Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts

Abstract

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure1 by providing human cardiomyocytes to support heart regeneration2. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment3,4,5,6,7. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models7, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

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Figure 1: Remuscularization of the infarcted macaque heart with human cardiomyocytes.
Figure 2: Human cardiomyocyte grafts mature with time from engraftment.
Figure 3: Blood vessels extend from the host coronary network into the graft.
Figure 4: Human cardiomyocytes are electrically coupled 1:1 to the infarcted host macaque heart after transplantation.
Figure 5: Ventricular arrhythmias after hESC-CM transplantation.

Change history

  • 11 June 2014

    Savannah M. Cook has been added to the author list and an incorrect grant number has been updated.

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Acknowledgements

We thank S. Dupras, B. Brown, D. Rocha, E. Wilson, C. English, J. Randolph-Habecker and T. Goodpaster for assistance with these experiments. This work was supported by National Institutes of Health grants P01HL094374, R01HL084642, U01HL100405 and P01GM081619 and an Institute of Translational Health Sciences/Primate Center Ignition Award. J.J.H.C. was supported by National Health and Medical Research Council of Australia Overseas Training and Australian-American Fulbright Commission Fellowships. X.Y. is supported by an American Heart Association post-doctoral scholarship 12POST11940060. J.J.W. is supported by an American Heart Association post-doctoral scholarship 12POST9330030. H.-P.K. is a Markey Molecular Medicine investigator and the recipient of the Jose Carreras/E.D. Thomas Chair for Cancer Research.

Author information

J.J.H.C., X.Y., C.W.D., E.M., L.P., H.R., H.-P.K., M.A.L. and C.E.M. designed the study. J.J.H.C. and E.M. performed mouse transplantation experiments. J.J.H.C. developed telemetry and analysed recordings. J.J.H.C., C.W.D., C.E.M., G.M.G., K.W.V., C.A.A., E.M. and V.N. performed macaque surgery and procedures. J.J.H.C., E.M., E.A.G. and C.E.H. performed echocardiography and E.M., E.G. and Y.-W.L. performed analysis. A.B. performed necropsies and non-cardiac histopathology. GCaMP3 visualization experiments were carried out and analysed by X.Y. and J.J.H.C. GCaMP3-expressing human ES cells were created by N.J.P., J.A.G. and B.V.B. hESC-CM production was by J.J.H.C., B.V.B., S.M.C., J.A.F. and M.A.L. Microcomputed tomography experiments were performed by J.J.W. and W.M.M. Jr. Immunohistochemistry was performed and analysed by J.J.H.C., V.M. and Y.-W.L. Figures were created by J.J.H.C. with assistance from X.Y., J.J.W., Y.-W.L., N.J.P. and V.M. The manuscript was written principally by J.J.H.C. and C.E.M.

Correspondence to Charles E. Murry.

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Competing interests

C.E.M. and M.A.L. are equity holders in BEAT BioTherapeutics.

Extended data figures and tables

Extended Data Figure 1 Cryopreservation does not affect hESC-CM engraftment.

a, Schematic representation of experimental design for cryopreservation testing experiments. b, Human genomes detected after injection of cryopreserved or non-cryopreserved hESC-CMs were not significantly different (P > 0.05, t-test). Mean ± s.e.m. is shown (n = 9 biological replicates) Experiment was performed once. NS, not significant.

Extended Data Figure 2 Creation and validation of the GCaMP3-expressing human ES-cell lines.

a, Targeting construct for ZFN engineering of GCaMP3 into the AAVS1 locus. The endogenous genomic probe and neomycin resistance gene probe binding sites used for Southern blotting are shown. b, Southern blot analysis demonstrates a single integration event by hybridization for neomycin resistance cassette (left) and heterozygous AAVS1 integration by genomic probe labelling (right).

Extended Data Figure 3 Chromosomal analysis of human ES cells modified to encode GCaMP3.

a, H7-GCaMP3 ES-cell line demonstrates an isochrome of the chromosome 20 long arm (arrow). b, RUES2-GCaMP3 ES-cell line shows normal karyotype.

Extended Data Figure 4 Flow cytometry for cardiomyocyte differentiation of human ES cells.

Representative histogram of hESC-CMs after differentiation shows 73% cTnT-expressing cells.

Extended Data Figure 5 Schematic representation of experimental design.

Myocardial infarction was created by advancing a balloon catheter into the distal left anterior descending artery and inflating it to create ischaemia (90 min) followed by reperfusion. The infarct was induced 14 days (D) before hESC-CM delivery via left thoracotomy. Immunosuppression using cyclosporine A, methylprednisolone and abatacept (T-cell co-stimulatory antagonizing fusion protein) was delivered 5 days before cell delivery and continued until animals were euthanized. Primary endpoints were (1) histologically based morphometric calculations of infarct and graft size with analysis of graft composition, and (2) ex vivo analysis of graft–host electromechanical coupling enabled by GCaMP3 fluorescence detection. Secondary endpoints were (1) detection of arrhythmias by telemetric electrocardiogram analysis, and (2) analysis of left ventricular functional change by trans-oesophageal echocardiography.

Extended Data Figure 6 Technique for hESC-CM injection to infarct region and border zones using ‘mattress’ suture strategy.

a, The macaque infarcted ventricular apex is seen as a blanched region (dotted line) during left thoracotomy. A total of 15 aliquots, each containing 100 μl of hESC-CM in pro-survival cocktail, were delivered through five epicardial puncture sites (arrows, note one further puncture site not seen is on posterior aspect). b, hESC-CM retention after injection was increased by use of a ‘mattress’ suture. Crosses indicate insertion points of suture with dotted lines representing path of suture (exaggerated size for diagrammatic representation). A needle tip was inserted into the resulting rectangular area and the suture was tightened after a series of three injections (altering the trajectory of the needle) but before withdrawal of needle tip. c, Quantification of India ink retention after injection into left ventricular myocardium of anaesthetized macaques with or without use of the mattress suture technique (n = 3 biological replicates each group). A trend favouring greater retention with the mattress suture is seen.

Extended Data Figure 7 Remuscularization of the infarcted macaque heart.

af, Single channels of confocal immunofluorescence shown in Fig. 1a–f. Macaque heart shown was subjected to myocardial infarction and transplantation of hESC-CMs 14 days before being euthanized. gl, Picrosirius Red staining of sections in close proximity to confocal immunofluorescence in af shows lack of fibrosis within hESC-CM grafts. Scale bars: 2,000 μm (a, f, g, l); 1,000 μm (b–e, h–k).

Extended Data Figure 8 Remuscularized infarct region is composed of engrafted cardiomyocytes that increase in size with time.

a, Quantification of the sarcomeric protein α-actinin expression in GFP-expressing grafts. The vast majority (>98%) of GFP-expressing cells co-expressed α-actinin. P3–P6 represent individual animals (n = 1) euthanized at 2 weeks (2 wk) 1 month (m) or 3 months after hESC-CM delivery. Five hundred to seven hundred cells were counted from three different graft regions of each heart. Percentage of GFP/α-actinin double-positive cells and GFP-positive/α-actinin-negative cells are shown as mean ± s.d. b, Normal curve from histograms showing the distribution of human ES-cell-derived cardiomyocyte diameters (graft) in monkey hearts 2 weeks, 1 or 3 months after cell delivery. cf, Individual histograms with superimposed normal curve of animals P3–P6 (as above).

Extended Data Figure 9 No evidence of human graft rejection.

ai, Representative low- (df) and high- (ac and gi) power magnification of hESC-CM graft 28 days after cell delivery to infarcted macaque heart. Representative low- (jk) and high- (l, m) power magnification of infarct region from control macaque 28 days after sham treatment. The hESC-CM graft is detected by anti-GFP primary antibody with 3,3′-diaminobenzidine (DAB) detection of secondary antibody (brown). Few CD3+ T lymphocytes or CD20+ B lymphocytes are seen surrounding the hESC-CM grafts. Comparable numbers of T and B cells are seen in control infarcts receiving no hESC-CM treatment. Boxed inset regions show areas of higher magnification.

Extended Data Figure 10 Summary of ventricular tachycardia and echocardiographic assessment of left ventricular function.

a, Table characterizing episodes of ventricular tachycardia after engraftment of hESC-CMs (detailed in Fig. 5). Note that P5 demonstrated no discernible sinus rhythm on telemetric recording of the ECG 14 days after hESC-CM delivery. Although QRS morphology varied, the tachyarrhythmia comprised sustained periods of stable monomorphic QRS morphology. LBBB, left bundle branch block; RBBB, right bundle branch block. bpm, beats per minute. b, Left ventricular function was assessed by trans-oesophageal echocardiography at the following time points: before myocardial infarct creation, before hESC-CM delivery (2 weeks after myocardial infarction) and before animals were euthanized (2, 4 or 12 weeks after myocardial infarction). P7 received no cells/vehicle only. All other animals received hESC-CMs. Results shown are for left ventricular ejection fraction calculated by two blinded cardiologists from the two-chamber view of the left ventricle. Note that this view best captures the infarcted antero-apical wall. The vehicle-treated control monkey showed a modest diminution in ejection fraction post-infarction. The cell-treated animals showed variable responses, with some having increased function and some having decreased function. Because of small group size, no statistical effects of hESC-CM therapy can be discerned. c, Table of antibodies used. DSHB, Developmental Studies Hybridoma Bank.

Related audio

Charles Murry discusses how his team regenerated monkey hearts using stem cell-derived cardiomyocytes.

Supplementary information

GCaMP3-expressing RUES2hESC-CMs exhibit robust fluorescence in vitro

Representative GCaMP3-expressing hESC-CMs were imaged using an inverted stereo-microscope with halogen and laser light sources. The video file shows the cardiomyocytes first by bright-field microscopy alone and then later with the green fluorescent signal. Note that the cells exhibit robust fluorescent transients with each contractile cycle. (MOV 4315 kb)

GCaMP3 expressing H7hESC-CMs exhibit robust fluorescence in vitro

Similar to Video S1, representative H7GCAMP3-hESC-CMs are shown first in bright field then with the green fluorescent signal. Note robust fluorescence with each cardiac cycle. (MOV 3327 kb)

Blood vessels extend from the host coronary network into the graft

3-dimensional rendering of microcomputed tomography (same heart shown in Extended Data Fig. 9) animated with rotation to enable better visualization of the vessels within the graft. Arteries feeding the graft are red, other vessels are gray in the uninjured cardiac tissue, or white within the graft. (MOV 22355 kb)

Modified Langendorff apparatus and GCAMP3 fluorescence imaging equipment

A harvested macaque heart is seen spontaneously beating after retrograde perfusion of with modified Tyrode’s solution. Electrodes are attached to the heart for electrocardiogram recording and analysis. Placement of the heart under a stereomicroscope with fluorescence imaging capabilities allows 2 visualization and analysis of epicardial fluorescence emitted from GCAMP3 expressing hESC-CM grafts. (MOV 23764 kb)

Intravital imaging of the infarcted macaque heart with GCaMP3-expressing hESC-CM grafts

A representative macaque heart with numerous GCaMP3-expressing hESC-CM graft regions, harvested at 28 days posttransplantation, mounted ex vivo on a Langendorff apparatus and mechanically arrested with 2,3-butanedione monoxime. This low power video (0.8x) shows several regions of GCaMP3-positive graft (distributed through infarct region and border zones) visible from the epicardial surface. All regions exhibited cyclic fluorescent transients that occurred in synchrony with QRS complexes of the host ECG, indicating 1:1 host-graft coupling. This video corresponds to Figure 2 b in main text. (MOV 6533 kb)

Higher power of intravital imaging of the infarcted macaque heart with GCaMP3-expressing hESC-CM graft

This video shows the same heart as video S3 at higher (2x) magnification and corresponds to Figure 2c-d in main text. (MOV 6527 kb)

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Chong, J., Yang, X., Don, C. et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510, 273–277 (2014). https://doi.org/10.1038/nature13233

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