Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair,

ARTICLE PREVIEW view full access options NATURE | LETTER Print Share/bookmark 日本語要約 Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts Yuji Shiba, Toshihito Gomibuchi, Tatsuichiro Seto, Yuko Wada, Hajime Ichimura, Yuki Tanaka, Tatsuki Ogasawara, Kenji Okada, Naoko Shiba, Kengo Sakamoto, Daisuke Ido, Takashi Shiina, Masamichi Ohkura, Junichi Nakai, Narumi Uno, Yasuhiro Kazuki, Mitsuo Oshimura, Itsunari Minami & Uichi Ikeda AffiliationsContributionsCorresponding author Nature 538, 388–391 (20 October 2016) doi:10.1038/nature19815 Received 07 October 2015 Accepted 31 August 2016 Published online 10 October 2016 Article tools Citation Reprints Rights & permissions Article metrics Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy1, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary. Subject terms: Induced pluripotent stem cells Stem-cell research At a glance Figures First | 1-3 of 14 | Last left Transplanted iPSC-CMs partially remuscularize infarcted cynomolgus monkey hearts. Figure 1 iPSC-CMs electrically couple with the host heart. Figure 2 Transplantation of iPSC-CMs improves cardiac contractile function. Figure 3 Electrical consequences of transplantation of iPSC-CMs. Figure 4 Characteristics of the HT4 haplotype. Extended Data Fig. 1 Generation of iPSCs from a MHC homologous cynomolgus monkey. Extended Data Fig. 2 Characteristics of G-CaMP7.09. Extended Data Fig. 3 Generation and purification of cynomolgus iPS cell-derived cardiomyocytes. Extended Data Fig. 4 Study protocol and design. Extended Data Fig. 5 Immune response following transplantation of iPS cell-derived cardiomyocytes. Extended Data Fig. 6 Macroscopic and microscopic analysis of iPSC-CM recipients. Extended Data Fig. 7 Summary of histological, mechanical and electrophysiological consequences. Extended Data Fig. 8 Additional electrical analysis of hearts transplanted with iPSC-CMs. Extended Data Fig. 9 Time course of left ventricular size and BNP levels. Extended Data Fig. 10 right Videos READ THE FULL ARTICLE Subscribe to Nature for full access: $199 Subscribe ReadCube Access*: $3.99 rent $9.99 buy *printing and sharing restrictions applyBuy/Rent now Purchase article full text and PDF: $32 Buy now Already a subscriber? Log in now or Register for online access. Additional access options: Use a document delivery service Login via OpenAthens Purchase a site license Institutional access References References• Author information• Extended data figures and tables• Supplementary information Deleidi, M., Hargus, G., Hallett, P., Osborn, T. & Isacson, O. Development of histocompatible primate-induced pluripotent stem cells for neural transplantation. Stem Cells 29, 1052–1063 (2011) CASPubMedArticle Laflamme, M. A. & Murry, C. E. Heart regeneration. 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Methods 50, 153–159 (2004) CASPubMedArticle Download references Author information References• Author information• Extended data figures and tables• Supplementary information Author footnotes These authors contributed equally to this work. Yuji Shiba & Toshihito Gomibuchi Affiliations Institute for Biomedical Sciences, Shinshu University, Matsumoto 390-8621, Japan Yuji Shiba Department of Cardiovascular Medicine, Shinshu University School of Medicine, Matsumoto 390-8621, Japan Yuji Shiba & Uichi Ikeda Department of Cardiovascular Surgery, Shinshu University School of Medicine, Matsumoto 390-8621, Japan Toshihito Gomibuchi, Tatsuichiro Seto, Yuko Wada, Hajime Ichimura, Yuki Tanaka, Tatsuki Ogasawara & Kenji Okada Department of Pediatrics, Shinshu University School of Medicine, Matsumoto 390-8621, Japan Naoko Shiba Ina Research Inc., Ina 399-4501, Japan Kengo Sakamoto & Daisuke Ido Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan Takashi Shiina Brain Science Institute, Saitama University, Saitama 338-8570, Japan Masamichi Ohkura & Junichi Nakai Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan Masamichi Ohkura & Junichi Nakai Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan Narumi Uno, Yasuhiro Kazuki & Mitsuo Oshimura Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan Itsunari Minami Contributions Y.S. designed the study. Y.S., T.G., T.Se., Y.W., H.I., Y.T., K.S. and D.I. performed all animal procedures. T.O., N.S. and Y.S. performed histological analysis. K.S. and D.I. analysed Holter ECGs. N.U., Y.K., and M.Os. performed karyotype analysis of iPSCs. T.Sh. analysed RNA sequences of cynomolgus MHC. K.O. and U.I. analysed all other data and provided administrative assistance. M.Oh. and J.N. generated the G-CaMP7.09 plasmid. In vitro fluorescent imaging studies were performed by I.M. The manuscript was written by Y.S., T.Sh., M.Oh., I.M. and N.U. Competing financial interests K.S. and D.I. are employees of Ina Research, where all animal procedures in this study were performed. The remaining authors have no competing interests to declare. Corresponding author Correspondence to: Yuji Shiba Reviewer Information Nature thanks T. Braun, K. Fukuda, T. Kamp and the other anonymous reviewer(s) for their contribution to the peer review of this work. Extended data figures and tables References• Author information• Extended data figures and tables• Supplementary information Extended Data Figures Extended Data Figure 1: Characteristics of the HT4 haplotype. (649 KB) a, b, Basic structure of MHC in HT4 haplotypes. One of the cynomolgus monkeys (DrpZ5-32B-C) is strictly a ‘homozygote’ that has the A-Hp7.2 and B-Hp2 haplotypes in the Mafa-class I region and the #7 haplotype in the Mafa-class II region on both chromosomes (tentatively named ‘HT4’). c, In vitro mixed lymphoid reactions (MLR) showed that when inactivated lymphocytes from a HT4-heterozygous monkey were cocultured with active lymphocytes from a HT4-homozygous monkey, proliferation was inhibited to the level of control (only inactivated cells) or autologous (inactivated and active cells from same animal). ‘MHC mismatched’ indicates two groups of lymphocytes from two different animals with different MHC types. **P < 0.01 versus control. n = 5 per group. Extended Data Figure 2: Generation of iPSCs from a MHC homologous cynomolgus monkey. (774 KB) Donor iPSCs were established from skin fibroblasts by transfection of episomal vectors carrying OCT4, KLF4, SOX2 and L-MYC. a, iPSCs form typical ES-cell-like colonies. Scale bar, 50 μm. b–e, iPSCs express pluripotent markers as assessed by immunofluorescence. Scale bars, 100 μm. f, Gene expression of pluripotent markers in the iPSCs is identical to that in cynomolgus ES cells. g–i, When transplanted into immunodeficient mice, the iPSCs gave rise to teratomas manifesting all three germ layers: endoderm (intestinal epithelium), mesoderm (cartilage) and ectoderm (squamous cells). j, After expansion, the iPSCs showed normal karyotype (42, XY). Extended Data Figure 3: Characteristics of G-CaMP7.09. (217 KB) a, Schematic structure of G-CaMP7.09. Mutations are indicated with respect to G-CaMP7. RSET and M13 are tags that encode hexahistidine and a target peptide for Ca2+-bound CaM derived from myosin light chain kinase, respectively. The amino-acid numbers of EGFP and CaM are indicated in parentheses. The dynamic range of G-CaMP7.09 (Fmax/Fmin) was 19.3 ± 2.52 (n = 3) and the Kd for Ca2+ was 212 ± 6.9 nM (n = 3). b–h, In vitro fluorescence transients of G-CaMP7.09-expressing cardiomyocytes. Data are representative of three independent experiments. b, Spontaneous contraction. Scale bar, 2 s. c, The firing rate of G-CaMP signals was reduced by treatment with ryanodine, a ryanodine receptor blocker. Scale bar, 2 s. d, Addition of the L-type calcium-channel blocker, nifedipine, resulted in cessation of fluorescent transients. Scale bar, 6 s. e, Treatment with an activator of the ryanodine receptor, caffeine, induced fluorescent transients in the G-CaMP7.09-expressing iPSC-CMs. Scale bar, 6s. f, G-CaMP7.09 transients were sustained for a few minutes after spontaneous contraction and stopped by 40 mM BDM. Scale bar, 1 s. g, After cessation of spontaneous fluorescent transients, iPSC-CMs on Parafilm were stretched but no fluorescent transient was detected. Scale bar, 10 s. h, Treatment with caffeine induced G-CaMP7.09 transients again. Scale bar, 5 s. Extended Data Figure 4: Generation and purification of cynomolgus iPS cell-derived cardiomyocytes. (404 KB) a, Pilot experiments showed that cultivation of iPSC-CMs in glucose-free medium for 72 h significantly enhances cardiac purity, **P < 0.01 versus 0 h, n = 4 for each time point. Data are representative of three independent experiments. b, iPSC-CMs express the cardiac-specific marker cTnT. Scale bar, 50 μm. c, d, After multiple attempts to generate cardiomyocytes for transplantation, 2 × 109 cardiomyocytes (cTnT-positive 83.8 ± 1.0% as indicated by flow-cytometric analysis) were prepared. e, f, The cardiomyocytes were positive for GFP. g, RT–PCR analysis indicated that cTnT mRNA expression in iPSC-CMs was detectable, but lower than in the adult heart. Data are representative of three independent experiments. Extended Data Figure 5: Study protocol and design. (132 KB) a, A monolayer of cultured undifferentiated cynomolgus monkey iPSCs on a Matrigel (MG)-coated dish was treated with Matrigel. The culture medium was replaced with serum-free medium supplemented with Matrigel and activin A (AA) on day 0. On day 1 after activation, the medium was replaced with medium containing BMP4 and basic fibroblast growth factor (bFGF), and cells were cultured until day 5. On day 14, cardiomyocytes were selected by cultivation in glucose-free medium for 3 days. b, Fourteen days before transplantation, 10 female monkeys were subjected to ischaemia/reperfusion injury. Either 4 × 108 iPSC-CMs reconstituted in a prosurvival cocktail (PSC) or the PSC vehicle was injected on day 0. Cardiac μCT and UCG were performed to evaluate cardiac contractile function before and after transplantation. Additionally, BNP was measured. Spontaneous arrhythmias were monitored by Holter electrocardiogram (ECG) on days −1, 7, 14 and every other week thereafter. On day 84, all animals were euthanized, and the hearts were excised and subjected to intravital G-CaMP imaging, followed by histological analysis. Extended Data Figure 6: Immune response following transplantation of iPS cell-derived cardiomyocytes. (1,724 KB) a, b, iPSC-CMs were transplanted into MHC-mismatched infarcted hearts (n = 2). Animals were euthanized and the hearts were collected at 4 weeks post-transplantation. Only a small portion of grafts (GFP) showed a severe infiltration of inflammatory cells, such as CD3+ T lymphocytes. c–i, Immunohistochemical analysis of recipients of iPSC-CMs or PSC vehicle 84 days post-transplantation. The sections were stained with antibodies against CD45 (leukocytes), CD20 (B lymphocytes), CD3 (T lymphocytes) and GFP (graft). Scale bars in a–i, 200 μm. Extended Data Figure 7: Macroscopic and microscopic analysis of iPSC-CM recipients. (1,332 KB) a–h, All recipients of iPSC-CMs received full necropsy after euthanasia. Neither macroscopic (a–d) nor microscopic (e–h) analysis revealed any evidence of tumour formation at 12 weeks post cell transplantation. Scale bars in a–d and e–h: 10 mm and 200 μm, respectively.i–p, Additional immunohistochemical analysis of cynomolgus hearts. i, Immunohistochemistry for GFP (brown) counterstained with fast green. Scale bar, 1 mm. j, k, Picrosirius red staining of a section in close proximity to the visual field in a shows partial remuscularisation of the scar (shown in red) by grafted cardiomyocytes. l–n, Different sections (lower by 5 mm towards the apex) showing the corresponding 2 grafts from Fig. 1b. Scale bar, 200 μm. m, n, Magnified images of the grafts, scale bar, 50 μm. Note the more direct contact zone of grafted cardiomyocytes with host myocardium. o, p, Additional examples of grafted cardiomyocytes in the scar and the border zone. Scale bars, 200 μm. Extended Data Figure 8: Summary of histological, mechanical and electrophysiological consequences. (366 KB) a, Animal characteristics with histological, mechanical and calcium imaging results. b, Correlation between ejection fraction (EF) and scar area relative to left ventricular area (LV). c, Correlation between ejection fraction and graft area relative to left ventricle. d, Summary of sustained ventricular tachycardia (VT), including number of VTs, maximum duration, and maximum heart rate (HR), in the recipients of iPSC-CMs. Extended Data Figure 9: Additional electrical analysis of hearts transplanted with iPSC-CMs. (305 KB) a, b, Activation map obtained from G-CaMP7.09 transients showing the interval (in ms) between the R wave of ECG and the peak of the G-CaMP7.09 fluorescent signal. c–f, Examples of sustained and non-sustained VT in recipients of iPSC-CMs. Arrows indicate P wave during VT, suggesting atrioventricular dissociation. Scale bar, 1 s. Extended Data Figure 10: Time course of left ventricular size and BNP levels. (196 KB) a–d, Left ventricular size was analysed before transplantation (Pre-Tx), 4 weeks post-transplantation (4 w post-Tx) and 12 weeks post-transplantation (12 w post-Tx) by echocardiography (a, b) and μCT (c, d). LVEDD: left ventricular end-diastolic dimension, LVESD: left ventricular end-systolic dimension, LVEDV: left ventricular end-diastolic volume, LVESV: left ventricular end-systolic volume. n = 5 per group. #P < 0.05; ##P < 0.01. * P < 0.05; **P < 0.01 versus Pre-TX. e, BNP was measured on days 0 (14 days after myocardial infarction), 28, 56 and 84. No significant difference was detected between recipients of iPSC-CMs and recipients of PSC vehicle at any time point. *P < 0.05 versus day 0. Supplementary information References• Author information• Extended data figures and tables• Supplementary information Video Video 1: In vitro fluorescent imaging of G-CaMP7.09-expressing cynomolgus iPSC-CMs (10.37 MB, Download) Monolayer-cultured cardiomyocytes exhibit robust fluorescent flashes in synchrony with their contraction. Video 2: In vitro experiments with G-CaMP7.09-expressing iPSC-CMs by using nifedipine, ryanodine, and caffeine (15.49 MB, Download) Representative video of G-CaMP7.09 fluorescent transients and contraction of cardiomyocytes before and after treatment with the L-type calcium-channel blocker nifedipine, the ryanodine receptor blocker ryanodine, and the ryanodine receptor activator caffeine. Video 3: In vitro G-CaMP7.09 transients sustained after cessation of spontaneous contraction of iPSC-CMs by BDM. (6.01 MB, Download) Treatment with 40 mM BDM resulted in cessation of spontaneous contraction of iPSC-CMs, but G-CaMP7.09 fluorescent transients were sustained for a few minutes. Video 4: Myocardial ischemia/reperfusion model in cynomolgus monkey (19.84 MB, Download) The myocardial infarction model was produced by 3 h of ischemia followed by reperfusion using polyethylene tubing 2 weeks before transplantation. Video 5: Intravital imaging of G-CaMP7.09-expressing iPSC-CMs in cynomolgus heart (9.49 MB, Download) G-CaMP7.09⁺ iPSC-CMs were transplanted into infarcted cynomolgus hearts. The hearts were excised and mechanically arrested ex vivo by perfusion with BDM on a Langendorff apparatus at 12 weeks post-transplantation. All graft regions exhibited cyclic changes in fluorescent intensity that occurred synchronously in a 1:1 relationship with the host ECG when the heart beat spontaneously or was electrically paced at rates from 3 to 5 Hz. Note that some, but not all hearts could be paced up to 5 Hz. Video 6: Cardiac contractile function assessed by mCT at 4 weeks post-transplantation (3.34 MB, Download) Cardiac function was assessed by mCT pre- and post-transplantation. The first and second segments of the video show left ventricular contractions of the short axis at the base and apex, respectively. Note that while contraction at the base is similar, that at the apex in iPSC-CM recipients looks better than that in PSC-vehicle recipients. The last segment represents a long-axis view of the left ventricle.