoethanol (M3148; Sigma), putrescine dihydrochloride (PUT001; BioShop), ethanolamine hydrochloride (E6133; Sigma), triiodothyronine (T6397; Sigma), recombinant insulin-like growth factor 1 (PHG0071; Gibco), recombinant leukemia inhibitory factor (SRP3316; Sigma), growth hormone (869008; Millipore Sigma), hydrocortisone (H0888, Sigma), recombinant neuregulin 1β2 (ab73753; Abcam), metformin hydrochloride (ICN15169101; MP Biomedicals), in a base of Medium 199 (M4350; Sigma). High-peforming controls used were iCell cardiomyocytes maintenance medium (CMM-100-120-001; Cellular Dynamics), cardiomyocyte maintenance medium (05020; STEMCELL Technologies), and RPMI 1640 (R8758; Sigma) with 1X B27 supplement (17504044; Gibco). An additional top-performing formulation from recent literature (“Feyen”) was prepared as previously described 21 and used in proteomic and metabolomic analyses as an exemplar of maturation progress.

For immunofluorescent staining, anti-α-actinin (ab9465; Abcam), anti-connexin 43 (ab217676; Abcam), anti-ryanodine receptor (ab2868; Abcam), anti-SERCA2 (ab137020; Abcam), anti-NCX1 (ab2869; Abcam), anti-DHPR (ab65266; Abcam), goat anti-mouse, Alexa Fluor™ Plus 488 (A32723, Invitrogen); goat anti-rabbit, Alexa Fluor™ 488 (A11008, Invitrogen), goat anti-mouse Alexa Fluor 568 (A11004; Invitrogen), donkey anti-rabbit, Alexa Fluor™ Plus 594 (A32754, Invitrogen) were used, as well as phalloidin-tetramethylrhodamine B isothiocyanate, (P1951; Sigma), and DAPI (62248; Thermo Scientific). For respirometry, a DMEM-based XF medium (103334; Agilent) was used, with oligomycin (O4876; Sigma), FCCP (15218; Cayman Chemical), antimycin A (A8674; Sigma), and rotenone (NC0779735; Cayman Chemical) used as inhibitors. Mitochondrial staining was performed using MitoTacker Deep Red FM (M22426; Invitrogen). Unless otherwise noted, all other materials were obtained from Sigma.

Cells
An iterative formulation search, and subsequent characterization of hiPSC-CM cultured in the chosen final maturation medium candidate (“C16”) was performed using the Personal Genome Project of Canada-17 hiPSC line (PGPC17), which have been extensively characterized across a wide range of differentiation pathways 51. Further testing for cross-cell line compatibility was performed on PGPC14 hiPSC, and on MYBPC3-knockout PGPC17 cells generated via CRISPR; both lines were also previously characterized as hiPSC-CMs 51.

Cells were frozen in clumps in mTeSR (STEMCELL Technologies) containing 10% DMSO, and thawed and cultured when ready for differentiation. Cells were cultured in mTeSR on Geltrex (Thermo-Fisher; LDEV-free, hESC-qualified; incubated at 1:100 in DMEM for 1 h on TCPS plates) and passaged in clumps using ReLeSR (STEMCELL Technologies), all according to manufacturer guidelines. Cells were passaged at least two times after thawing before being expanded for differentiation. PSC-CM differentiation was performed using the Cardiomyocyte Differentiation Kit (STEMCELL Technologies) according to manufacturer directions, with cell seeding densities optimized for each cell line (between 4-8 x 105 cells well-1 in a 12-well dish, previously coated with 1:100 Matrigel (Corning) in DMEM). Cells were cultured post-differentiation until d18 post induction before reseeding for maturation experiments. Obtained monolayers in 12-well format were dissociated within 1 week of differentiation by incubation for 1 h at 37°C in 1 mL Hanks buffer, containing 200 U mL-1 Collagenase Type II (Worthington), with 0.5 mL TryPLE Select (Gibco) added for 15 additional minutes. Cells were centrifuged 5 min at 300 x g, resuspended, and plated in RPMI + B27 for 1 d culture before applying treatments. For monolayer imaging, cells were plated in 96-well polymer coverslip-bottom imaging plates (89626; ibidi) and treated as above. Non-screening treatments were applied to monolayers for 6 weeks; all monolayer cultures were performed in the absence of antibiotics.

Formulation screening
Soluble factors used in the iterative screening and final C16 formulation (Supplementary Table 1) were selected with the goal of providing a range of substrates, cofactors, and hormones that would not necessarily be created by a maturing or mature CM but which would be conducive to increased biosynthesis and hypertrophy, oxidative metabolism to fuel highly-energetically demanding physiological processes, or activation of pathways implicated in maturation of myocardium or other functional tissues in vivo. In all, 169 unique formulations were queried over 4 generations of varying size, using the Seahorse XFe96 mitochondrial stress test to interrogate metabolic function. Cells were dissociated from differentiation wells and seeded at 8 x 104 cells well-1 in CM support medium on Matrigel-coated XFe96 monolayer plates (Agilent Technologies). Culture medium was changed the next day to the well’s respective treatment. Cells were then cultured 21 days in 60 µL medium well-1, with full medium changes every second day for 3 weeks in either duplicates of a formulation candidate, or one of four wells of control Cardiomyocyte Maintenance Medium (05020; STEMCELL Technologies). The four corner wells of the plate were additionally left unseeded as internal measurement controls according to standard XFe96 manufacturer guidelines.

Respirometry
Cells were equilibrated 45 min before metabolic characterization in initial volume 150 µL Seahorse XF base medium (103334-100, Agilent Technologies) containing additional (in mmol L-1) glucose (5), pyruvate (1), glutamine (1), sodium lactate (5), and 1X Chemically Defined Lipid, to best supply the oxidative substrate flexibility of mature CMs 76. A mitochondrial stress test was performed with sequential injections of 25 µL each (final concentrations): oligomycin (2.5 µmol L-1), carbonyl cyanide-4-phenylhydrazone (FCCP; 1 µmol L-1 for iterative testing, or either 0.20, 0.40, 0.50, 0.65, 0.80, or 1.00 µmol L-1 for characterization of hiPSC-CMs cultured in C16 medium), antimycin A and rotenone (2.5 µmol L-1 each). Due to the high specific oxidative flux of CMs, a modified measurement protocol to avoid hypoxia was employed 77; cells were measured for 2 rather than 3 cycles at each step, with the minimum measurement time of 2 min.

Respirometric measurements were normalized to cell numbers per well. Each well was fixed in 2% formalin for 5 min, washed 3 times with Ca2+ and Mg2+-free PBS, stained with Hoechst (1 µg mL-1) in PBS without for 5 min, washed 3 additional times, and imaged using an IX71 inverted widefield fluorescent microscope (Olympus Corporation) with a FITC filter. Cell quantifications were performed by performing an automated count of nuclei using ImageJ 1.52p (NIH), by sequential use of the standard Otsu threshold, watershed segmentation, and particle count functions. Normalized per-cell oxidation rates (OCR) and uncoupled:control ratios (UCR) were compared between treatments using a repeated measures ANOVA and Dunnett’s multiple comparisons post-hoc test for high-performing comparative treatments vs. the C16 formulation where indicated, to account for batch effects of the respirometric assay.

Immunofluorescence, confocal microscopy, and morphometric analysis
Cell immunofluorescence for manual analysis
Cultured cells were fixed with 2% paraformaldehyde for 10 min at room temperature, followed by 90% ice-cold methanol for 10 min, followed by permeabilization buffer (0.5% Triton X-100, 0.2% Tween-20 in PBS) for 30 min at 4 °C. Blocking buffer (5% FBS in permeabilization buffer) was then added and incubated for 1 h at room temperature. Cells were incubated with primary antibodies (α-actinin 1:500, Cx43 1:500) in blocking buffer overnight at 4 °C; incubation in rhodamine-red-conjugated phalloidin (Sigma) was 1 h at RT. Fluorophore-conjugated secondary antibody staining (anti-rabbit AlexaFluor® 488, anti-mouse AlexaFluor® 594, anti-mouse AlexaFluor® 647; Molecular Probes; 1:800) was performed at room temperature for 1 h in the dark. Nuclear counterstaining was performed using 1 μg mL−1 DAPI at room temperature for 15 min in the dark. Images were acquired using an FV3000 confocal microscope with 405, 488, 561, and 640 nm lasers.

Cell immunofluorescence for quantitative analysis
Cultured cells were cultured in 96-well polymer coverslip-bottom imaging plates. At 3 weeks of culture, cells were washed in PBS with calcium and magnesium (PBS+/+) and incubated at 37°C in 500 nmol L-1 MitoTacker Deep Red FM dye for 40 minutes. The dye was prepared according to manufacturer instructions by solubilizing in anhydrous DMSO to a 1 mM stock, then diluting to 500 nM in either Cardiomyocyte Maintenance Media or C16 media. The cells were then washed in PBS+/+ and fixed in 4% paraformaldehyde for 10 minutes at RT prior to washing 3 times in PBS+/+ for 5 minutes, then permeabilized and blocked with 10% normal goat serum (NGS) in 0.5% Triton X-100/0.2% Tween-20 for 90 minutes at RT. They were then incubated with primary mouse anti-α-actinin (1:500) and primary rabbit anti-connexin 43 (1:800) in 5% NGS in 0.5% Triton X-100/0.2% Tween-20, overnight at 4°C on an orbital platform. The next day, they were washed 3x in PBS+/+, then incubated with secondary Alexa Fluor 568–conjugated goat anti-mouse IgG (1:200) and secondary Alexa Fluor 488–conjugated goat anti-rabbit IgG (1:200), for 90 minutes at RT on an orbital platform. Then, after washing again in PBS+/+ 3 times for 5 minutes each time, cells were incubated with DAPI counterstain (1:1000) for 5 minutes at RT. Finally, cells were washed in PBS+/+ twice, then in distilled water once, and mounted in a drop per well of Aqua-Mount (Epredia 13800). At this point, cells were sealed and stored at 4°C away from light for up to 3 months. Confocal images were obtained using an Olympus FluoView 3000 laser scanning confocal microscope (Olympus Corporation). Acquisition followed in 3 sites in each of 10 wells per media treatment condition of each of the 4 channels (DAPI, AF488, AF568, and AF647) using a 60X/1.35 NA oil immersion objective. Images were saved in files appended with plate, well, and site metadata.

Automated image morphometric analysis
Images for automated analysis, as acquired above, were analyzed with CellProfiler78. A custom CellProfiler pipeline was built based on the CellPainting assay79. The pipeline imported all images and applied an illumination correction filter. Each image was labeled with plate, well, and site information by extracting its metadata from original files. The DAPI channel was first used to identify nuclei through a global Otsu thresholding method. The AF568 (α-actinin) channel was used next to identify cell borders through a global thresholding method minimizing cross-entropy. A threshold correction factor from 0.25-0.4 and a regularization factor of 0.005 were also applied through trial and error to give the best cell segmentation results. Cells touching the edges of the image were then excluded from subsequent analysis. 1047 parameters were calculated and recorded for each cell. Notable parameters included cell area, perimeter, eccentricity, form factor, orientation, co-localization of α-actinin and connexin-43, and α-actinin intensity. These parameters quantified for each cell in each image (10 images per media treatment condition) were exported to a comma-separated values (CSV) file for statistical analysis. All the above-mentioned parameters were generated per cell as technical replicates to generate 1 biological replicate per well except cell orientation, for which the average circular variance per biological replicate was taken instead. Normality was tested using a D’Agostino and Pearson test, and conditions compared using a 1-way ANOVA with Tukey’s post-hoc test.

Sarcomere spacing measurement and analysis
In each of the image stacks acquired (5 wells per treatment condition × 3 sites per well), the AF568 α-actinin channel was used to manually measure sarcomere spacing. Representative myofibrils (5 per image) were selected and the average distance between peaks were calculated automatically detected using the ImageJ integrated ridge detection plugin, and sarcomere length was determined by measuring the distance between intensity peaks between adjacent filaments. One cell was selected per image and the lengths of 5 sarcomeres for that cell were acquired. Equality of variance was tested using the D’Agostino and Pearson test, and conditions compared using a one-way ANOVA with Tukey’s post-hoc test.

Monolayer transmission electron microscopy
hiPSC-CMs were fixed with 4% paraformaldehyde in PBS and 1% glutaraldehyde for 1 hour at room temperature, fresh fixative was added, and samples were incubated overnight at 4°C. Samples were washed with PBS, and subsequently incubated in 1% osmium tetroxide in 0.1 mol L-1 sodium cacodylate buffer for 1 hour at room temperature and washed. Samples were then dehydrated with a graded ethanol series (30%, 50%, 70%, 90%, 100%). Samples were resin-embedded by increasing concentrations of Epon resin mixed with propylene oxide in the following sequence (propylene oxide: Epon resin): 1:0 (30 min), 2:1 (2 h, agitated), 1:2 (3 h, agitated), 0:1 (overnight), 0:1 (2 h, agitated), and polymerized for 48 hours at 40°C. Samples were sectioned at 80 nm using a Leica Reichert Ultracut E microtome and stained using 5% uranyl acetate and 5% Reynold's lead citrate, prior to imaging with a Talos L120C TEM (Thermo) and a BM-ICeta camera.

Optical contractile tracking of hiPSC-CM monolayers
Contractile kinetics were assessed from phase-contrast videos recorded at ~20 fps with a 40X objective, using a particle-image velocimetry package for ImageJ 80 to quantify time-resolved displacement over the contractile cycle relative to the relaxed state. Individual traces from biological replicates were fitted with a quartic regression using Prism 9 (GraphPad Software Inc., San Diego, USA).

Electrophysiological characterization
Intracellular recordings
Action potentials (APs) were record in single hiPSC-CMs or small clusters of hiPSC-CMs (n = 14 for C16 and n = 30 for RPMI + B27) using sharp microelectrodes of resistances between 40 to 90 MΩ (filled with 3 mol L-1 KCl) with an Axopatch 200B amplifier. Intracellular voltages were digitized at a sample rate of 10 kHz (Digidata 1440 A plus pCLAMP 10.3, Molecular Devices). Voltage recording was performed at room temperature and perfused with modified Tyrode’s solution (pH 7.4) containing (in mmol L-1): NaCl (120), KCl (5) CaCl (2), MgCl (1), Na HPO (0.84), MgSO (0.28), KH PO (0.22), NaHCO (27), and glucose (5.6) 55.

Patch-clamp recordings
Voltage-clamp measurements were used to record whole-cell currents in single isolated hiPSC-CMs using the patch-clamp technique at room temperature. The hardware was the same as for the AP recordings. The pipette solution (pH 7.2 adjusted with KOH) contained (in mmol L-1): K-gluconate (150), EGTA (5), HEPES (10), and MgATP (5). The series resistance of the pipettes was 3-4 MΩ. The external bath solution (pH 7.35-7.4 with NaOH) contained (in mmol L-1): NaCl (148), KCl (5.4), MgCl (1.0), CaCl (1.8), NaH PO (0.4), glucose (5.5), and HEPES (15). The external perfusion solutions also contained nifedipine (5 μmol L-1) to block possible overlapping Ca2+ currents. IK1 was assessed by using voltage-steps from -115 mV to -20 mV (in 5 mV intervals) were applied for 500 ms from a membrane voltage of -60 mV. Currents were recorded at sampling rates of 10 kHz and analyzed using Clampfit 10.7 (Molecular Devices). Current-voltage (I-V) relationships were generated using peak currents as a function of membrane voltage. Currents were also recorded following the perfusion of BaCl (100-400 μmol L-1), which is a potent and selective blocker of IK1 at such low concentrations 81. Normality was not tested due to sample size limitations and treatments were compared using a two-tailed t-test or one-way ANOVA with Tukey’s post hoc test, as appropriate.

Ca2+ imaging
Cells cultured in 12-well plate format were dissociated as described above and replated in single-cell format on 96-well plates with a #1.5 coverslip bottom treated with 1:100 Matrigel, and cultured in their treatment medium to recover for 2 days before imaging. Frozen dry vials of 50 µg Fluo-4 AM (Thermo-Fisher) were reconstituted with 50 µL DMSO containing 20% (w/v) Pluronic F-127. Cells were treated with 50 µL well-1 Fluo-4 AM solution, diluted 1:100 in Tyrode’s solution, for 30 min at RT. Cells were rinsed twice with Tyrode’s solution and maintained up to 2 h thereafter in 100 µL Tyrode’s solution, containing 5 µmol L-1 S-blebbistatin (Toronto Research Chemicals) to prevent movement artifact.

Ca2+ imaging was performed on a FluoView FV3000 (FV3000; Olympus Corporation) confocal microscope heated to 37 °C. Linescan measurements with 488 nm excitation and collection from 505-550 nm at 1% laser power and c.a. 500 V at the HSD were taken at the 20X objective and 2X zoom with spatial and temporal resolutions of 0.62 µm and c.a. 1.2-1.8 ms, respectively. Cells were subjected to steady-state stimulation at 1 Hz monophasic square-wave impulses of 2 ms and 40 V at approximately 0.6 cm using custom-made graphite electrodes connected to an S48 physiological stimulator (Grass Technologies, Warwick, RI). For monolayer SR capacity experiments, cells were paced at 1 Hz at steady-state before consecutive additions of verapamil (20 µmol L-1) and caffeine (10 mmol L-1). Fractional SR load upon addition of caffeine was normalized to steady-state transient amplitudes for each cell; comparisons did not assume equal variance and were analyzed by Welch’s t-test. Fluo-4 fluorescence measurements were taken using a CCD camera at c.a. 30 Hz framerate on an IX71 inverted epifluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a FITC filter cube. Transients were manually extracted using ImageJ. Epifluorescent SR capacity transients were directly reported and interpreted. Transient confocal linescans were smoothed with a 7-frame rolling-average filter and analyzed for kinetics, using a custom MATLAB script. Kinetics parameters assessed included time from baseline to peak fluorescence, and times from peak fluorescence to a decay of 10%, 25%, 50%, and 75% peak magnitudes. Kinetic metrics were compared between treatments using a Brown-Forsythe ANOVA and Dunnett’s multiple comparisons post-hoc test for high-performing comparative treatments vs. the C16 formulation where indicated, to account for unequal variances between treatments.

Microtissue maturation
Culture and functional benchmarking
Microtissues were created according to established methods 32. Briefly, hiPSC-CMs mixed with primary human fibroblasts were cast in fibrin gels within embossed polystyrene molds and suspended between flexible poly(octamethylene maleate (anhydride) citrate) (POMaC) posts to allow for contraction against resistance. All strips were cultured in 10 cm cell culture dishes, containing 10 mL of medium changed twice weekly, with 0.1% penicillin-streptomycin. The C16 medium used for monolayer culture was found to be incompatible with tissue function due to toxicity to primary cardiac fibroblasts (as found in monolayer fibroblast culture); a modified version without 2-deoxyglucose allowed continued tissue function and was used as the working version of C16 for all Biowire II experiments. Strips of up to 8 functional tissues were cultured 1 week in RPMI+B27 medium, to allow compaction, before assignments to treatments in either modified C16 2-deoxyglucose was omitted for co-culture applications, as it was found to be toxic to primary fibroblasts at working concentrations) or continued RPMI + B27 media, and with or without electrical field stimulation (10 V cm-1 at 1 cm, 1 Hz monophasic square wave pulses for 3 ms) via two graphite electrodes connected to an S48 physiological stimulator (Grass Technologies, USA). Tissues were assessed for function at 0, 7, 14, and 21 d of treatment for baseline paced diastolic stress, peak systolic twitch stress, and stimulation force-frequency relationship up to 6 Hz using an IX71 microscope at 37 °C. Polymeric wires were separately assessed for stiffness using a MicroSquisher (CellScale, Waterloo, Canada) to calculate absolute tissue diastolic and active forces. Twitch forces were calculated as the difference between diastolic and active forces. Stresses were derived from forces normalized to calculated cross-sectional tissue area (an ellipse of 5:3 aspect ratio at the tissue midpoint between polymeric wires based on existing cross-sectional imaging 32). Stress values were first analyzed using a 3-way repeated measures ANOVA (Medium x Stimulation x Time) to assess interactions between the effects of medium versus stimulation on systolic stress. If the 3-way interaction analyses failed to find significance, the were repeated by 2-way repeated measures ANOVA combined with a within-timepoints Tukey’s HSD, as post-hoc test. Sphericity was not assumed and the Geisser-Greenhouse correction was applied when indicated.

Transmission electron microscopy
Microtissues were rinsed 3x with PBS before fixation in 4% paraformaldehyde and 1% glutaraldehyde in PBS. Secondary fixation was in 1% OsO in 0.1 mol L-1 sodium cacodylate buffer. Specimens were dried in a series of 50%, 70%, 90%, and 3x 100% EtOH, then infiltrated in 50% and 70% (2 h steps) and 100% (overnight) Spurr resin. Samples were sectioned at 70 nm, stained with uranyl acetate and lead citrate, and imaged with a T20 transmission electron microscope (Thermo) and a 4k CCD (Gatan).

13C metabolic flux analysis
Cells were incubated 24 h in one of three substrate-labeled culture media. The base medium consisted of glucose-free DMEM, containing 0.1% w/v Fraction V bovine serum albumin, 50 µmol L-1 carnitine, and 1X Insulin-Transferrin-Selenium supplement (Gibco). Medium also contained 10 mmol L-1 glucose, 3 mmol L-1 lactate, and 120 µmol L-1 palmitate; in any well, one of these three carbon sources was U-13C labeled while the remaining two were untagged. After incubation, cells were quickly washed twice in PBS and extracted with a 2:2:1 (v:v:v) mixture of acetonitrile, methanol, and water, all mass spectrometry grade and pre-cooled to -80°C. Cells were scraped from plates and the slurry kept at -80°C until analysis. Analysis was performed by LC-MS by The Metabolomics Innovation Centre at McGill University according to previously-established methods 82, and isotopomer relative balances were measured for metabolites of interest with each label.

Protein extraction and global proteomic profiling
Cells were washed 3x in PBS, scraped from 12-well plates, and centrifuged 5 min at 200 x g. Pellets were flash-frozen and stored at -80°C until extraction. Protein extraction was in 8 M urea with 5x3 s sonication bursts at 30% power on ice. Protein concentrations were assessed by BCA assay (Pierce). Samples were reduced in 2.5 mmol L-1 dithiothreitol (DTT) 1 h at 37°C, before alkylation in 5 mmol L-1 iodoacetamide for 30 min at RT in the dark. Finally, samples were diluted 1:10 in 50 mM ammonium bicarbonate before the addition of 1:50 protein:protein by mass of sequencing-grade trypsin (Promega) for overnight digestion at 37 °C, followed by the terminating addition of formic acid at a final concentration of 1%. Mass spectrometry and analysis of proteomic samples were performed on three independent samples for each media treatment as previously described 83, detailed below.

Chromatography, DDA Mass Spectrometry, Search and Data Processing Methods
For each sample, 1 μg of total protein equivalent was loaded on C18 tips (Evosep, EV-2001) using manufacturer instructions and subjected to in-line chromatography using the Evosep One (EV-1000) instrument 15SPD LC protocol. This involved an 88-minute gradient using the Endurance 15 cm analytical column (EV-1106) and a stainless-steel emitter (EV-1086), with a spray voltage of 1.9 kV. For DDA MS data acquisition, the Q Exactive Plus instrument (Thermo) was used, with each cycle involving one full MS scan (at 70,000 resolution) followed by top 15 selected MS/MS scans at 17,500 resolution. The full MS scan range was set to 400-1500 m/z. The AGC target was set to 1e6 and 40 ms maximum injection time for full MS scans and 1e5 AGC target and 50 ms maximum injection time for MS/MS scans. MS/MS scans were performed with a normalized collision energy of 27 and isolation window of 1.6 m/z in centroid mode with an acquisition mass range between 200 and 2000 m/z. Generated RAW files were searched using the MaxQuant (v2.0.3.0) search engine. The data was searched using the canonical and isoform protein sequence FASTA files from UNIPROT (UP000005640_9606, February 2021 release). Three missed cleavage events and variable modifications for methionine oxidation and N-terminal acetylation were allowed with carbamidomethylation of cysteines set as a fixed modification. The output was filtered based on a 0.01 precursor FDR, with the ‘match between runs (MBR)’ feature enabled. The remaining search parameters were left at default settings and are in the search data and RAW mass spectrometry files deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD036639. Search results were processed using Perseus version 1.6.15.0. LFQ values were used for quantitative analyses. Principal component analysis (PCA) and head-to-head comparisons were performed on the set of 1869 proteins that had valid (greater than 0 LFQ intensity) values in all three samples from at least one experimental treatment with data imputed from normal distribution (width = 0.3, downshift = 1.8).

RNA sequencing
Sample Preparation
Total RNA was extracted from hiPSC-CM monolayers using a Qiagen RNeasy Micro kit and prepared for sequencing at 100 ng with a Stranded Total RNA Prep kit (Illumina Inc., USA), and bulk-sequenced at the Princess Margaret Genomics Center (Toronto, Ontario) using 100 bp paired-end reads at a depth of 40 million reads. Biowire II tissues were extracted using a PicoPure™ kit (Thermo-Fisher), prepared with a SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian kit (Takara Bio Inc., Japan) with 0.75ng and bulk-sequenced using 100 bp paired-end reads at a depth of 50 million reads. Reads were aligned to the human reference transcriptome assembly (GR.Ch38) using Salmon 84.

Data Analysis
All RNAseq data analyses were performed in R. Gene-level read counts were obtained using the Tximeta 85 package. For the meta-analysis, ComBat-seq 86 was used to correct for batch effects introduced by the inclusion of samples from different publications. For principal component and T-distributed stochastic neighbor embedding (tSNE) analyses, the raw count matrix was adjusted using the variance stabilizing transformation implemented in the DESeq2 package. PCA and PC-associated GO term enrichment was performed using the pcaExplorer 87 package. tSNE analysis was performed using the Rtsne package, and clusters were determined from tSNE analysis using the Louvain method 88, implemented in the bluster package. Differential expression was determined using the DESeq2 89 package, and Benjamin-Hochberg adjusted p-values < 0.05 were considered significant. Gene set enrichment analysis (GSEA) was performed using the GAGE package 90, and terms with Benjamini-Hochberg adjusted p-values < 0.05 were considered significant. GO term similarity was determined using the kappa similarity index, and clusters were identified by hierarchical clustering. Pathway enrichment analysis was performed using the GAGE package 90, and pathway diagrams were generated using Pathview 91. All plots were produced using the ggplot2 and ComplexHeatmap packages.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the present study, Callaghan et al. presented a new formula for hPSC-CM maturation media and demonstrated that it promotes more mature hPSC-CM functional and morphological properties. More specifically, cells appear to have increased size and improved striations, improved calcium handling, electrophysiological and contractile properties, and better aerobic metabolism. The authors have done a significant amount of revision work, and their culture medium appears to convincingly promote a more mature hPSC-CM phenotype.\nHowever, there are three main residual concerns regarding their manuscript. (1) The transcriptomic data on treated cells is rather controversial at times, especially as it relates to sarcomeric genes. (2) the authors do not provide any evidence using hPSCs from a disease model to prove the applicability of their approach. (3) The main criticism of this work involves the comparisons of hPSC-CMs in C16 vs other previously published maturation media. Although I do understand that repeating all experiments and analyses of cells grown in the Feyen et al. media protocol (which admittedly has had a more consistent effect on maturation properties) is quite burdensome, this would significantly strengthen the novelty and significance of this work. I think the authors should at least perform morphometric/structural analyses and calcium transient recordings that are critical for the functional maturation of hPSC-CMs comparing C16 with the Feyen medium. Moreover, and given the discrepancy in the expression of sarcomeric proteins, it will be useful if the authors could compare contractile properties of hPSC-CMs in Feyen vs C-16 media.\nMinor comments:\nIndividual values should be added in each graph in Fig. 3n-u to assist the readers and reviewers interpret the number of experiments performed.", "reviewer_2": "The manuscript has substantially improved since its last version submitted to NBT. There are, however, still some points to address:\n1. This revised statement is still too negative and general on the current state-of-the-art: “However, despite growing acceptance, current iPSC-CMs remain phenotypically immature in all aspects of CM-specific physiology…” There are multiple studies showing conditions for positive force-frequency for example, well-organized sarcomeres, t-tubules, action potentials of adult cardiomyocyte magnitude, etc. Perhaps defining “mature” would help: postnatal is what most studies achieve with some physiological parameters “adult-like” although none typical of aged cells. Please modify.\n2. Regarding the comments on the low and variable n in the electrophysiology data: this hardly seems to have increased. Just a different method of statistical analysis has been used: the only text revision is “Data were analyzed by non-parametric Mann-Whitney U-test.”\nOtherwise, the work will be a useful addition to the literature in this area although the question of whether the algorithm is relevant for the maturation of other cell types remains open. You could conclude that the algorithm has demonstrated its value for CM maturation and therefore its value is complete."}, "author_response": "Manuscript ID: NCOMMS-24-61115-T\nTitle: Advanced physiological maturation of iPSC-derived human cardiomyocytes using\nan algorithm-directed optimization of defined media components\nRESPONSE TO REVIEWERS\nThank you to the editor and reviewers for their careful and constructive review of our manuscript.\nWe believe that, having fully considered all comments and with the addition of new experiments,\nwe have strengthened the manuscript to make it suitable for publication. We have included directly\nbelow a point-by-point response to the reviewer comments, including a summary of all of our\nchanges to the manuscript in accepting the reviewers’ suggestions.\nReviewer comments are provided in blue below, with our responses in black and changes to the\ntext in the manuscript in red.\nREVIEWER #1:\nRemarks to the Author:\nThe manuscript has substantially improved since its last version submitted to NBT. There are,\nhowever, still some points to address:\n1. This revised statement is still too negative and general on the current state-of-the-art:\n“However, despite growing acceptance, current iPSC-CMs remain phenotypically immature in\nall aspects of CM-specific physiology…” There are multiple studies showing conditions for\npositive force-frequency for example, well-organized sarcomeres, t-tubules, action potentials of\nadult cardiomyocyte magnitude, etc. Perhaps defining “mature” would help: postnatal is what\nmost studies achieve with some physiological parameters “adult-like” although none typical of\naged cells. Please modify.\nWe have included additional context summarizing important advancements in iPSC-CM maturity\nby parameter in the introductory paragraph to highlight both the range and magnitude of change\nof morphological and functional metrics of interest, while still highlighting the gap between\nmaturing PSC-CMs and adult CMs in situ (Line 23):\nBoth disease phenotypes and drug responses are emergent composites of\ninteractions across multiple aspects of physiology 8,9, necessitating higher-fidelity\nin vitro models of myocardium. Important advances have been made in evoking\nphysiological maturation in both iPSC-CM monolayers and organoids in\ntranslational and preclinical applications 10–12, including transcriptomic or proteomic\nprofiles 13–30, morphometrics 13,15,27,29–31,16,18–22,25,26, metabolic profile 13,14,28–31,15,16,20–\n23,26,27, Ca2+ handling 16,19,29–31,20–27, electrophysiology 15,16,20,23,24,26,27,29–31, and\ndisease modeling 13,16,24,26,27,29,30,32,33. However, despite the advancements of many\nphysiological parameters of current iPSC-CMs to neonatal levels or beyond, they\nare remain phenotypically immature in all most aspects of CM-specific physiology;\nthey do not recapitulate the contractile force, morphology, electrophysiology, Ca2+\nhandling characteristics, or metabolic profile associated with adult myocardium 34,\nand are not satisfactorily predictive of patient responses to pharmaceuticals 35.\n2. Regarding the comments on the low and variable n in the electrophysiology data: this hardly\nseems to have increased. Just a different method of statistical analysis has been used: the only\ntext revision is “Data were analyzed by non-parametric Mann-Whitney U-test.”\nWe completed new experiments in which I measurements were made with larger sample sizes\nK1\n(Figure 2g). In keeping with Reviewer 2’s suggestion of comparing additional metrics against\nthe high-efficacy maturation medium of Feyen et al, we have additionally included this treatment\nin the experiment. These independent studies confirm our original findings (Line 133):\nMeasurement of I density slope between -115 and -95 mV further demonstrated\nK1\nsignificant differences between C16-treated cells to those treated with RPMI + B27\nor the high-performing medium of Feyen et al. 29 (Figure 2g).\n3. Otherwise, the work will be a useful addition to the literature in this area although the question\nof whether the algorithm is relevant for the maturation of other cell types remains open. You\ncould conclude that the algorithm has demonstrated its value for CM maturation and therefore\nits value is complete.\nWe thank the reviewer for their appreciation of the value of our work and for their constructive\nfeedback to improve it.\nREVIEWER #2:\nRemarks to the Author:\nIn the present study, Callaghan et al. presented a new formula for hPSC-CM maturation media\nand demonstrated that it promotes more mature hPSC-CM functional and morphological\nproperties. More specifically, cells appear to have increased size and improved striations,\nimproved calcium handling, electrophysiological and contractile properties, and better aerobic\nmetabolism. The authors have done a significant amount of revision work, and their culture\nmedium appears to convincingly promote a more mature hPSC-CM phenotype.\nHowever, there are three main residual concerns regarding their manuscript:\n(1) The transcriptomic data on treated cells is rather controversial at times, especially as it\nrelates to sarcomeric genes.\nWe agree that some of transcriptomic data, including certain sarcomeric genes, do not\ncompletely mirror the more immediately relevant proteomic or functional metrics. We discussed\nthis on Line 275:\nFurthermore, the transcriptional downregulation of key traditional markers of iPSC-CM\nmaturity such as MYH7 and TNNI3 in the C16 treatment was not reflected in the\nexpression of their protein products, suggesting that transcriptional assessment without\ncorresponding protein or functional verification may not be sufficient in determining the\nmaturation status of advanced iPSC-CM cultures.\nWe speculate that given the electrophysiological basis for non-spontaneous contraction in iPSC-\nCMs treated with C16 for 3 weeks, it is possible that decreased cyclic mechanical strain\ncontributed to lower turnover and therefore gene expression, despite the stability of\ncorresponding protein expression. We have added a short passage to this effect in the\ndiscussion (Line 278):\nAdditionally, given the electrophysiological basis for non-spontaneous contraction\nin the C16-treated iPSC-CMs, it is possible that decreased cyclic mechanical strain\ncontributed to lower turnover and therefore gene expression, despite the stability of\ncorresponding protein expression.\n(2) the authors do not provide any evidence using hPSCs from a disease model to prove the\napplicability of their approach.\nWe include in the Results (Line 100) and Supplementary Figure 1 a demonstration of differential\nfunction of a disease model based on CRISPR knockout of MYBPC3, which shows modified\ncontractile kinetics that vary based on maturation state, demonstrating the importance of iPSC-\nCM maturity in modeling cardiomyopathy.\nOptical contractile traces, obtained using particle image velocimetry, demonstrated…\npathologically slowed and biphasic contraction in a MYBPC3-KO CRISPR-generated\nPGPC17 mutant iPSC line (Figure S1g).\n(3) The main criticism of this work involves the comparisons of hPSC-CMs in C16 vs other\npreviously published maturation media. Although I do understand that repeating all experiments\nand analyses of cells grown in the Feyen et al. media protocol (which admittedly has had a\nmore consistent effect on maturation properties) is quite burdensome, this would significantly\nstrengthen the novelty and significance of this work. I think the authors should at least perform\nmorphometric/structural analyses and calcium transient recordings that are critical for the\nfunctional maturation of hPSC-CMs comparing C16 with the Feyen medium. Moreover, and\ngiven the discrepancy in the expression of sarcomeric proteins, it will be useful if the authors\ncould compare contractile properties of hPSC-CMs in Feyen vs C-16 media.\nTo complement our existing global proteomic data that comprehensively compare the Feyen\nmedium to C16 and other control media (Fig. 5), we performed new experiments to compare\nmorphometric and structural parameters including the Feyen medium (Figure 1f/h, Figure S1f)\n(Line 90):\nCompared to treatment with both STEMCELL and a leading high-efficacy maturation\nformulation by Feyen et al. 29, iPSC-CM monolayers treated with C16 medium exhibited\nincreased spontaneous alignment (circular variance p < 0.0001), cell elongation\n(eccentricity p = 0.0019), and intracellular organization (α-actinin:connexin-43 (Cx43)\ncolocalization coefficient p < 0.0001; Figure 1f), while confocal microscopy demonstrated\nthat C16 medium enhanced myofibrillar density, α-actinin striation, and Cx43 junctional\nexpression (Figure 1g). C16-treated cells demonstrated clear sarcomeric striations under\nphase-contrast in C16-treated monolayers (Figure S1d-e) with increased sarcomere\nlength (p = 0.0003; Figure 1h), as well as increased hypertrophy (projected area p <\n0.0001; perimeter p < 0.0001) and anisotropy (form factor p < 0.0001), but not α-actinin\nintegrated density (p = 0.91) (Figure S1f).\nIn expanding our electrophysiological experiments per the suggestions of Reviewer 1, we have\nnow included iPSC-CMs treated in parallel with the Feyen medium (Figure 2g). As the reviewer\nis aware, the medium of Feyen et al. is highly effective at arresting cell proliferation and in\nadvancing multiple metrics of maturity. We were unfortunately unable to replicate the extent of\nthe absolute magnitude of I current reported in the Feyen et al. paper (c.a. -7.5 pA/pF at -115\nK1\nmV), despite very favourable cell hypertrophy, morphological changes, contractility, and\nmetabolic performance. This discrepancy could be due to differences in cell lines, replating\ntechnique, or electrophysiological technique. Regardless, we were able to demonstrate outsized\nperformance of the Feyen medium relative to maintenance media and of C16 relative to both,\nwhich is included as Figure 2g as voltage-dependent slope of current density (Line 130):\nMeasurement of IK1 density slope between -115 and -95 mV further demonstrated\nsignificant differences between C16-treated cells to those treated with RPMI + B27 or\nthe high-performing medium of Feyen et al. 29 (Figure 2g).\nMinor comments:\nIndividual values should be added in each graph in Fig. 3n-u to assist the readers and reviewers\ninterpret the number of experiments performed.\nWe have added the individual values as suggested."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of my concerns. Although I remain somewhat skeptical about the comparisons of their approach to the Feyen et al. protocol and the novelty of their work, I do think that their methodology can likely have applications for the in vitro maturation of hPSC-CMs.", "reviewer_2": "The authors have made some useful additions to the manuscript which brings some improvement.\nRegarding points of reviewer 1:\n1. There are still some overstatements: eg \"and are not satisfactorily predictive of patient responses to pharmaceuticals (ref 35). There are multiple reports such as the CiPA study, showing for arrhythmias, monolayer hiPSC-CMs are predictive of drug responses. In addition Saleem et al (doi: 10.1093/toxsci/kfaa058) showed in Engineered Heart Tissues that contraction responses to drugs are >80% predictive of responses in humans cf ±70% in routinely-used primary rabbit cardiomyocytes. There are multiple other studies of this type. This would seem (more than) satisfactory especially as here neither an extensive screen of test compounds nor multiple diseased hiPSC-CMs have been examined.\nMinor points: phenotypically immature IN most aspects : IN has been deleted in error\nPlease use the conventional hiPSC and hiPSC-CMs for human iPSCs, throughout, as also reviewer 2. iPSC would normally refer to mouse iPSCs."}, "author_response": ""}]

Decision letter