1. Volume 24, Issue 11, Pages 1949-1964 (November 2016)
Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs 
Clementina Sitzia, Andrea Farini, Luciana Jardim, Paola Razini, Marzia Belicchi, Letizia Cassinelli, Chiara Villa, Silvia Erratico, Daniele Parolini, Pamela Bella, Joao Carlos da Silva Bizario, Luis Garcia, Marcelo Dias-Baruffi, Mirella Meregalli, Yvan Torrente 
Molecular Therapy 
Volume 24, Issue 11, Pages (November 2016)
DOI: /mt
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Characterization of 133+musSCs. (a) In vivo characterization of 133+musSCs (green) around untreated Golden Retriever muscular dystrophy (GRMD) muscle fibers (laminin in violet). (b) Fluorescence-activated cell sorting (FACS) analysis of muscle derived cells from homogenized tibialis cranialis showed CD133 and CD34 coexpression (c,d) FACS characterization of freshly isolated CD133+musSCs: median purity value (95%); CD133 and CD34 coexpression (>50%), and CXCR4 antigen expression (2.3%). No expression of CD45 antigen. (e) 133+musSCs 24 hours after cell sorting, and (f) in proliferation medium for 7 days. (g) Vascular structures of dystrophic 133+musSCs in endothelial differentiation medium for 21 days. (h) Pax7 (green) and desmin (red) coexpression of CD133+musSCs in muscle differentiation medium. (i,j) Desmin positive (green) fused myoblasts, desmin (green), and Myosin Heavy Chain (red) positive completely differentiated myotubes derived from 133+musSCs. (k) 133+muscSC proliferation rate during 4 weeks of culture. (l) Viability and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay showed cell viability as a percentage of viable cells on total cells (n = 3 replicates). (m) FACS immunophenotyping of LVex musSCs and (n-) in vitro culture optical images. (o) Optical images of LVex musSCs in colture in differentiation medium. (p) LVex musSCs myotube desmin expression (green). Nuclei were costained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) for all the confocal images. All images were captured with a confocal microscope Leica TCS SP2 (Leica, Germany): 20× magnification. (q,r) Proliferation rate during 4 weeks of culture and (r) viability assessed by MTT assay of LVex musSCs demonstrating no change of cell behaviour after lentiviral transduction. (s) Fluorescent in situ hybridization (FISH) analysis of LVex musSCs for the expression of U7 probe (red), showing the presence of vector in nuclei (DAPI) of transduced cells (t) and no signal could be observed in not-infected cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
In vivo analysis of tibialisis cranialis muscle biopsies. (a) Azan Mallory (AM) images and dystrophin immunostaining of tibialis cranialis sections from each severe phenotype dog injected with LVex musSCs at T6, T12, and TS. (b) Histograms representing the time course of dystrophin expression as a percentage of total myofibers. (c) Histograms representing myofiber mean diameters.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
In vivo analysis of vastus lateralis muscle biopsies. (a) Azan Mallory (AM) images and dystrophin immunostaining of vastus lateralis sections from each severe phenotype dog injected with LVex6-8 cell treated at T6, T12 and TS. (b) Histograms representing the time course of dystrophin expression as a percentage of total myofibers. (c) Histograms representing myofiber mean diameters.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Efficacy characterization of 133+musSCs. (a) Fluorescent in situ hybridization (FISH) analysis of 5′TEX615 probe (red) demonstrated the presence of muscle fibers with peripheral U7 positive nuclei (arrows) on muscles isolated from LVex6-8 cell treated injected dogs. (b,c) Serial sections of dystrophin (green) positive muscle fibers expressing positive U7 nuclei (arrows; red) in LVex6-8 cell treated GRMD dogs muscles. Magnification 40X for all the images. (d) WB analysis of cell-treated GRMD dogs muscle biopsies showed no detectable dystrophin expression through the time points. (e) WB evaluation of skipped dystrophin expression on muscular biopsies from LVex6-8cell-treated GRMD mild and severe phenotype dogs: no dystrophin expression at T0; detection of skipped dystrophin in all dogs at T12; mild phenotype dog dystrophin expression at TS. Each muscular biopsy was loaded with 70 µg of proteins and a muscle from a control dog was used as positive control (each gel was loaded with serial dilution of control dog; Lane 1:70 µg, Lane 2:35 μg, Lane 3:17.5 μg, and Lane 4:8.75 μg to allow dystrophin quantification). (f) RT-PCR analysis and sequencing of exon skipping product in dystrophin-positive fibers (laser-dissected) from muscular biopsies of LVex6-8cell-treated dogs at T12 and (g) at TS. M: molecular weight marker; T01 and T02: mild phenotype dogs; NT: not treated GRMD dog; T03, T04, and T05: severe clinical phenotype dogs. PCR product sequencing confirmed ligation between the exons after the skipping: (i) no skipping (*), (ii) exon 5 and 9 in frame skipping (**), and (iii) exon 5 and 10 in frame skipping (***). GRMD, Golden Retriever muscular dystrophy; RT-PCR, reverse transcription-polymerase chain reaction; SCs, stem cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Evolution of SCT and 6MWT test before and after the treatment. Graphs representing longitudinal evolution of SCT and 6MWT functional test for mild (a) and severe dogs (b): cell-treated GRMD (green curves) and LVex6-8cell-treated GRMD (red curves) compared with mean value (dark curve) ± SD (dot curves) of untreated colony GRMD dogs. A dark arrow indicates cell injection time. Of note, early on there was an important improvement of SCT and 6MWT in severe LVex6-8cell-treated dogs, unexpectedly followed by a rapid decrease in the curves after the third infusion. GRMD, Golden Retriever muscular; 6WMT, six-minute walking test; SCT, stair climbing test.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Immune response analysis. (a) Survival rate of untreated and treated Golden Retriever muscular dystrophy (GRMD) dogs. Age-matched severe and mild untreated dogs (dark lines) were compared with LVex6-8cell-treated (red) and cell-treated (green) dogs. It is remarkable that LVex6-8cell-treated GRMD dogs shown an increased survival time compared both with mild and severe untreated colony GRMD dogs, and to cell-treated GRMD dogs. (b) Staining of tibialis cranialis from GRMD T03, T04, and T05 dogs showed cellular infiltrates (DAPI) surrounding dystrophin (green) positive muscle fibers. (c) CD3 positive cells (red) surrounding the basal lamina of dystrophin positive myofibers (green) in T03 muscular biopsies. Immunofluorescence staining magnification showed a characteristic CD3 staining of muscle infiltrating positive cells (red). (c′) Absence of CD3+ cell in T01 muscular biopsy. Histogram representing CD3+ cells count per field on different sections from all treated dogs. (d) Up-regulation of MHC I and II positive myofibers in GRMD T03 dog in comparison to basal expression in U01 untreated GRMD dog. (e) CD4 positive T memory cells resident in the inguinal lymph node of GRMD T03, T04,T05, inguinal lymph node stained with secondary antibody alone was used as negative control and absence of CD4+ reactive clusters in U01 untreated GRMD dog. (f) Peripheral blood mononuclear cells (PBMCs) from all treated dogs, were stimulated either with autologous 133+musSCs (red) or LVex musSCs (blue) or with protein extracts from transplanted muscles (green) that expressed high amounts of dystrophin: at T12 weak immunogenicity of lymphocytes from all dogs to autologous CD133+ cells or to LVex musSCs and to dystrophin expressing muscle, at TS GRMD T03, T04, and T05 lymphocytes showed a strong reaction against dystrophin positive muscle and a comparable reaction with C01, C02, C03, and T01, T02 GRMD dogs against transduced or untreated cells, indicating the occurrence of an antidystrophin T cell immune response after the third infusion only in severe LVex6-8cell treated GRMD dogs.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Immune response characterization. IFNγ-ELISpot analysis of PBMCs at TS of severe LVex6-8cell treated GRMD dogs showing a significant positive reaction to a pool of dystrophin peptides (cells + DYS) while no positive reaction was observed in mild LVex6-8cell treated GRMD dogs T01 and T02. Representatives image of significant wells were shown. (b) ELISA of serum IFNγ levels during the treatment and the follow up, infusions were shown by arrows. Severe LVex6-8cell treated GRMD dogs showed a progressive increase in IFNγ levels till death. (c) WB analysis of specific serum IgG antibody against dystrophin. The different lanes contain muscle homogenate from healthy dog expressing full-length dystrophin (1, 3, 5, 7, and 9), from healthy mice (2, 4, 6, and 8) The blots were stained using the NCL-DYS 1 and NCL-DYS 2 that recognizes the carboxyl terminus and the Rod domain of canine and murine dystrophin, serum from severe dog or serum from mild dog at different time point (T0, T12, and TS). The band of ∼427 kDa that corresponds to dystrophin appears in the monoclonal antibody-positive control (lane 1 and 2) and in the immunoreactive serum of severe LVex6-8 cell treated GRMD dogs. ELISPOT, enzyme-linked immunosorbent spot; GRMD, Golden Retriever muscular dystrophy; PBMCs, peripheral blood mononuclear cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions