Volume 22, Issue 1, Pages 91-103.e5 (January 2018) Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction  Kazutaka Miyamoto, Mizuha Akiyama, Fumiya Tamura, Mari Isomi, Hiroyuki Yamakawa, Taketaro Sadahiro, Naoto Muraoka, Hidenori Kojima, Sho Haginiwa, Shota Kurotsu, Hidenori Tani, Li Wang, Li Qian, Makoto Inoue, Yoshinori Ide, Junko Kurokawa, Tsunehisa Yamamoto, Tomohisa Seki, Ryo Aeba, Hiroyuki Yamagishi, Keiichi Fukuda, Masaki Ieda  Cell Stem Cell  Volume 22, Issue 1, Pages 91-103.e5 (January 2018) DOI: 10.1016/j.stem.2017.11.010 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 SeV-GMT Promotes the Efficiency and Speed of Cardiac Reprogramming in MEFs (A and B) FACS analyses for cTnT expression in pMX-G/M/T-, pMX-MGT-, and SeV-GMT-transduced MEFs at 1 week after transduction (A). MOI for each infection was as indicated. Quantitative data are shown in (B) (n = 3 independent triplicate experiments). (C) Immunocytochemistry for αMHC-GFP, cTnT, and DAPI. The pMX-G/M/T-, pMX-MGT-, and SeV-GMT-transduced MEFs expressed αMHC-GFP and cTnT after 4 weeks. High-magnification views in insets show the sarcomeric organization. (D) Immunocytochemistry for αMHC-GFP, cTnT, and DAPI in MEFs transduced with pMX-G/M/T, pMX-MGT, or SeV-GMT after 1, 2, and 4 weeks. Only merged pictures are shown. Note that sarcomeric structures were clearly detected as of 1 week in the SeV-GMT-induced iCMs (high-magnification views in insets). (E) Time courses of immunopositive cells for αMHC-GFP and cTnT (n = 7 independent triplicate experiments). (F) Western blot analyses for cTnT expression in pMX-G/M/T-, pMX-MGT-, or SeV-GMT-transduced MEFs. (G) Relative mRNA expression levels were determined in pMX-G/M/T-, pMX-MGT-, or SeV-GMT-transduced MEFs by qRT-PCR after 1 week (n = 3 independent triplicate experiments). (H–J) Spontaneous Ca2+ oscillations in the SeV-GMT-induced iCMs at 4 weeks after transduction. Maximum and minimum concentrations of Ca2+ signals are shown in the upper panels, and the Rhod-3 intensity trace is shown in the lower panel in (H). Percentages of Ca2+ oscillation+ cells were calculated as the number of Ca2+ oscillation+ cells divided by the numbers of initial fibroblasts (I) and total cells at the time of assay (J), respectively (n = 3 independent triplicate experiments). (K–M) Percentage of beating cells was calculated as the number of beating cells divided by the number of initial fibroblasts (K, L; n = 3 independent triplicate experiments). Time course of spontaneously beating cells after transduction of mock, pMX-G/M/T, pMX-MGT, or SeV-GMT is shown in (K), and the number of spontaneously beating cells after 6 weeks in three independent triplicate experiments is shown in (L). Percentage of beating cells calculated as the number of beating cells divided by the number of total cells after 6 weeks is shown in (M) (n = 3 independent triplicate experiments). All data are presented as the means ± SD. ∗p < 0.05, ∗∗p < 0.01; versus the relevant control. NS, not significant. ND, not detected. Scale bars represent 100 μm. See also Figures S1 and S2 and Movie S1. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Functional Analyses of SeV-GMT-Induced iCMs (A–C) Action potentials (APs) of single beating SeV-GMT-iCMs were recorded after 2–4 weeks of transduction. Representative APs of iCMs are shown (nodal- [A], atrial- [B], and ventricular-like iCMs [C]). Enlarged views of single action potentials are shown to the right of the tracing. Electrically stimulated action potentials are shown at the right ends in (B) and (C). (D) Summary of the measured AP parameters. MDP, maximum diastolic potential; AMP, amplitude; dV/dt, maximum rate of rise of AP; APD50, AP duration at 50% of repolarization; APD90, AP duration at 90% of repolarization. Atrial-like APs were most frequently recorded in the SeV-GMT-iCMs (n = 14). (E and F) Recorded Ca2+ transients showing effects of 2 μM isoproterenol (Isp), 5 μM carbachol (Cch), or 1 μM verapamil (Vp) on the calcium-transient frequency and the relative fluorescence intensity (F/F0) of SeV-GMT-iCMs (E). Quantitative data are shown in (F) (n = 28 [Isp], 27 [Cch], 29 [Vp]). All data are presented as the means ± SD.∗p < 0.05, ∗∗p < 0.01; versus the relevant control. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 SeV-GMT Promotes Cardiac Reprogramming in a Dose-Dependent Manner without Genomic Integration (A) FACS analyses and immunocytochemistry for GFP expression in MEFs transduced with pMX-GFP or SeV-GFP at an MOI of 20 after 1 week. (B) Immunocytochemistry for GATA4 and MEF2C expression in MEFs transduced with pMX-G/M/T or SeV-GMT after 1 week. (C) Western blot analyses for GMT protein expression in MEFs transduced with pMX-G/M/T or SeV-GMT at an MOI of 20 after 2, 4, and 6 days (M, marker; Con, control). (D) Western blot analyses for GMT protein expression in MEFs transduced with pMX-MGT at an MOI of 20 or SeV-GMT at an MOI of 1–50 after 2 days. (E and F) Immunocytochemistry for αMHC-GFP, α-actinin, cTnT, and DAPI at 4 weeks after transduction (E). SeV-GMT infection induced cardiac protein expression in MEFs in a dose-dependent manner, peaking at an MOI of 20. Quantitative data of immunopositive cells for cTnT and α-actinin are shown in (F) (n = 5 independent triplicate experiments). (G) The number of beating cells also increased in SeV-GMT-transduced MEFs in a dose-dependent manner (n = 3 independent triplicate experiments). (H) PCR analyses for GMT transgenes and endogenous Gapdh in genomic DNA from SeV-GMT (MOI 20) and pMX-G/M/T-infected MEFs. All data are presented as the means ± SD. ∗p < 0.05, ∗∗p < 0.01; versus the relevant control. NS, not significant. Scale bars represent 100 μm. See also Figure S3 and Tables S1 and S2. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Sendai Viral Particles Do Not Promote Cardiac Reprogramming or Induce iCM Proliferation (A) Western blot analyses for GMT expression in MEFs infected with SeV-GFP, pMX-G/M/T with or without SeV-GFP, and SeV-GMT after 2 days. Note that addition of SeV-GFP to pMX-G/M/T reduced GMT protein expression. (B and C) Immunocytochemistry for α-actinin, cTnT, and DAPI staining in MEFs infected with SeV-GFP and pMX-G/M/T with or without SeV-GFP (B). Quantitative data are shown in (C) (n = 3 independent triplicate experiments). (D) qRT-PCR analyses for cardiac gene expression at 1 week after transduction (n = 3 independent triplicate experiments). (E) Spontaneously beating iCMs at 6 weeks after transduction (n = 3 independent triplicate experiments). Addition of SeV-GFP to pMX-G/M/T reduced beating iCMs. (F) Schematic representation of EdU treatment during cardiac reprogramming. (G) Immunocytochemistry for cTnT in mock-, pMX-G/M/T-, and SeV-GMT-transduced MEFs cultured for 4 weeks, coupled with 2 weeks of EdU incorporation. The majority of cTnT-positive cells were negative for EdU. (H) FACS analysis of EdU incorporation in mock-, pMX-G/M/T-, and SeV-GMT-transduced MEFs at 4 weeks after transduction. All data are presented as the means ± SD. ∗p < 0.05, ∗∗p < 0.01; versus the relevant control. ND, not detected. Scale bars represent 100 μm. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 SeV Vectors Induce Cardiac Reprogramming in Postnatal Mouse Tail-Tip Fibroblasts and HCFs (A and B) FACS analyses for cTnT expression in postnatal mouse TTFs transduced with pMX-G/H/M/T or SeV-GMT/H (MOI 100) after 1 week (A). Quantitative data are shown in (B) (n = 3 independent triplicate experiments). (C) qRT-PCR analyses for cardiac gene expression at 1 week after transduction in TTFs (n = 3 independent triplicate experiments). (D and E) Immunocytochemistry of αMHC-GFP, α-actinin, cTnT, and DAPI staining in SeV-GMT/H- transduced TTFs after 6 weeks (D). Quantitative data are shown in (E) (n = 3 independent triplicate experiments). (F and G) Spontaneous Ca2+ oscillations in SeV-GMT/H-iCMs at 6 weeks after transduction (F). Maximum and minimum concentrations of Ca2+ are shown in the upper panels, and the Rhod-3 intensity trace is shown in the lower panel. Quantitative data are shown in (G) (n = 3 independent triplicate experiments). (H) Spontaneously beating iCMs at 6 weeks after transduction (n = 3 independent triplicate experiments). (I and J) FACS analyses for cTnT expression in the SeV-GMT/Mesp1/Myocd (GMTMM) or SeV-GMTMM/miR133-transduced HCFs at 10 days after transduction (I). Quantitative data are shown in (J) (n = 3 independent triplicate experiments). (K) Immunocytochemistry for α-actinin and DAPI staining in the SeV-GMTMM/miR133-transduced HCFs at 6 weeks after transduction. High-magnification views in insets show the sarcomeric organization. (L) qRT-PCR analyses for cardiac gene expression at 1 week after transduction in HCFs (n = 3 independent triplicate experiments). All data are presented as the means ± SD. ∗p < 0.05, ∗∗p < 0.01; versus the relevant control. ND, not detected. Scale bars represent 100 μm. See also Figures S3–S5, Movie S2, and Movie S3. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Lineage Tracing Demonstrates Efficient In Vivo Cardiac Reprogramming by SeV-GMT (A) SeV-GFP was injected into the ICR mouse infarct heart. GFP expression was localized to the infarct/border regions after 7 days. MI, myocardial infarction. (B) IHC for GFP, α-actinin, and DAPI staining 1 week after injection of SeV-GFP into MI hearts. Note that GFP+ cells were localized beside the cardiomyocytes at the infarct/border areas. (C and D) IHC for GFP, α-actinin, vimentin, collagen 1, and DAPI in the SeV-GFP-injected mouse MI hearts after 1 week (C). GFP+ cells were immunopositive for vimentin and collagen 1, but not for α-actinin. Ratios of α-actinin+ cardiomyocytes (CM), collagen 1+ CF (Fibro), and others to GFP+ cells are shown in (D) (n = 5). (E) Schematic diagram showing the genetic fate mapping method to trace the lineage of resident CFs using Tcf21iCre/R26-tdTomato mice. (F) Section of the Tcf21iCre/R26-tdTomato mouse heart 7 days after MI. (G) IHC for tdTomato, cTnT, and DAPI 1 week after MI. The tdTomato+ cells were not co-localized with cTnT. (H–J) IHC for tdTomato, cTnT, and DAPI 1 week after MI in Tcf21iCre/R26-tdTomato mouse hearts. Mock, pMX-MGT, and SeV-GMT were directly injected into the infarct myocardium. High-magnification views in insets show the sarcomeric organization and arrowheads indicate iCMs (H). z stack image for tdTomato and cTnT double-positive iCMs is shown in (I). Quantitative analyses are shown in (J) (n = 3). All data are presented as the means ± SD. ∗p < 0.05, ∗∗p < 0.01; versus the relevant control. ND, not detected. Scale bars represent 50 μm. See also Figure S6. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 In Vivo Delivery of SeV-GMT Improves Cardiac Function after Myocardial Infarction (A–C) IHC for GFP, α-actinin, and DAPI in SeV-GFP-, pMX-MGT/GFP-, or SeV-GMT/GFP-injected NOD-SCID mouse infarct hearts 4 weeks post-surgery. High-magnification views in insets show the sarcomeric organization, and arrowheads indicate iCMs (A). z stack image for GFP and α-actinin double-positive cells is shown in (B). Quantitative analyses are shown in (C) (n = 10). (D–F) Ejection fraction (EF) and fractional shortening (FS) of the left ventricle were serially quantified by echocardiography in mice injected with SeV-GFP, pMX-MGT, and SeV-GMT (D and E) (n = 15). Cardiac function was improved with pMX-MGT and SeV-GMT after 4 weeks (F) (n = 15). (G) Comparison of fibrosis areas among the SeV-GFP, pMX-MGT, and SeV-GMT injection groups 4 weeks after MI. Fibrosis was evaluated at three levels (L1–L3) by Azan staining. Representative histology data and quantitative analyses for the fibrotic area at each level are shown (n = 5). (H and I) IHC for collagen 1, cTnT, and DAPI in SeV-GFP, pMX-MGT, or SeV-GMT-injected NOD-SCID mouse hearts 4 weeks after MI (H). Quantitative analyses for collagen 1-positive areas are shown in (I) (n = 5). All data are presented as the means ± SD. ∗p < 0.05, ∗∗p < 0.01; versus the relevant control. ND, not detected. Scale bars represent 50 μm. See also Figure S6. Cell Stem Cell 2018 22, 91-103.e5DOI: (10.1016/j.stem.2017.11.010) Copyright © 2017 Elsevier Inc. Terms and Conditions