Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair

doi:10.1161/CIRCULATIONAHA.116.024145

. 2017 May 9;135(19):1832-1847.

doi: 10.1161/CIRCULATIONAHA.116.024145. Epub 2017 Feb 6.

Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair

Malte Tiburcy¹, James E Hudson¹, Paul Balfanz¹, Susanne Schlick¹, Tim Meyer¹, Mei-Ling Chang Liao¹, Elif Levent¹, Farah Raad¹, Sebastian Zeidler¹, Edgar Wingender¹, Johannes Riegler¹, Mouer Wang¹, Joseph D Gold¹, Izhak Kehat¹, Erich Wettwer¹, Ursula Ravens¹, Pieterjan Dierickx¹, Linda W van Laake¹, Marie Jose Goumans¹, Sara Khadjeh¹, Karl Toischer¹, Gerd Hasenfuss¹, Larry A Couture¹, Andreas Unger¹, Wolfgang A Linke¹, Toshiyuki Araki¹, Benjamin Neel¹, Gordon Keller¹, Lior Gepstein¹, Joseph C Wu¹, Wolfram-Hubertus Zimmermann²

Affiliations

¹ From Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wettwer, W.-H.Z.); German Center for Cardiovascular Research (DZHK), partner site Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wingender, W.A.L., W.-H.Z.); Institute of Bioinformatics, University Medical Center Göttingen, Germany (S.Z., E. Wingender); Stanford Cardiovascular Institute (J.R., M.W., J.D.G., J.C.W.) and Department of Radiology (J.D.G., J.C.W.), Molecular Imaging Program, Stanford University School of Medicine, CA; The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Technion-Israel Institute of Technology, Haifa (I.K., L.G.); Institute of Pharmacology and Toxicology, Technical University Dresden, Germany (E. Wettwer, U.R.); University Medical Center Utrecht and Hubrecht Institute, The Netherlands (P.D., L.W.v.L.); Leiden University Medical Center, The Netherlands (M.J.G.); Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany (S.K., K.T., G.H., W.A.L.); Center for Applied Technology, Beckman Research Institute, City of Hope, Duarte, CA (L.A.C.); Department of Cardiovascular Physiology, Institute of Physiology, Ruhr University Bochum, Bochum, Germany (A.U., W.A.L.); New Laura and Isaac Perlmutter Cancer Center at New York University Langone (T.A., B.N.); and McEwen Centre for Regenerative Medicine, Toronto, Canada (G.K.). The current address for Dr Hudson is Laboratory for Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Australia.
² From Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wettwer, W.-H.Z.); German Center for Cardiovascular Research (DZHK), partner site Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wingender, W.A.L., W.-H.Z.); Institute of Bioinformatics, University Medical Center Göttingen, Germany (S.Z., E. Wingender); Stanford Cardiovascular Institute (J.R., M.W., J.D.G., J.C.W.) and Department of Radiology (J.D.G., J.C.W.), Molecular Imaging Program, Stanford University School of Medicine, CA; The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Technion-Israel Institute of Technology, Haifa (I.K., L.G.); Institute of Pharmacology and Toxicology, Technical University Dresden, Germany (E. Wettwer, U.R.); University Medical Center Utrecht and Hubrecht Institute, The Netherlands (P.D., L.W.v.L.); Leiden University Medical Center, The Netherlands (M.J.G.); Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany (S.K., K.T., G.H., W.A.L.); Center for Applied Technology, Beckman Research Institute, City of Hope, Duarte, CA (L.A.C.); Department of Cardiovascular Physiology, Institute of Physiology, Ruhr University Bochum, Bochum, Germany (A.U., W.A.L.); New Laura and Isaac Perlmutter Cancer Center at New York University Langone (T.A., B.N.); and McEwen Centre for Regenerative Medicine, Toronto, Canada (G.K.). The current address for Dr Hudson is Laboratory for Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Australia. w.zimmermann@med.uni-goettingen.de.

PMID: 28167635
PMCID: PMC5501412
DOI: 10.1161/CIRCULATIONAHA.116.024145

Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair

Malte Tiburcy et al. Circulation. 2017.

. 2017 May 9;135(19):1832-1847.

doi: 10.1161/CIRCULATIONAHA.116.024145. Epub 2017 Feb 6.

Authors

Affiliations

¹ From Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wettwer, W.-H.Z.); German Center for Cardiovascular Research (DZHK), partner site Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wingender, W.A.L., W.-H.Z.); Institute of Bioinformatics, University Medical Center Göttingen, Germany (S.Z., E. Wingender); Stanford Cardiovascular Institute (J.R., M.W., J.D.G., J.C.W.) and Department of Radiology (J.D.G., J.C.W.), Molecular Imaging Program, Stanford University School of Medicine, CA; The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Technion-Israel Institute of Technology, Haifa (I.K., L.G.); Institute of Pharmacology and Toxicology, Technical University Dresden, Germany (E. Wettwer, U.R.); University Medical Center Utrecht and Hubrecht Institute, The Netherlands (P.D., L.W.v.L.); Leiden University Medical Center, The Netherlands (M.J.G.); Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany (S.K., K.T., G.H., W.A.L.); Center for Applied Technology, Beckman Research Institute, City of Hope, Duarte, CA (L.A.C.); Department of Cardiovascular Physiology, Institute of Physiology, Ruhr University Bochum, Bochum, Germany (A.U., W.A.L.); New Laura and Isaac Perlmutter Cancer Center at New York University Langone (T.A., B.N.); and McEwen Centre for Regenerative Medicine, Toronto, Canada (G.K.). The current address for Dr Hudson is Laboratory for Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Australia.
² From Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wettwer, W.-H.Z.); German Center for Cardiovascular Research (DZHK), partner site Göttingen, Germany (M.T., J.E.H., P.B., S.S., T.M., M.-L.C.L., E.L., F.R., S.Z., E. Wingender, W.A.L., W.-H.Z.); Institute of Bioinformatics, University Medical Center Göttingen, Germany (S.Z., E. Wingender); Stanford Cardiovascular Institute (J.R., M.W., J.D.G., J.C.W.) and Department of Radiology (J.D.G., J.C.W.), Molecular Imaging Program, Stanford University School of Medicine, CA; The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Technion-Israel Institute of Technology, Haifa (I.K., L.G.); Institute of Pharmacology and Toxicology, Technical University Dresden, Germany (E. Wettwer, U.R.); University Medical Center Utrecht and Hubrecht Institute, The Netherlands (P.D., L.W.v.L.); Leiden University Medical Center, The Netherlands (M.J.G.); Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Germany (S.K., K.T., G.H., W.A.L.); Center for Applied Technology, Beckman Research Institute, City of Hope, Duarte, CA (L.A.C.); Department of Cardiovascular Physiology, Institute of Physiology, Ruhr University Bochum, Bochum, Germany (A.U., W.A.L.); New Laura and Isaac Perlmutter Cancer Center at New York University Langone (T.A., B.N.); and McEwen Centre for Regenerative Medicine, Toronto, Canada (G.K.). The current address for Dr Hudson is Laboratory for Cardiac Regeneration, School of Biomedical Sciences, The University of Queensland, Australia. w.zimmermann@med.uni-goettingen.de.

PMID: 28167635
PMCID: PMC5501412
DOI: 10.1161/CIRCULATIONAHA.116.024145

Abstract

Background: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions.

Methods: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM.

Results: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β₁- and β₂-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair.

Conclusions: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.

Keywords: heart failure; models, cardiovascular; regeneration; stem cells; tissue engineering.

PubMed Disclaimer

Conflict of interest statement

Conflict of Interest Disclosures

A patent concerning serum-free EHM generation for applications in drug screens and heart repair has been filed by the University of Goettingen with M.T., J.H., and W.H.Z. listed as inventors. W.H.Z. is founder and scientific advisor of myriamed GmbH and Repairon GmbH.

Figures

**Figure 1. Defining human EHM**
(a) EHM generation is characterized by two phases: EHM consolidation for 3 days (left panel: casting mold with 4 EHMs; **inset**: magnification of EHM in mold) and EHM maturation for at least 7 days under mechanical load (right panel: EHM on flexible PDMS holders). Bars: 5 mm (left panel), 1 mm (right panel). (b) Force of contraction (FOC; normalized to maximal FOC) in relation to output cardiomyocyte percentage (actinin⁺ cells) of EHM made from HES2-RFP, HES2, and hiPS-BJ lines. Blue square indicates an EHM sample constructed from SIRPA2A-selected cardiomyocytes. Grey area indicates optimal cardiomyocyte percentage across indicated lines (mean±SD). (c) Purification of cardiomyocytes for defined EHM generation. Quantification of cardiomyocyte purity (actinin⁺ cells) before and after enrichment by metabolic selection; n=8, p<0.05 by two-tailed, paired Students t-test. (d) Macroscopic appearance of EHM with >92% CM (CM EHM) and EHM with >92% CM supplemented with HFF (70:30% CM+HFF EHM). Immunostaining for actinin (green), f-actin (red), and nuclei (blue) in CM EHM (middle panel) and CM+HFF EHM (right panel). Bars: 5 mm (left panel), 50 μm (middle and right panels). (e) Titration of the optimal CM:HFF-ratio. Output CM percentage and force per CM in 2 week old EHM made with indicated input cell ratios of purified CMs and HFFs. Colors indicate the input CM:HFF ratio of respective EHMs (each circle represents one individual EHM with an additional empty circle indicating the mean±SEM of the respective groups). (f) Force of contraction (FOC) recorded under increasing calcium concentrations and electrical stimulation at 1.5 Hz in 4 week EHM constructed according to the undefined Starting Protocol (n=19; Table 1) and defined, Serum-free Protocol (n=59; Table 1); pooled data from EHM generated from different ESC and iPSC lines (please refer also to Supplementary Figure 4 for detailed information); *p<0.05 by 2-way ANOVA with Tukey’s multiple comparisons *post hoc* test.

**Figure 2. Morphological and functional maturation of EHM**
(a) Immunostaining of isolated cardiomyocyte from 4 week EHM (hiPS-G1); top panel: myosin heavy chain (green); middle panel: brightfield image with nucleus labelled with Hoechst (blue; bottom panel: overlay; bar: 20 μm. (b) Electron micrographs of 4 week EHM (hiPS-G1), low power (left panel, bar: 2.5 μm) and high power magnification (right panel: characteristic sarcomere structures are labelled; Mito: mitchondria; bar: 1 μm). (c) FOC per cross sectional area (CSA) of serum-free EHM from HES2 and hiPS-G1 at the indicated time points in culture; n=12/14/8 for weeks 2/4/6 in HES2 EHM and n=7/10/8 for weeks 2/4/8 in hiPS-G1 EHM *p<0.05 by 2-way ANOVA with Tukey’s multiple comparison *post hoc* test. (d) Force-frequency-response of hiPS-G1-EHM (at 4 weeks in culture) generated according to the Starting Protocol (red; n=8) and defined, Serum-free Protocol (black; n=21). ^§p<0.05 vs 1 Hz of the respective group by 2-way ANOVA with Tukey’s multiple comparison *post hoc* test; *p<0.05 by 2-way repeated measures ANOVA followed by Sidak’s multiple comparison test. (e) Representative force traces recorded from hiPS-G1-EHM (at 4 weeks in culture) at 1.5 Hz stimulation with an intermittent stimulation pause (10 sec); enhanced FOC at the reintroduction of electrical stimulation, i.e., post-rest potentiation, is characteristic for cardiomyocytes with mature intracellular calcium storage and release (the dotted line marks pre-pause baseline maximal FOC. (f) Representative action potentials recorded by impaling electrode measurements in EHM developed under the Starting Protocol (HES3) and the Serum-free Protocol (HES2); the table summarizes data recorded from together 51 independent action potential recordings; values in parentheses indicate maximally negative RMP and fastest dV/dt_max recorded in the respective groups.

**Figure 3. Molecular maturation of serum-free EHM**
(a) Strategy to determine cardiomyocyte and fibroblast transcriptomes from RNAseq data obtained from purified pluripotent stem cell-derived (PSC) cardiomyocytes (n=3 hES2 RFP, n=3 iCell CM, n=3 hiPS-G1) and primary fibroblasts (n=3 HFF, n=3 human cardiac fibroblasts, n=3 human gingiva fibroblasts). (b) RPKM values of the 29 most abundantly expressed transcripts in PSC-derived cardiomyocyte and primary fibroblasts. (c) Heatmap of cardiomyocyte transcripts in 22 day old cardiomyocyte monolayer cultures (2D D22), 60 day old cardiomyocyte monolayer cultures (2D D60), 6 week old EHM (note that cardiomyocyte “age” in these EHM was similar to 2D D60 cultures), fetal heart, and adult heart. Boxed areas indicate cardiomyocyte maturation genes; “adult”: increasing expression with development (upper box), and “embryonic”: decreasing expression with development (lower box). (d) Histogram of cardiomyocyte gene expression level (RPKM) compared to fetal heart as reference. Comparison of 22 day old cardiomyocyte monolayer cultures (2D, grey box) as starting point, 60 day old cardiomyocyte monolayer cultures (2D, blue box), and 6 week EHM cultures (red box). (e) Venn diagram and corresponding list of differentially expressed cardiomyocyte maturation genes with specific regulation in EHM, 60 day old cardiomyocyte monolayer cultures (2D), or both (overlap in Venn diagram; p<0.05 corrected for multiple testing by Benjamini-Hochberg method).

**Figure 4. Modeling heart failure in color-coded EHM**
(a) Effect of 7 day treatment with indicated concentrations (in μmol/L) of norepinephrine (NE) or endothelin-1 (ET-1) on FOC of EHM; *p<0.05 vs. Control by 2-way ANOVA with Tukey’s multiple comparison *post hoc* test, n=8–10 per Control and NE groups, n=4 for ET-1 group. (b) Inotropic response to acute isoprenaline (ISO) stimulation in EHM previously exposed to 7 day NE or ET-1 at the indicated concentrations (same EHM as in a); *p<0.05 vs. Ctr by 1-way ANOVA with Tukey’s multiple comparison *post hoc* test. (c) **Left panel**: macroscopic view of color-coded EHM (RFP⁺-CM: red, GFP⁺-Fib: green); scale bar: 1 cm; **middle panel**: cross section of color-coded EHM (red: actinin⁺-CM, green: GFP⁺-Fib); scale bar: 500 μm, **inset**: magnification - scale bar: 50 μm; **right panel:** flow cytometry of RFP⁺-CM and GFP⁺-Fib after enzymatic dispersion of color-coded EHM. (d) CM size measured by determination of RFP median fluorescence intensity (MFI, please refer to Supplementary Figure 9 for experimental details); *p<0.05 vs. Ctr by 1-way ANOVA with Tukey’s multiple comparison *post hoc* test. (e) Cell type distribution in color-coded EHM assessed by total cell quantification after enzymatic dispersion and subsequent flow cytometry for the separation of RFP⁺-CM and GFP⁺-Fib (from same EHM as in a); *p<0.05 for cardiomyocyte number vs. Ctr by 1-way ANOVA with Tukey’s multiple comparison *post hoc* test. (f) NT-proBNP secretion per CM into the culture medium (n=3/group). (g) Maximal FOC, response to ISO, CM viability, and CM size in comparison to control (dashed line) in EHM treated with 1 μmol/L NE with and without preincubation with 5 μmol/L metoprolol (Met) or 5 μmol/L phenoxybenzamine (Phen); *p<0.05 vs. Ctr by 1-way ANOVA with Tukey’s multiple comparison *post hoc* test (n=4–10/group).

**Figure 5. Scaling of EHM for heart repair**
(a) Technical drawings of the EHM patch manufacturing devices: (**top left**) 3D-printed patch holder with flexible poles; (**top right**) inverted patch holder positioned in hexagonal casting mold; (**bottom**) top view on patch holder for small and large EHM patch with dimensions in mm. (b) Display of different EHM designs (from left to right): small (1.5×10⁶ cells/500 μl) and big (2.5×10⁶ cells/900 μl) loops, fusion of five big loops according to technology reported earlier for rat, small (10×10⁶ cells/2 ml) and clinical-sized large (40×10⁶ cells/8 ml) patch. (c) Overview and 90° projections of an immunostained (f-actin in green) small EHM patch (image stitched together from 24× 850×850 μm tiles); boxed areas magnified on right for a demonstration of cell orientation. Bars: 5 mm (overview) and 1 mm (magnifications). (d) Explanted rat heart 4 weeks after epicardial implantation of an EHM patch in a RNU rat; bar: 1 cm. (e) Overview of human EHM on rat heart, immunostaining of human MYH7 (red), dashed line outlines the human EHM; bar: 500 μm. (f) Immunostaining of human EHM 107 days after implantation, cardiac troponin T (red), sarcomeric actinin (green), nuclei (blue); bar: 100 μm. (g) Immunostaining of CD31 (white) and human specific β1-integrin (red); bar: 500 μm.

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Comment in

One Stride Forward: Maturation and Scalable Production of Engineered Human Myocardium.
Yang X, Murry CE. Yang X, et al. Circulation. 2017 May 9;135(19):1848-1850. doi: 10.1161/CIRCULATIONAHA.117.024751. Circulation. 2017. PMID: 28483831 Free PMC article. No abstract available.

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References

1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. - PubMed
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1. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 2008;453:524–528. - PubMed
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[1] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. - PubMed

[2] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. - PubMed

[3] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. - PubMed

[4] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. - PubMed

[5] Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 2008;453:524–528. - PubMed

[6] Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 2008;453:524–528. - PubMed

[7] Burridge PW, Keller G, Gold JD, Wu JC. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell. 2012;10:16–28. - PMC - PubMed

[8] Burridge PW, Keller G, Gold JD, Wu JC. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell. 2012;10:16–28. - PMC - PubMed

[9] Lian X, Bao X, Zilberter M, Westman M, Fisahn A, Hsiao C, Hazeltine LB, Dunn KK, Kamp TJ, Palecek SP. Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods. 2015;12:595–596. - PMC - PubMed

[10] Lian X, Bao X, Zilberter M, Westman M, Fisahn A, Hsiao C, Hazeltine LB, Dunn KK, Kamp TJ, Palecek SP. Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods. 2015;12:595–596. - PMC - PubMed

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Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair

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Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair

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