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Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Naturevolume 556pages239243 (2018) | Download Citation

Abstract

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1,2,3,4,5,6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force–frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.

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Acknowledgements

The authors acknowledge funding support from the National Institutes of Health of the USA (NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); Columbia University MD/PhD program (S.P.M., T.C.); University of Minho MD/PhD program (D.T.); Japan Society for the Promotion of Science fellowship (K.M.); and Columbia University Stem Cell Initiative (D.S., L.S., M.Y.). We thank S. Duncan and B. Conklin for providing human iPSCs, M. B. Bouchard for assistance with image and video analysis, and L. Cohen-Gould for transmission electron microscopy services.

Reviewer information

Nature thanks T. Kamp and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Laboratory for Stem Cells and Tissue Engineering, Department of Biomedical Engineering, Columbia University, New York, NY, USA

    • Kacey Ronaldson-Bouchard
    • , Stephen P. Ma
    • , Keith Yeager
    • , Timothy Chen
    • , Dario Sirabella
    • , Diogo Teles
    •  & Gordana Vunjak-Novakovic
  2. Department of Rehabilitation and Regenerative Medicine, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA

    • LouJin Song
    • , Kumi Morikawa
    •  & Masayuki Yazawa
  3. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

    • Diogo Teles
  4. ICVS/3B’s, PT Government Associate Laboratory, Braga–Guimarães, Portugal

    • Diogo Teles
  5. Department of Medicine, Columbia University, New York, NY, USA

    • Gordana Vunjak-Novakovic

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Contributions

K.R.-B. and G.V.-N. designed the study. K.R.-B., K.Y. and G.V.-N. designed the tissue culture platform. K.R.-B., S.P.M., T.C. and D.T. cultured all tissue types with different stimulation conditions, and performed real-time and end-point assessments of tissue properties (including gene expression, histomorphology, ultrastructure, distributions of cardiac proteins, contractile behaviour and calcium handling). K.R.-B., S.P.M., T.C. and D.T. independently replicated the entire process of tissue cultivation and assessment. D.S. and L.S. expanded iPSCs and derived cardiomyocytes. K.R.-B. performed immunostaining for the presence of T-tubules. M.Y., K.M. and L.S. conducted single-cell dissociation and electrophysiology experiments. K.R.-B., S.P.M., T.C., M.Y. and G.V.-N. interpreted data and wrote the manuscript.

Competing interests

G.V.-N. and K.R.-B. are co-founders of TARA Biosystems, a Columbia University spin-off that is commercializing the use of bioengineered human cardiac tissue for drug testing.

Corresponding author

Correspondence to Gordana Vunjak-Novakovic.

Extended data figures and tables

  1. Extended Data Fig. 1 Experimental design and overall appearance of cardiac tissues.

    a, Schematic of the pillars (purple) placed via interlocking mating components between the bioreactor wells (grey) and pillar lid (yellow) with tissues (pink) formed around the pillars, and electrodes (black) placed perpendicular to the cardiac tissues. A glass slide (blue) is epoxied to the bottom of the bioreactor to enable image acquisition. b, A schematic of the assembled bioreactor. c, Photographs of the cardiac tissues cultured within the bioreactor. d, The tissue pillar. e, Increase in the electrical stimulation frequency throughout the intensity training regime. fh, Photographs of the tissues attached to pillars at the end of four-week cultivation: side view (f, g), bottom view (h). Scale bars, 500 µm. ij, Immunofluorescence in serial sections of the early-stage intensity-trained tissue. The dotted yellow and red lines in g, i, j indicate corresponding pillar placement within the tissue. Scale bars, 500 µm. kp, Immunofluorescence in serial sections of the early-stage intensity-trained tissue in i. WGA, green; α-actinin, pink; nuclei, blue. Scale bars, il, 500 µm; m, 100 µm; n, 20 µm; o, p, 50 µm. Images were selected to include landmark features that facilitate localization and comparisons. Similar results were obtained from three independent experiments.

  2. Extended Data Fig. 2 Enhanced gene expression and conduction in intensity-trained cardiac tissues over time.

    a, Quantitative gene expression in FCTs and C2A iPSC cardiac tissues after two weeks of culture, as determined by RT–PCR; shown as fold change relative to late-stage tissues at the start of stimulation. b, Quantitative gene expression in early-stage cardiac tissues, normalized to GAPDH, from three different iPSC lines as determined by RT–PCR after four weeks of culture. n = 12 biologically independent samples per group; mean ± 95% CI; no significant difference between the cell lines by two-way ANOVA. cf, Representative conduction velocity activation maps for early-stage control (c), late-stage intensity-trained (d) and early-stage intensity-trained (e) cardiac tissues, and surrogate of conduction velocity in early-stage and late-stage C2A iPSC cardiac tissues after four weeks of culture, assessed by calcium propagation (f). Mean ± s.e.m., n = 4–5 biologically independent samples per group. g, h, Representative immunofluorescence of gap junction (connexin-43 (Cx43), green) expression in early-stage intensity-trained iPSC cardiac tissue after four weeks of culture, at low (g; scale bar, 10 µm) and high magnification (h; scale bar, 5 µm). cTnT, red; nuclei (DAPI), blue. Similar results were obtained from four independent experiments.

  3. Extended Data Fig. 3 Electrophysiological characterization of human engineered cardiomyocytes.

    a, Representative traces of action potentials in early-stage control (n = 9), late-stage intensity-trained (n = 9) and early-stage intensity-trained (n = 14) groups. n is the number of biologically independent samples obtained during two independent experiments. b, Representative traces of IK1 current for the early-stage intensity-trained group using voltage-clamp mode. cf, Electrophysiology data after four weeks of culture showing the resting membrane potential (c), peak amplitude (d), duration of action potential (e) and upstroke velocity (f) obtained in two independent experiments, resulting in biologically independent data from early-stage control (n = 9), late-stage intensity-trained (n = 9) and early-stage intensity-trained (n = 14) groups. **P < 0.01, *P < 0.05 using one-way ANOVA Bartlett’s test with multiple comparison. n.s., not significant. gi, Representative continuous organ bath force recordings under electrical pacing from 1–6 Hz from three biologically independent early-stage intensity trained tissues (C2A cells) from one experiment. j, k, Representative continuous recordings from an early-stage intensity-trained tissue (C2A cells) under electrical pacing from 1–6 Hz of calcium (j) and surrogate force (k) as determined by tissue displacement, normalized to 1 Hz.

  4. Extended Data Fig. 4 Enhanced maturation and synchronicity of cardiac tissues in response to training regime as a function of time.

    ac, Representative contraction profiles of FCT (a), early-stage (b) and late-stage cardiac tissues (c) over time (C2A cell line). d, Frequency of contractions in cardiac tissues over four weeks of culture. n = 35 biologically independent samples over 16 independent experiments; mean ± 95% CI, *P < 0.05 compared to control group by two-way ANOVA with Tukey’s HSD test. Early-stage intensity-trained tissue shows significant differences versus other training regimes by two-way ANOVA with Tukey’s HSD test. e, Characterization of cardiac cell population within cardiac tissues (C2A line) after four weeks of culture by fluorescence-activated cell sorting (FACS) analysis. f, g, Characterization of cells isolated from early-stage intensity-trained cardiac tissues (C2A line) by FACS analysis after four weeks of culture: cardiac cells (f; cTnT), and supporting fibroblast cells and endothelial cells (g; vimentin and von Willebrand Factor(vWF), respectively). hj, Representative immunofluorescence of whole tissues showing the enhanced cardiac ultrastructure (α-actinin, green; cTnT, red; nuclei, blue) in early-stage cardiac tissues from the C2A line (h), WTC11 cell line (i), and IMR90 cell line (j) after four weeks of culture. Scale bars, 5 µm; experiment repeated independently 14 times with similar results. k, Representative immunofluorescence showing the cell population in a histological section from early-stage cardiac tissue (C2A line) after four weeks of culture. cTnT, green; vimentin, red; nuclei, blue. Scale bar, 50 µm; experiment repeated independently two times with similar results.

  5. Extended Data Fig. 5 Physiological hypertrophy within cardiac tissues enhances contractility.

    ac, Physiological hypertrophy of cardiomyocytes cultured in the electromechanically conditioned cardiac tissue format increases as a function of time and training regime beyond FCT levels, as shown by cell elongation ratio (a) and sarcomere length (b). n = 326 biological replicates from 15 independent samples in one experiment. c, This enables the change in area while being electrically paced at 1 Hz, an indirect measure of fractional shortening, to similarly increase beyond FCT levels as a function of time and training regime. Data represent the ratio of the change in area for a given time point and the change in area at day 6. n = 6 biologically independent samples per group; mean ± 95% CI; *P < 0.05 compared to FCT group at week four by ANOVA with Tukey’s HSD test; line above graph indicates P < 0.05 compared to other training regimes by two-way ANOVA with Tukey’s HSD test. d, The enhanced cardiac ultrastructure in intensity-trained early-stage cardiac tissues is documented by the quantification of sarcomere distribution in cardiac tissues. n = 12 biologically independent samples per group, mean ± 95% CI. e, f, Representative immunofluorescence of gap junction (connexin-43 (Cx43), white) in early-stage iPSC cardiac tissue (β-myosin heavy chain (β-MHC), green; cTnT, red; nuclei (DAPI), blue) (e) and cardiac ultrastructure in early-stage iPSC cardiac tissue (α-actinin, green; cTnT, red; nuclei (DAPI), blue) (f) after four weeks of culture. Scale bar, 50 µm; experiment repeated independently three times with similar results. g, α-Actinin immunofluorescence (white) in cardiac tissues after four weeks of culture. Scale bar, 10 µm; experiment repeated independently two times with similar results.

  6. Extended Data Fig. 6 Enhanced ultrastructural properties of cardiac tissues following intensity training.

    a, Representative transmission electron microscopy images for FCTs, adult cardiac tissue, and early-stage cardiac tissues (C2A line) using different electromechanical conditioning protocols, after four weeks of culture. Scale bar, 500 nm. b, TEM images of intensity-trained early-stage cardiac tissues (C2A line) after four weeks of culture showing details of various ultrastructural elements, Scale bar, 500 nm. Similar results to those in a and b were obtained independently with the following cells or treatments: FCT (n = 8), adult (n = 2), control (n = 3), constant (n = 3), intensity-trained (n = 4).

  7. Extended Data Fig. 7 Intensity training of cardiac tissues derived from early-stage hiPS-CMs is required to enhance mitochondrial development.

    a, Representative immunofluorescence showing ultrastructural proteins WGA (green), α-actinin (red), mitochondria (blue) and oxidative phosphorylation (yellow) for early-stage cardiac tissues (C2A cell line) at different culture times during exposure to the intensity-training electromechanical-conditioning regime. Scale bar, 20 µm. b, Representative immunofluorescence showing ultrastructural proteins WGA (green), α-actinin (red), mitochondria (blue) and oxidative phosphorylation (yellow) in cardiac tissues cultured with intensity training for four weeks from early-stage hiPS-CMs (C2A cell line), late-stage hiPS-CMs (C2A cell line) and GW19 FCT. Scale bar, 20 µm. Similar results to those in a and b were obtained independently from the following experiments: FCT (n = 5), early-stage intensity-trained (n = 3), late-stage intensity-trained (n = 3). c, d, Representative TEM images for early-stage (c) and late-stage cardiac tissues (d) (C2A cell line) after two weeks of exposure to the intensity-training electromechanical-conditioning regime. Scale bar, 1 µm; experiment not repeated independently.

  8. Extended Data Fig. 8 Formation of T-tubules in early-stage intensity-trained cardiac tissues.

    ae, Axial tissue cross-sections from intensity-trained cardiac tissues (C2A line) after four weeks of culture showing T-tubules (WGA, green) and nuclei (DAPI, blue) at low magnification (a; scale bar, 100 µm), medium magnification (b and c; scale bar, 10 µm) and high magnification (d and e; scale bar, 5 µm). f, g, Axial tissue cross-sections showing T-tubules (WGA, green), actin (red) and DAPI (blue) in intensity-trained cardiac tissues (C2A line) after four weeks of culture (f) and GW19 FCT (g). Scale bar, 10 µm. h, Immunofluorescence of paraffin-embedded and sectioned cardiac tissues from three different iPSC cell lines (C2A, WTC11, IMR90) after four weeks of intensity training showing the formation of T-tubules (confirmed by both WGA staining and di-8-ANEPPS staining), and striated ultrastructure (actin). Scale bar, 10 µm. Similar results to those in ah were obtained in a minimum of four independent experiments.

  9. Extended Data Fig. 9 Intensity training upregulates cardiac maturation in early-stage tissues through enhanced calcium handling.

    a, b, Intensity training promotes T-tubule formation in early-stage hiPS-CM tissues, as demonstrated by immunofluorescence of ryanodine 2 receptor (RYR2, green), bridging integrator 1 (BIN1, blue) and T-tubule staining (di-8-ANEPPs, red). Scale bar, 10 µm. c, Expression of ATP2A2 and SLC8A1 genes, which are responsible for maintaining proper calcium homeostasis, in early-stage tissues as determined by RT–PCR and normalized to GAPDH over four weeks of culture with the designated stimulation regime. Independent biological replicates per group: FCT, n = 8; control, n = 6; constant, n = 6; intensity-trained, n = 14; adult, n = 1. Mean ± 95% CI, *P < 0.05 versus FCT group at week four by ANOVA with Tukey’s HSD test. Line over graph indicates P < 0.05 compared to other training regimes by two-way ANOVA with Tukey’s HSD test. d, Relaxation times in early-stage tissues as characterized by the full-width half-maximum (FWHM) values and the decay time (90% of the time from the maximal peak of the calcium transient). Independent biological replicates per group: FCT, n = 8; C2A, n = 12; WTC11, n = 6; IMR90, n = 6. Mean ± 95% CI; *P < 0.05 versus FCT group by ANOVA with Tukey’s HSD. Line over graph indicates P < 0.05 between cell lines by two-way ANOVA. e, Representative calcium traces of early-stage tissues treated with 1 µM nifedipine. f, g, Representative traces of calcium release after stimulation with 5 mM caffeine in early-stage tissues and FCTs treated with 1 mM verapamil (f) or 2 µM thapsigargin (g). h, Representative traces of calcium release after stimulation with 5 mM caffeine for early-stage tissues and FCTs. i, Calcium spikes detected by fluorescent calcium dyes in early and late-stage tissues (C2A line) after four weeks of culture at two calcium concentrations. j, Intensity-trained early-stage but not late-stage tissues (C2A line) after four weeks of culture respond to ryanodine (1 µmol l−1). k, The force–frequency relationship of early-stage intensity-trained cardiac tissues (C2A line) after four weeks of culture, treated with the RYR2 blocker ryanodine (1 µM) or the SERCA2a blocker thapsigargin (1 µM). Directly measured force data; n = 13 biologically independent samples for intensity group and n = 3 biologically independent samples for other groups. Mean ± 95% CI, line over graph indicates P < 0.05 by two-way ANOVA.

  10. Extended Data Fig. 10 Intensity training in early-stage tissues enables physiologically relevant drug responses and the development of a pathological hypertrophy disease model.

    a, b, Calcium intensity measurements (a) and relaxation time obtained by measuring the time from the peak to 90% of the relaxation (R90) during electrical pacing (b) at 1 Hz in early-stage intensity-trained tissues (C2A line) after four weeks of culture with increasing doses of isoproterenol. n = 20 biological replicates from six independent experiments. Mean ± 95% CI; *P < 0.05 versus baseline response by ANOVA with Tukey’s HSD test. c, Cell area over four weeks of culture for the designated stimulation regime. n = 10 biological replicates from five independent experiments. Mean ± 95% CI; line above graph indicates P < 0.05 compared to other training regimes by two-way ANOVA with Tukey’s HSD test. d, Frequency of contractions in healthy (C2A) and hypertrophic (HCM) heart tissues over four weeks of culture. n = 12 independent biological samples from five independent experiments. Mean ± 95% CI. e, Relaxation times in early-stage tissues (C2A line) and early-stage hypertrophy tissues (HCM) as characterized by FWHM values and the decay time (90% of the time from the maximal peak of the calcium transient). n = 20 biological replicates from four independent experiments. Mean ± 95% CI; line above graphs indicate P < 0.05 compared to other training regimes by two-way ANOVA with Tukey’s HSD test. f, Early-stage intensity-trained hypertrophy tissues exhibit impaired FDAR, as shown for each stimulation frequency by individual traces of calcium peaks.

Supplementary information

  1. Reporting Summary

  2. Supplementary Data

    This file contains detailed sample sizes and the number of independent experiments performed for data presented in the main figures

  3. Supplementary Data

    This file contains a detailed list of primers used

  4. Video 1: Early-stage intensity trained cardiac tissues after 4 weeks of culture

  5. Video 2: Late-stage intensity trained cardiac tissues after 4 weeks of culture

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