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

Figures

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  Fig. 1. Iterative development and performance of hiPSC-CM maturation medium candidates relative to existing high-performing control formulations. a Visual representation of High Dimensional, Differential Evolution (HD-DE) algorithm workflow; from a quasi-random distribution of vectors (formulations) in an extensive but discrete solution space, low-performing candidates are discontinued while high-performing candidates are kept and iterated using mutation and crossover operations. b Ranked performance of all tested formulation candidates (n = 169 unique vectors over 204 instances) against STEMCELL Technologies Cardiomyocyte Maintenance Medium (dashed line), using respirometric uncoupled-control ratio as an objective metric. c Soluble additive factor doses (y-axes) by their tested levels (x-axes) included in the HD-DE optimization, demonstrating exponential coverage of solution space over 17 dimensions. d Final composition of C16 hiPSC-CM maturation medium by factor (orange), superimposed over factor levels of the top 10 formulation candidates in the optimization phase (gray violin plots), and analogous reference levels, where applicable, from the literature (dark gray) or in the commonly…

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  Fig. 2. Electrophysiological enhancement of hiPSC-CMs matured using C16 medium. a Sample traces of intracellular electrode recordings of hiPSC-CMs treated with RPMI + B27 (left) or C16 maturation medium (right) for 6 weeks, in neat Tyrode’s solution, followed by sequential supplementation with 100 and 400 mol L−1 BaCl2, before eventual washout. b Minimum diastolic potentials (resting membrane potentials) from intracellular recording in cell sheets, with or without 400 µM BaCl2 perfusion (n = 31 and 7 biological replicates for RPMI + B27 without and with BaCl2, and 16 and 3 biological replicates for C16 without and with BaCl2). c Spontaneous action potential firing rates measured during action potential recordings, with or without 400 µmol L−1 BaCl2 perfusion (n = 8 and 3 biological replicates, respectively, for RPMI + B27 and C16 treatments). d Inward rectifier (IK1) currents in neat Tyrode’s and supplemented with 400 µmol L−1 BaCl2; inset indicates voltage protocol and scale. Voltage sensitivity curves (e) of IK1 (inset demonstrates positive component of IK1 at high voltage) and IK1 current density at −115 mV (f) (n = 8 and 4, respectively). g IK1 current density slope between −115 and…

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  Fig. 3. Functional enhancement of hiPSC-CMs matured in C16 medium. a Representative line-scanning Fluo-4 traces during steady-state Ca2+ transients at 1 Hz pacing of single hiPSC-CMs treated 6 weeks with C16 maturation medium compared to high-performing control hiPSC-CM media (iCell Cardiomyocytes Maintenance Medium, RPMI + B27, and STEMCELL Cardiomyocyte Maintenance Medium); kinetic metrics isolated from trace sets include time to peak (b), and times to 10% (c), 25% (d), and 50% (e) decay from peak fluorescence, respectively. Comparisons between high-performing comparative treatments vs. the C16 formulation by Brown-Forsythe ANOVA to account for unequal variances between treatments and Dunnett’s multiple comparisons post-hoc test. n = 54 individual cells for C16, n = 47 for iCell, n = 38 for RPMI, and n = 37 for STEMCELL. Fluo-4-enabled Ca2+ traces of C16 (f) and STEMCELL (g)-treated cultures after 6 weeks with steady-state pacing at 1 Hz, followed by sequential additions of verapamil (20 µmol L−1) and caffeine (10 mmol L−1). h Fractional Fluo-4-enabled sarcoplasmic reticulum (SR)-derived calcium load upon addition of caffeine normalized to steady-state transient amplitudes for each…

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  Fig. 4. Transcriptomic profiling of matured hiPSC-CMs and RNA sequencing-enabled meta-analysis to ex vivo tissues and historical maturation experiments. a Principal component analysis of hiPSC-CMs cultured in C16 medium or a high-performing control formulation for 6 weeks, or at d20 of differentiation and prior to treatment (control). Colors denote different sample groups. b–j Heatmaps displaying expression of select genes from the GO terms; top 5 upregulated (red) and downregulated (blue) genes within C16-treated cells per GO term, listed with their respective Z scores. n = 3 biological replicates per treatment. Head-to-head treatment comparisons found in Supplementary Figs. 2, 3. k, l Meta-analysis of bulk RNA sequencing of hiPSC-CM in vitro maturation compared to native CMs at fetal, infant, and adult life stages, including tSNE (k) and principal component analysis (l). Sample descriptions found in Supplementary Table 3; validation found in Supplementary Fig. 4, principal component attribution in Supplementary Fig. 5, and cluster analysis in Supplementary Fig. 6.

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  Fig. 5. Global proteomic profiling of C16-treated hiPSC-CMs relative to high-performing control formulations. a Principal component analysis of differential protein expression in hiPSC-CMs after 6 weeks of treatment in C16, iCell, RPMI + B27, or STEMCELL media, as well as a top-performing formulation from the literature21. b Heat map demonstrating stratification of treatments by protein regulation. c Over-representation analysis of the top 5 GO terms differentially expressed in C16-treated hiPSC-CMs compared to other treatments for each cluster in panel (b); GO term size (in number of gene products) represented both by marker size and size on X-axis.

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  Fig. 6. Contractile enhancement of engineered myocardial tissues cultured in C16 maturation medium. a Decrease in tissue contractile spontaneity after 3 weeks of culture in C16 media (n = 4 independent experiments, each comprising 5–8 individual tissues representing technical replicates). b Progressive absolute twitch force increased over 3 weeks of Biowire II culture in C16 or RPMI + B27 medium formulations, with or without continuous electrical field stimulation (1 Hz). c Positive force-frequency relationship of contractile displacement of polymeric beams summatively increases with both medium- and electrical stimulation-mediated maturation of tissues after 3 weeks of tissue treatment in C16 or RPMI + B27 media; measurements are normalized to 1 Hz stimulation of the relevant treatment condition (dotted line). For panels 6b and 6c, n = 9 biological replicates, except n = 11 for the C16 + stimulation condition. d Confocal microscopy reveals differential expression of SERCA2a (magenta) and DHPR (yellow) in electrically-stimulated Biowires cultured for 3 weeks in C16 or RPMI + B27 media. e TEM of longitudinal and transverse sections of C16-treated Biowires induces sarcomeric development…