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Human embryonic stem cell–derived cardiomyocytes restore function in infarcted hearts of non-human primates

Nature Biotechnology volume 36, pages 597–605 (2018)Cite this article

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An Erratum to this article was published on 01 October 2018

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Abstract

Pluripotent stem cell–derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell–derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9% vs. 2.5 ± 0.8% in controls, and by 3 months there was an additional 12.4% improvement in treated vs. a 3.5% decline in controls. Grafts averaged 11.6% of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99% ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

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Figure 1: Effects of hESC-CM transplantation on cardiac function.
Figure 2: Analysis of arrhythmias.
Figure 3: Structural assessment of infarct, graft size, and graft composition.
Figure 4: Graft maturation, integration, vascularization, and proliferation.
Figure 5: Visualization of grafts by MRI.

Change history

  • 18 July 2018

    In the version of this article initially published, NIH grant P51 OD010425 was omitted. The error has been corrected in the HTML and PDF versions of the article.

  • 01 October 2018

    Nat. Biotechnol. 36, 597–605 (2018); published online 2 July 2018; corrected after print 18 July 2018 In the version of this article initially published, NIH grant P51 OD010425 was omitted. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

These studies were supported in part by NIH Grants R01HL128362, R01 HL084642, P01HL094374, and a grant from the Fondation Leducq Transatlantic Network of Excellence (all to C.E.M.), and grant P51 OD010425 from the NIH Office of Research Infrastructure Programs to the Washington National Primate Research Center. These studies also were supported by the UW Medicine Heart Regeneration Program, the Washington Research Foundation, and a gift from Mike and Lynn Garvey. The manufacture of cells provided by City of Hope was funded in part through the National Heart Lung and Blood Institute's Production Assistance for Cell Therapies (PACT). We also acknowledge the support of the Cell Analysis Facility Flow Cytometry and Imaging Core in the Department of Immunology at the University of Washington. We thank the Garvey Imaging Core for assistance with microscopy. We are indebted to the dedicated staff of the Washington National Primate Research Center for supporting many aspects of this study. We thank M. Laflamme for helpful discussions, W.-Z. Zhu for support with electrophysiological studies, and P. Swanson for consultation on the renal tumor. We thank J. Maki and G. Wilson for help with cardiac MRI protocol.

Author information

Author notes

  1. Yen-Wen Liu, Larry Couture & Stephanie A Tuck

    Present address: Present addresses: Division of Cardiology, Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan (Y.-W.L.); Orbsen Therapeutics, LTD, Orbsen Buildings, National University of Ireland Galway, Galway, Ireland (L.C.); Uptake Medical Technologies, Seattle, Washington, USA. (S.A.T.).,

  2. Yen-Wen Liu, Billy Chen and Xiulan Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA

    Yen-Wen Liu, Billy Chen, Xiulan Yang, James A Fugate, Faith A Kalucki, Akiko Futakuchi-Tsuchida, Stephanie A Tuck, Hiroshi Tsuchida, Anna V Naumova, Sarah K Dupras, Dale W Hailey, Hans Reinecke, Lil Pabon, Benjamin H Fryer, W Robb MacLellan, R Scott Thies & Charles E Murry

  2. Center for Cardiovascular Biology, University of Washington, Seattle, Washington, USA

    Yen-Wen Liu, Billy Chen, Xiulan Yang, James A Fugate, Faith A Kalucki, Akiko Futakuchi-Tsuchida, Stephanie A Tuck, Hiroshi Tsuchida, Anna V Naumova, Sarah K Dupras, Hans Reinecke, Lil Pabon, Benjamin H Fryer, W Robb MacLellan, R Scott Thies & Charles E Murry

  3. Division of Cardiology, Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan

    Yen-Wen Liu

  4. Department of Medicine/Cardiology, University of Washington, Seattle, Washington, USA

    Billy Chen, Creighton W Don, Zachary L Steinberg, Stephen P Seslar, James Lee, W Robb MacLellan & Charles E Murry

  5. Department of Pathology, University of Washington, Seattle, Washington, USA

    Xiulan Yang, James A Fugate, Faith A Kalucki, Akiko Futakuchi-Tsuchida, Stephanie A Tuck, Hiroshi Tsuchida, Sarah K Dupras, Hans Reinecke, Lil Pabon, Benjamin H Fryer, R Scott Thies & Charles E Murry

  6. City of Hope, Beckman Research Institute, Duarte, California, USA

    Larry Couture

  7. Washington National Primate Research Center, University of Washington, Seattle, Washington, USA

    Keith W Vogel, Clifford A Astley, Audrey Baldessari & Jason Ogle

  8. Department of Pediatrics, University of Washington, Seattle Children's Hospital, Seattle, Washington, USA

    Stephen P Seslar

  9. Department of Radiology, University of Washington, Seattle, Washington, USA

    Anna V Naumova & Milly S Lyu

  10. Research Institute of Biology and Biophysics, National Research Tomsk State University, Tomsk, Russia

    Anna V Naumova

  11. Department of Bioengineering, University of Washington, Seattle, Washington, USA

    Charles E Murry

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Contributions

Y.-W.L. designed experiments, conducted animal studies, analyzed data and edited the manuscript. B.C. designed experiments, conducted animal studies, performed histology studies, analyzed data and edited the manuscript. X.Y. performed histology, histomorphometry, analyzed data, prepared figures, and edited the manuscript. J.A.F. prepared cells for transplantation and edited the manuscript. F.A.K. prepared cells for transplantation and edited the manuscript. A.F.-T. prepared cells for transplantation and edited the manuscript. L.C. supervised cell manufacturing. K.W.V. designed experiments, conducted animal studies, and provided veterinary care. C.A.A. designed experiments and conducted animal studies. A.B. performed necropsies and provided pathology consultation. J.O. conducted animal studies and edited the manuscript. C.W.D. designed experiments, conducted animal studies and edited the manuscript. Z.L.S. conducted animal studies and edited the manuscript. S.P.S. designed experiments, conducted electrophysiology studies, analyzed data and edited the manuscript. S.A.T. conducted animal studies, analyzed data and edited the manuscript. H.T. conducted animal studies and edited the manuscript. A.V.N. performed MRI studies, analyzed data and edited the manuscript. S.K.D. performed histology. M.S.L. analyzed MRI scans. J.L. analyzed MRI scans and edited the manuscript. D.W.H. advised on and performed microscopy. H.R. designed experiments, performed histology, conducted molecular analyses, analyzed data, and prepared figures. L.P. designed experiments, analyzed data and edited the manuscript. B.H.F. prepared cells for transplantation. WR.M. designed experiments, analyzed data and edited the manuscript. R.S.T. designed experiments, conducted animal studies, oversaw preparation of cells for transplantation, analyzed data, prepared figures and edited the manuscript. C.E.M. supervised all components of this study, designed experiments, performed animal experiments, analyzed data, obtained funding for the study, prepared figures and wrote the manuscript.

Corresponding author

Correspondence to Charles E Murry.

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Competing interests

C.E.M., R.S.T., and W.R.M. are scientific founders and equity holders in Cytocardia.

Integrated supplementary information

Supplementary Figure 1 Flow cytometry for assessment of cardiomyocyte purity.

(a) Forward and side scatter with main population of cells indicated. (b) Isotype control antibody showing minimal staining in M2 gate. (c) cTnT antibody showing 98.7% of cells expressing this epitope. (d) Overlay of isotype control and cTnT antibodies showing clear spectral separation of the two preparations. This experiment was repeated in 4 biologically independent cell preparation runs, with purities ranging from 86-99% cTnT-positive. PE, phycoerythrin.

Supplementary Figure 2 Flow diagram for animal assignment and inclusion in MRI and electrophysiology (EP) studies.

This figure depicts the experimental flow, mortality at each stage of the study, assignment into various experimental protocols, exclusion for technical limitations and final group sizes for MRI and electrophysiological (EP) analyses.

Supplementary Figure 3 Inter-observer variance in MRI and EKG telemetry.

(a) Regression plot for determination of LVEF by two MRI observers blinded to treatment. There is a high correlation coefficient (R2 = 0.92; p < 0.001) with a slope close to 1.0. (b) Bland and Altman analysis showing that there was no difference of LVEF from observer 1 (Y axis) and observer 2 (X axis) (p = 0.94). (c) Regression plot for quantification of ventricular arrhythmias by two observers. There is a high correlation coefficient (R2=0.98) with a slope close to 1.0. (d) Bland and Altman analysis showing that there was no difference for ventricular arrhythmia duration between the two observers.

Supplementary Figure 4 Regional contractile function (percent LV wall systolic thickening) in the non-infarcted wall at baseline and at 4 weeks post-treatment.

There was no difference between groups at any time or within groups at different times. Each point represents one heart. Data were obtained from 4 biologically independent control animals and 5 biologically independent hESC-CM treated animals. Group data represent means ± SEM.

Supplementary Figure 5 GCaMP3 transgene and amiodarone are not necessary for arrhythmias.

The small infarct protocol (90 min occlusion of distal LAD) was used here. This model demonstrates minimal arrhythmias before transplantation and consistent arrhythmias after transplantation. Control group (not shown) had minimal arrhythmias throughout. (a) Omission of GCaMP transgene in engrafted hESC-CM did not diminish the ventricular arrhythmias. Data are from 4 biologically independent GCaMP transgenic hESC-CM animals and 1 wild-type hESC-CM treated animal. (b) Withholding amiodarone did not diminish the ventricular arrhythmias. Data are from 4 biologically independent animals receiving hESC-CM in the presence of amiodarone and from 1 hESC-CM treated animal that did not receive amiodarone.

Supplementary Figure 6 Analysis of tumor origin identified in one macaque kidney.

All data in this figure were obtained from one hESC-CM treated animal that developed a renal tumor. (a) Hematoxylin and eosin stained section of kidney containing carcinoma cells within a lymphatic vessel. A glomerulus (Glom) is visible to the right of the tumor. Scale bar, 50 μm. (b) High magnification image of the region boxed in (a), shows that the carcinoma cells have formed tubules and have eosinophilic proteinaceous material in the lumens. Scale bar, 50 μm. (c) PCR genotyping of tumor. DNA extracted from a histological slide containing kidney tissue and tumor was analyzed by PCR using primers specific for human mitochondria (high copy number per cell) and macaque chromosome 3. Human mitochondrial primers generated an amplicon in purified human DNA but not in the kidney tumor sample or monkey DNA sample. Macaque chromosome 3 primers gave amplicons in the monkey and kidney tumor samples, but not in purified human DNA. (d) In situ hybridization positive control sample of hESC-CM graft in macaque heart. The human graft nuclei hybridize intensely with the probe (red), whereas host nuclei are negative. Individual channels are shown below. DAPI counterstain. Scale bar, 50 μm. (e) Negative control for in situ hybridization study on an adjacent section of the tumor where the human-specific probe was omitted. The proteinaceous material in the glandular lumens is intensely autofluorescent (red), but the tumor nuclei are negative. Scale bar, 50 μm. (f) In situ hybridization study of tumor on adjacent section shows autofluorescent proteinacous material in the glandular lumens (red), but the nuclei are unstained. This indicates the tumor is not of human origin. Scale bar, 50 μm.

Supplementary Figure 7 Infarct shrinkage from baseline to 1 month post-treatment.

Infarct size was determined serially in 8 macaques by MRI using delayed Gd enhancement. Both groups show infarct shrinkage, but significantly greater scar contraction occurred in hESC-CM-treated hearts. Data are from 5 biologically independent hESC-CM treated and 3 control animals.

Supplementary Figure 8 Validation of human-specific monoclonal antibodies to cardiac troponin I (cTnI) and slow skeletal troponin I (ssTnI).

(a) Archival section from macaque heart containing an hESC-CM graft that expresses GFP. The graft region is readily identifiable by the brown immunoperoxidase reaction product using anti-GFP immunostaining. (b) Adjacent section from the same tissue block stained by DNA in situ hybridization for human-specific pan-centromeric sequences (red). The human graft is readily identifiable in the background of the primate heart and shows good spatial correspondence with the anti-GFP staining shown in (a). (c) Adjacent section stained with human cTnI antibody (brown) shows graft region that corresponds well with anti-GFP and human pan-centromeric probe in situ hybridization. (d) Adjacent section stained with ssTnI antibody (brown) shows graft region that corresponds well with anti-GFP, human pan-centromeric probe in situ hybridization and human cTnI immunostaining. Note that, although this antibody also labels monkey ssTnI, this isoform is not expressed by the adult monkey heart. These validation studies were performed on a single hESC-CM treated heart.

Supplementary Figure 9 Low magnification whole-slide scans from 4 hearts receiving hESC-CM grafts, stained for human cTnI (brown).

Apical section is on the left and basal section is on the right. Extensive areas of remuscularization are present in all hearts. These experiments were performed on 4 biologically independent hESC-CM treated hearts, and data from each heart are shown here. Scale bar, 5 mm.

Supplementary Figure 10 Maturation of adherens and gap junctions in hESC-CM grafts.

(a) At 1 month post-engraftment, Connexin43+ gap junctions (green) are punctate and often circumferentially distributed around the graft cardiomyocytes (ssTnI, red). This experiment was repeated in 2 biologically independent hESC-CM treated hearts with similar results. (b) At 3 months post-engraftment, many Connexin43+ gap junctions have polarized to intercalated disks, although in other locations (not shown) the staining was still circumferential. This experiment was repeated in 2 biologically independent hESC-CM treated hearts with similar results. (c) At 1 month post-engraftment, pan-cadherin+ adherens junctions (green) are punctate and distributed around the graft cardiomyocytes (ssTnI, red). This experiment was repeated in 2 biologically independent hESC-CM treated hearts with similar results. (d) At 3 months post-engraftment, cadherin+ adherens junctions have polarized to the intercalated disks of graft cardiomyocytes. This experiment was repeated in 2 biologically independent hESC-CM treated hearts with similar results. Scale bar a-d, 25 μm.

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Liu, YW., Chen, B., Yang, X. et al. Human embryonic stem cell–derived cardiomyocytes restore function in infarcted hearts of non-human primates. Nat Biotechnol 36, 597–605 (2018). https://doi.org/10.1038/nbt.4162

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