Volume 20, Issue 3, Pages 407-414.e4 (March 2017) Injury-Induced Senescence Enables In Vivo Reprogramming in Skeletal Muscle  Aurélie Chiche, Isabelle Le Roux, Mathieu von Joest, Hiroshi Sakai, Sabela Búa Aguín, Coralie Cazin, Rana Salam, Laurence Fiette, Olinda Alegria, Patricia Flamant, Shahragim Tajbakhsh, Han Li  Cell Stem Cell  Volume 20, Issue 3, Pages 407-414.e4 (March 2017) DOI: 10.1016/j.stem.2016.11.020 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2017 20, 407-414.e4DOI: (10.1016/j.stem.2016.11.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Injury-Enabled In Vivo Reprogramming (A) Scheme of the experiments. (B) Panels left to right represent transverse TA cryosections after histological staining with H&E and immunofluorescence with anti-laminin, anti-Nanog, and anti-Pax7 antibodies. Large dashed circle highlights Nanog+ cell; arrowheads show Pax7+ cells (high magnification in last panel). DPI, days post-injury. (C) Survival curve of Dmdmdx-βgeo;i4F-A mice (n = 14) compared to Dmdmdx-βgeo mice (n = 3) treated with DOX. (i) Analysis of TA muscles (parts of whole chart). (D) Histological section of a teratoma derived from the muscle of a Dmdmdx-βgeo;i4F-A mouse. H&E-stained sections revealed features of (from left to right): ectoderm (squamous epithelium with keratinization and neural tissues shown in the top and middle panels, respectively); mesoderm (striated muscle and cartilage shown in the top and middle panels, respectively); and endoderm (respiratory ciliated epithelium and columnar epithelium shown in the top and middle panels, respectively). Immunohistochemical stainings confirmed the pathological analyses (bottom row, from left to right), anti-neuron-specific Class III β-tubulin (βIII) (TUJ1), SMA (smooth muscle actin), and GATA4. Scale bars: for (B), 50 μm for H&E image, 25 μm for immunofluorescence images, and 20 μm for immunohistochemistry; for (D), 25 μm. See also Figure S1. Cell Stem Cell 2017 20, 407-414.e4DOI: (10.1016/j.stem.2016.11.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Satellite Cells Are Major Cells of Origin for In Vivo Reprogramming in Muscle (A) In vitro reprogramming efficiency of SCs and FAPs compared to mouse skin fibroblasts (MSFs). Representative image of iPSC colony from SCs of Pax7CT2;R26mT/mG; i4F-A mice (n = 4 mice). Statistical significance was assessed by the two-tailed Student’s t test: ∗p < 0.05. (B) Survival curve of Pax7CT2;R26mT/mG;i4F-A mice (n = 7) compared to Pax7CT2;R26mT/mG mice (n = 3). (i) Characterization of TA muscles (parts of whole chart). (C) Immunofluorescence using anti-GFP and anti-Pax7 antibodies on TA cryosections showing teratoma formation in Pax7CT2;R26mT/mG;i4F-A mouse. White arrowheads point to Pax7+ cells, white arrows show Pax7−/mGFP+ cells. Scale bars, 100 μm. All data correspond to the average ± SEM. See also Figure S2. Cell Stem Cell 2017 20, 407-414.e4DOI: (10.1016/j.stem.2016.11.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Cellular Senescence Associates with In Vivo Reprogramming (A) SAβGal staining combined with immunohistochemistry using anti-Nanog antibody. Low (left) and high (right) magnification panels are shown. Black arrowheads point to SAβGal+ cells. (B) Quantification and correlation of SAβGal+ and Nanog+ cells from the same section (n = 9 mice; value represents the average of two TAs per mouse). (C) Expression of the indicated genes in injured muscle measured by qRT-PCR. Values are relative to the expression of these genes in the non-injured TA from the same mouse (n = 9 mice; nine TAs per condition). Data correspond to the average ± SD. For each assay, qRT-PCR values were obtained in duplicate or triplicate. (D) Scheme of the experiments; Histological staining with H&E (left panels) and SAβGal staining on the TA muscle (right panels). Black arrowheads point to SAβGal+ cells (inset, high magnification). DPI, days post-injury. (E) Quantification of SAβGal+ cells in three different experimental settings: injured TAs with DOX, non-injured TAs with DOX, and injured TAs without DOX (n = 3 per group; two TAs per mouse). Statistical significance was assessed by the two-tailed Student’s t test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. For all histological, immunohistochemistry stainings, data were collected from transverse TA muscle cryosections of adult mice. Scale bars: for (A), 100 μm for the left panel and 25 μm for the right panel; for (D), 100 μm. All data correspond to the average ± SD. See also Figure S3. Cell Stem Cell 2017 20, 407-414.e4DOI: (10.1016/j.stem.2016.11.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Cellular Senescence Promotes Injury-Induced In Vivo Reprogramming (A) Survival curve of young i4F-B mice treated with DOX (n = 6), old i4F-B mice treated with DOX (n = 4), and old i4F-B mice without DOX (n = 4) (p < 0.05). (B) Quantification of SAβGal+ and Nanog+ cells in locally irradiated (IR) TAs compared to non-irradiated control TAs of the same mouse (n = 5). (C) Quantification of SAβGal+ and Nanog+ cells in TAs from ABT263-treated mice (n = 4) compared to vehicle control (n = 4). (D) Quantification of SAβGal+ and Nanog+ cells in TAs from GCV-treated mice (n = 7) compared to vehicle control (n = 7). (E) Survival curve of mice treated with IL-6 (n = 8) versus IgG (n = 6) by intraperitoneal injection. (F) In vitro reprogramming efficiency of SCs co-cultured on non-senescent (NSen) compared to senescent cells (Sen) and treatment of various dosage of IL-6 blocking antibody or IL-6 recombinant protein (n = 4 mice). All data correspond to the average ± SEM. Statistical significance was assessed by the two-tailed Student’s t test: ∗p < 0.05, ∗∗p < 0.01. See also Figure S4. Cell Stem Cell 2017 20, 407-414.e4DOI: (10.1016/j.stem.2016.11.020) Copyright © 2016 Elsevier Inc. Terms and Conditions