Volume 17, Issue 11, Pages 1948-1958 (November 2009) Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities  Karine Vauchez, Jean-Pierre Marolleau, Michel Schmid, Patricia Khattar, Alain Chapel, Cyril Catelain, Séverine Lecourt, Jérôme Larghéro, Marc Fiszman, Jean-Thomas Vilquin  Molecular Therapy  Volume 17, Issue 11, Pages 1948-1958 (November 2009) DOI: 10.1038/mt.2009.204 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Immunohistological and flow cytometric characterization of ALDH+ cells in human skeletal muscle. (a) Immunohistofluorescence localization. An aldehyde dehydrogenase-positive (ALDH1+) cell (green, nucleus in blue) located outside muscle fibers delineated by laminin labeling (red, arrow heads) is in an endomysial position (merge, arrow). Bar = 10 µm. (b) Representative flow cytometric analysis of ALDH activity. Baseline fluorescence established in the presence of Aldefluor substrate and the inhibitor diethylaminobenzaldehyde. Gate 1 (G1) was defined to exclude debris (first panel). Based on G1, ALDHbr cells presented a shift of fluorescence defining the population in Gate 2 (G2) (second panel). Based on G2, SMALD+/CD34+ (G3) and SMALD+/CD34− (G4) were clearly discriminated by the level of allophycocyanin-conjugated CD34 labeling (third and fourth panels). (c) Illustrative dot plot phenotypic characterizations of SMALD sub-populations based on G2. Percentages relate to the total number of cells in G1. SMALD, skeletal muscle aldehyde dehydrogenase. Molecular Therapy 2009 17, 1948-1958DOI: (10.1038/mt.2009.204) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Cell sorting and phenotypic analysis in vitro. (a) Flow cytometric analysis, gating, and sorting of dissociated muscle sub-populations. Cells were labeled for aldehyde dehydrogenase (ALDH) activity and CD34 expression. The shift of fluorescence defined the population in G1 (left panel) presenting high ALDH activity. The following gates defined the sub-populations on the basis of ALDH activity and allophycocyanin-conjugated CD34 labeling. G2: SMALD+/CD34+, G3: SMALD+/CD34−, G4: SMALD−/CD34+, and G5: SMALD−/CD34− cells (right panel). G2 and G3 were first determined on G1. (b) Immunocytofluorecence analysis of SMALD+/CD34− and SMALD+/CD34+. SMALD+ cells were labeled for ALDH activity (green) and extracellular markers (PE-conjugated, red) 18 hour (D0) and 6 days (D6) after plating. Nuclei were stained by DAPI (blue). SMALD+/CD34−: some cells only expressed CD44 on D0, whereas numerous cells expressed CD44, CD56, and CD146 on D6. SMALD+/CD34+: the cells only expressed CD34 on D0, whereas on D6 numerous cells expressed CD15 or CD44, but not CD34. Controls were obtained on D0 and D6 by omission of the primary antibodies. Bar = 10 µm. SMALD, skeletal muscle aldehyde dehydrogenase. Molecular Therapy 2009 17, 1948-1958DOI: (10.1038/mt.2009.204) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 In vitro analysis of SMALD+/CD34− and SMALD+/CD34+-derived cells. (a) Phase contrast pictures of SMALD+/CD34− and SMALD+/CD34+ cells expanded in proliferation and shifted to differentiation media. SMALD+/CD34− derived cells differentiated into myotubes (phase contrast) expressing fast myosin heavy-chain isoform (b,e; red; nuclei stained in blue). SMALD+/CD34+ derived cells accumulated lipid droplets of various sizes (c,f; droplets stained by Oil Red). Bars = 25 µm (a–e), 8 µm (f). (b) Fluoresence-activated cell sorting phenotypic characterizations for CD56 and CD15 membrane markers on expanded SMALD+/CD34− and SMALD+/CD34+ populations. (c) Quantitative comparisons between populations regarding CD56 or CD15 expression. Most SMALD+/CD34−-derived cells expressed CD56 in culture, but not CD15, whereas half SMALD+/CD34+-derived cells expressed CD15 but never CD56. CD56+ cells also originated from nonsorted and SMALD−/CD34− fractions. n, numbers of biopsies analyzed. SMALD, skeletal muscle aldehyde dehydrogenase. Molecular Therapy 2009 17, 1948-1958DOI: (10.1038/mt.2009.204) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Establishment and use of the predictor percentage (PP). (a) Initial fluoresence-activated cell sorting analysis of Aldefluor and CD34-allophycocyanin labeling on dissociated muscle cells (n = 20 biopsies) enabled calculation of PP value for each biopsy, before expansion in culture. Upon phenotyping (CD56, CD15), cultures were divided in two groups according to respective proportions of markers: [56+ > 15+] and [56+ < 15+]. In each group, biopsies (circles) were ranked as functions of their initial PP, with median PP indicated by black dash. The difference between groups was significant (P < 0.01). At the threshold PP value of 25.5% (dashed line) the outcome of a culture was equivalent regarding the yield in CD56+ and CD15+ cells. (b) Illustration of calculated outcome probabilities at established PP values. Below 25.5%, the probability is higher to produce more CD15+ cells than CD56+ cells. Above 25.5%, the probability is higher to produce more CD56+ cells. Molecular Therapy 2009 17, 1948-1958DOI: (10.1038/mt.2009.204) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Participation of SMALD+/CD34− and SMALD+/CD34+ sorted cells to muscle regeneration in vivo. Numerous SMALD+/CD34− cells were identified at respective injection sites by human nuclei (lamin A/C, red), human mitochondrial COX2 (green) and basal lamina delineation (laminin, blue) on transverse (a–r) sections. In s-u (oblique sections), blue DAPI staining of nuclei was illustrated instead of laminin staining. Only a few SMALD+/CD34+ cells were observed at injection sites (a–c, g–i) as labeled for COX2 (asterisks) and lamin A/C (arrows), and remained in the endomysium. In contrast, SMALD+/CD34− cells participated to muscle formation as illustrated by human mitochondria labeling inside muscle fibers indicative of the fusion between donor cells and recipient fibers or the formation de novo of muscle fibers (d–f, j–u, asterisks). Human nuclei were observed inside fibers (arrowheads) or in the endomysium (arrows). Bars = 50 µm (a–f), 10 µm (g–o, s–u), 20 µm (p–r). SMALD, skeletal muscle aldehyde dehydrogenase. Molecular Therapy 2009 17, 1948-1958DOI: (10.1038/mt.2009.204) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Localization of SMALD+/CD34− cells upon transplantation. SMALD+/CD34− cells were identified at injection sites by human nuclei (lamin A/C, red), dystrophin (green), and basal lamina (laminin, blue) delineations on transverse sections. Human nuclei were observed inside fibers (asterisks) or in the endomysium (arrows; a–f, j–l). Some fibers were surrounded by laminin, but did not express detectable levels of dystrophin, and may be regenerating fibers (d–f, j–l). A few human nuclei were located below basal lamina of muscle fibers (stained by laminin), but still outside them (delineated by dystrophin), hence in a putative satellite cell position (arrowheads; g–i, m–o). Bars = 15 µm (a–i), 8 µm (j–o). SMALD, skeletal muscle aldehyde dehydrogenase. Molecular Therapy 2009 17, 1948-1958DOI: (10.1038/mt.2009.204) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions