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Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips

Nature Biomedical Engineering volume 6, pages 372–388 (2022)Cite this article

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Abstract

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells’ action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells’ mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300–450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

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Fig. 1: Optimized hiPSC-CM MPS.
Fig. 2: Action-potential characterization of matured cardiac MPS.
Fig. 3: Mitochondrial morphology of MPS treated with MM.
Fig. 4: Inotropic response of MM-treated MPS.
Fig. 5: Gene expression analysis of monolayers and MPS treated with lipid-based MM.
Fig. 6: Mathematical modelling of the contribution of individual currents and calcium handling machinery to the action potential of monolayers and MPS.
Fig. 7: Pro-arrhythmia pharmacology of matured WTC cardiac MPS.
Fig. 8: Pro-arrhythmia pharmacology of matured SCVI20 cardiac MPS.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. All raw and analysed datasets generated during the study are available from the corresponding author on request. Source data are provided with this paper.

Code availability

The microphysiological systems and monolayer physiology were analysed with an open-source motion-tracking software available for download at huebschlab.wustl.edu. Calcium transients, action-potential waveforms and sarcomere regularity were analysed with in-house code that is available on request from the corresponding author.

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Acknowledgements

This work was funded in part by the California Institute for Regenerative Medicine DISC2-10090 (K.E.H.), NIH-NHLBI HL130417 (K.E.H.), NIH-NIGMS R35GM1195855 (E.W.M.), NIH-NIGMS T32GM066698 (S.C.B.), the Research Council of Norway INTPART Project 249885, the SUURPh programme funded by the Norwegian Ministry of Education and Research, and the Peder Sather Center for Advanced Study (UC Berkeley). We thank M. West (UC Berkeley) for assistance with image analysis and flow cytometry; S. Weber (Technische Universität Dresden) and S. Renschler (Washington University in St. Louis) for helpful advice on RNA isolation, cDNA amplification and data analysis; Y. Rudy, J. Nerbonne, J. Silva and J. Cui (Washington University in St. Louis) for critical discussions on action potential acquisition, mathematical modelling and data analysis; B. Conklin (Gladstone Institutes, San Francisco, USA) for technical advice on the WTC iPSC line; J. Wu (Stanford University) and the Stanford University Cardiovascular BioBank for providing the SCVI20 and SCVI273 iPSC lines and providing technical advice regarding these lines; and the Barcelona Stem Cell Institute for providing the G15.AO line.

Author information

Author notes

  1. Nathaniel Huebsch

    Present address: Department of Biomedical Engineering, The Washington University in Saint Louis, Saint Louis, MO, USA

  2. These authors contributed equally: Nathaniel Huebsch, Berenice Charrez.

Authors and Affiliations

  1. Department of Bioengineering and California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, CA, USA

    Nathaniel Huebsch, Berenice Charrez, Gabriel Neiman, Brian Siemons, Verena Charwat, David Cleres, Felipe T. Lee-Montiel, Nicholas C. Jeffreys, Nikhil Deveshwar & Kevin E. Healy

  2. Department of Chemistry, University of California at Berkeley, Berkeley, CA, USA

    Steven C. Boggess & Evan W. Miller

  3. Simula Research Laboratory, Oslo, Norway

    Samuel Wall, Karoline H. Jæger, Åshild Telle & Aslak Tveito

  4. Department of Pharmacology, School of Medicine, University of California, Davis, CA, USA

    Andrew G. Edwards

  5. Department of Pathology, New York University Langone Health and Medical School, New York, NY, USA

    Jonathan Serrano & Matija Snuderl

  6. Department of Nutritional Sciences and Toxicology, University of California at Berkeley, Berkeley, CA, USA

    Andreas Stahl

  7. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA

    Evan W. Miller

  8. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA

    Evan W. Miller

  9. Department of Materials Science and Engineering, University of California at Berkeley, Berkeley, CA, USA

    Kevin E. Healy

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Contributions

N.H., B.C., G.N., A.G.E. and K.E.H. designed experimental studies. N.H., B.C., G.N., B.S., S.C.B., V.C., F.T.L.-M., N.C.J. and N.D. performed experimental studies and iPSC cultures. S.W., K.H.J., Å. Telle and A. Tveito designed and executed computational modelling studies to predict molecular changes in voltage and calcium handling underlying MM-induced physiology changes. B.C. and D.C. created software routines to automate analysis of the force produced by cardiac microtissues. J.S. and M.S. designed and assisted in gene expression analysis studies. K.E.H., A.G.E. and S.W. guided drug-probe-based studies on cardiomyocyte electrophysiology and calcium handling in MPS. E.W.M. and S.C.B. synthesized BeRST-1 dye for action potential analysis. A.S. advised on cell metabolism. K.E.H., N.H., A.G.E. and B.C. wrote the paper. K.E.H. supervised and funded the research.

Corresponding author

Correspondence to Kevin E. Healy.

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Competing interests

K.E.H., A.E., N.H., S.W., B.S. and V.C. have a financial relationship with Organos Inc., and hence may benefit from the commercialization of the results of this research. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Development and characterization of iPSC-stromal cells.

A) Differentiation tree depicting the lineage of iPSC-cardiomyocytes (hiPS-CM) and the isogenic hiPSC-derived stromal cell population (hiPSC-SC). Specific biomarkers (blue) were verified by qRT-PCR. B) Brewer plot identifying gene expression patterns over the course of differentiation of hiPSC into hiPSC-SC, with hiPSC-CM included on the plot for comparison. C) Immunofluorescence molecular characterization of hiPSC-SC. These hiPSC-SC were markedly positive for all stromal markers shown, while markers of smooth muscle (Calponin) and endothelial cells (CD31) were not detected. iPS-SC also produce key ECM proteins: Laminin, Fibronectin and Collagen IV, while substantial Collagen I was not detected. D) Representative fluorescence micrograph of cardiac MPS, which shows highly organized, aligned cardiomyocytes stained for Sarcomeric α-Actinin (ACTN2, green). Cells were counterstained for nuclei with DAPI (blue) in all fluorescence micrographs. Scale bars: C) 500 µm D) left panel, 20 µm and right panel, 10 µm.

Source data

Extended Data Fig. 2 Design-of-experiments (DoE) based screens identify maturation media for hiPSC-CM microphysiological systems.

A) Approach used for initial screen. Computational motion capture is performed on bright-field videos of contracting cardiac MPS, giving the beating interval (defined as the distance between peaks in motion speed for contraction and relaxation, which approximates the interval over which displacement occurs). The knock-in reporter, GCaMP6f, is used to monitor the timing (rate corrected Full-Width-Half-Maximum, FWHM and amplitude of calcium transients in MPS. B-F) Results from representative L9 Taguchi Array experiments, depicting B) beating interval, C) spontaneous beating frequency, and D) tissue beating prevalence, all obtained from motion tracking analysis, along with calcium transient E) FWHM and F) amplitude, obtained from analysis of GCaMP6f fluorescence. Based on 1-way ANOVA tests, glucose levels significantly impact beating interval (p < 0.05) along with spontaneous beat rate, beating prevalence at 1hz, and GCaMP amplitude at 1Hz (p < 0.01). BSA levels had a significant impact on beating interval (p < 0.05) and GCaMP FWHM at 1Hz (p < 0.01). MPS were cultured for ten days prior to analysis for the L9 experiments. G-H) Comparison of MPS cultured in standard media (red) to MPS cultured in glucose-free media during L9 studies (black), and MPS cultured in the final Maturation Media (blue) when assessed for (H) beating prevalence at 1hz or (I) beating interval. Data: B-F: plot of mean ± SEM, n = 9 MPS per group; G-H: all data points with mean ± SD, n = 12 for SM group, n = 10 for MM group, and n = 3-4 for all other groups, except for beating interval in media 8, which could only be calculated in n = 1 sample (no other samples cultured in this media exhibited either spontaneous or paced beating). p values reflect values obtained from post-hoc Holm Sidak test after confirming global significance across groups with 1-way ANOVA.

Source data

Extended Data Fig. 3 Additional information about the media screen and representative 2D traces of MM-treated monolayers.

A-B) Representative action-potential tracings for 2D monolayers of A) WTC and B) SCVI20 cell lines cultured for one week in Maturation Media (MM). C-D) Quantification of action potential time from 20% above baseline to peak (Upstroke80) in C) WTC MPS (n = 5 per group) and 2D monolayers (n = 21 per group) and D) SCVI20 MPS (n = 60-63 per group). E-F) Quantification of C) beat-rate corrected cAPD80 and (MM: n = 16, SM: n = 10 per group); D) background corrected calcium amplitude (F/F0) in SCVI273 MPS (MM: n = 20, SM: n = 25 per group). G-H) Analysis of changes in G) cAPD80 (MM: n = 13, SM: n = 12 per group) and H) contractile prevalence in MM-pre-treated MPS that resulted from modulating the levels of palmitate and albumin in MM (MM: n = 9, SM: n = 12 per group). Removing both palmitate and albumin from MM (M1) resulted in MPS with APD80 that were significantly higher than APD80 of MM-treated MPS, and which were no different from APD80 of SM-treated MPS. Removal of Palmitate alone (M2), or of albumin alone (M3) led to a new medium that exhibited APD80 significantly less than MM (p < 0.05). Compared to MM, M2 treated MPS exhibited slightly reduced beating prevalence, whereas this metric was enhanced for M3 treated MPS, although these changes were not statistically significant. Slight reduction of the albumin content of MM from 2.5% to 1% (M4) did not have significant effects on APD80 or prevalence of motion in MPS, compared to those treated with MM. All data: plot of all points with mean ± SD. p values from post-Hoc Holm-Bonferonni test after confirming global significance with 1-way ANOVA.

Source data

Extended Data Fig. 4 Action-potential duration and beat rate in MPS of four genotypes.

A) Randomly selected subset of data on rate-corrected APD80 (cAPD80) and B) Spontaneous beat rate for a representative set of MPS for all four genotypes, pre-treated for 10 days with either SM or MM. p values from t-test with Welch’s correction for non-constant standard deviation.

Source data

Extended Data Fig. 5 Extended mitochondrial and metabolic analysis of MPS and 2D iPSC-CM monolayers.

A-D) Representative images of A-B) SCVI20 and C-D) G15.AO MPS after treatment for 10 days with either (A,C) MM or (B,D) SM. MPS are stained for Mitotracker (red) or anti-mitochondrial antibodies (green) with Draq5 nuclear counterstain (blue). E) Representative Oxygen Consumption Rate (OCR) tracings of SCVI20 iPSC-CM monolayers after culture in MM (blue) or SM (red) for 10 days. F,G) Quantification of reserve OCR (change in OCR from baseline with FCCP treatment) and total ATP capacity indicate a shift toward ß-oxidation with MM in 2D monolayers (n = 4 independent wells per group). H-K) Representative images of 2D H,I) WTC and J,K) SCVI20 iPSC-CM monolayers stained with MitoTrackerRed (left; red) and anti-mitochondrial antibodies (right; green). Scale bars: 20μm.

Source data

Extended Data Fig. 6 Expression and localization of sarcomere proteins in MM-treated MPS.

A-B) Fourier domain-based quantification of sarcomeric order in MPS treated with SM versus MM for (A) WTC or (B) SCVI20 cell line treated with MM (blue) or SM (red) for 10 days (n = 9 independent MPS per group). C,D) Quantification of protein expression (antibody staining with analysis by a condition-blinded user) for C) MYH7 (n = 8 independent MPS per group; WTC, open squares and circles; SCVI20, closed squares and circles) and D) MLC-2v (n = 3 independent MPS per group; SCVI20) in MPS treated for ten days with MM (blue) or SM (red). E-G) Representative micrographs of E) WTC and F,G) SCVI20 MPS after staining for E,F) MYH7 and G) MLC-2v. Error bars represent mean ± SD. Antibody staining in green, with blue Draq5 nuclear counterstain. Scale bars: 20μm.

Source data

Extended Data Fig. 7 Representative action-potential changes in MPS exposed to verapamil, flecainide and alfuzosin after MM pre-treatment.

Representative, intensity normalized action-potential traces are depicted for (A,C,E) WTC and (B,D,F) SCVI MPS pre-treated with MM and exposed to escalating doses of (A,B) verapamil, (C,D) flecainide, or (E,F) alfuzosin.

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Huebsch, N., Charrez, B., Neiman, G. et al. Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips. Nat. Biomed. Eng 6, 372–388 (2022). https://doi.org/10.1038/s41551-022-00884-4

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