Myoblasts and Embryonic Stem Cells Differentially Engraft in a Mouse Model of Genetic Dilated Cardiomyopathy
Authors: Cyril Catelain, Stéphanie Riveron, Aurélie Papadopoulos, Nathalie Mougenot, Adeline Jacquet, Karine Vauchez, Erica Yada, Michel Pucéat, Marc Fiszman, Gillian Butler-Browne, Gisèle Bonne, Jean-Thomas Vilquin
Journal: Molecular Therapy (2013)

The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy. The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy. Cell therapies are progressively emerging as promising tools for the treatment of heart failure. In an attempt to achieve cardiac cell-based replacement therapy in the setting of postischemic cardiomyopathies (ICM), a variety of adult cell types have been tested up to preclinical stages in small and large animal models, including skeletal myoblasts (Mb), muscle-derived stem cells, adipose-derived stem cells, bone marrow mononuclear cells, hematopoietic stem cells, circulating endothelial progenitors, mesenchymal stem cells, smooth muscle cells, cardiac stem cells, and most of these approaches have demonstrated some degree of efficacy.1Durrani S Konoplyannikov M Ashraf M Haider KH Skeletal myoblasts for cardiac repair.Regen Med. 2010; 5: 919-932Crossref PubMed Scopus (62) Google Scholar,2Okada M Payne TR Drowley L Jankowski RJ Momoi N Beckman S et al.Human skeletal muscle cells with a slow adhesion rate after isolation and an enhanced stress resistance improve function of ischemic hearts.Mol Ther. 2012; 20: 138-145Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,3Léobon B Roncalli J Joffre C Mazo M Boisson M Barreau C et al.Adipose-derived cardiomyogenic cells: in vitro expansion and functional improvement in a mouse model of myocardial infarction.Cardiovasc Res. 2009; 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A few preclinical studies have been carried out using Mb,14Ohno N Fedak PW Weisel RD Mickle DA Fujii T Li RK Transplantation of cryopreserved muscle cells in dilated cardiomyopathy: effects on left ventricular geometry and function.J Thorac Cardiovasc Surg. 2003; 126: 1537-1548Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar,15Pouly J Hagège AA Vilquin JT Bissery A Rouche A Bruneval P et al.Does the functional efficacy of skeletal myoblast transplantation extend to nonischemic cardiomyopathy?.Circulation. 2004; 110: 1626-1631Crossref PubMed Scopus (67) Google Scholar smooth muscle cells or ventricular heart cells16Yoo KJ Li RK Weisel RD Mickle DA Tomita S Ohno N et al.Smooth muscle cells transplantation is better than heart cells transplantation for improvement of heart function in dilated cardiomyopathy.Yonsei Med J. 2002; 43: 296-303Crossref PubMed Scopus (14) Google Scholar in cardiomyopathic hamsters, or mesenchymal stem cells,17Chen M Fan ZC Liu XJ Deng JL Zhang L Rao L et al.Effects of autologous stem cell transplantation on ventricular electrophysiology in doxorubicin-induced heart failure.Cell Biol Int. 2006; 30: 576-582Crossref PubMed Scopus (37) Google Scholar mixed mesenchymal stem cells and Mb,18Guarita-Souza LC Francisco JC Simeoni R Faria-Neto JR de Carvalho KA Benefit of stem cells and skeletal myoblast cells in dilated cardiomyopathies.World J Cardiol. 2011; 3: 93-97Crossref PubMed Google Scholar or bone marrow cells in rat models of DCM.19Ishida M Tomita S Nakatani T Fukuhara S Hamamoto M Nagaya N et al.Bone marrow mononuclear cell transplantation had beneficial effects on doxorubicin-induced cardiomyopathy.J Heart Lung Transplant. 2004; 23: 436-445Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Among those studies, Mb seem to have the best potential of integration in the dilated myocardium, and represent a "gold standard" for cell-based therapy, although these cells are not able to differentiate into cardiomyocyte lineage. In contrast, embryonic stem cells (ESC) are pluripotent and can be readily committed towards the cardiogenic lineage in vitro. There is also increasing evidence that cardiac-committed ESC can engraft into the scar tissue within the infarcted myocardium and differentiate into cardiomyocytes, thereby operating a regeneration of the myocardium, eliminating fibrotic scar tissue, and promoting sustained improvement of left ventricular function.7Liu J Zhang Z Liu Y Guo C Gong Y Yang S et al.Generation, characterization, and potential therapeutic applications of cardiomyocytes from various stem cells.Stem Cells Dev. 2012; 21: 2095-2110Crossref PubMed Scopus (23) Google Scholar,8Menasché P Embryonic stem cells for severe heart failure: why and how?.J Cardiovasc Transl Res. 2012; 5: 555-565Crossref PubMed Scopus (10) Google Scholar,11Ebelt H Jungblut M Zhang Y Kubin T Kostin S Technau A et al.Cellular cardiomyoplasty: improvement of left ventricular function correlates with the release of cardioactive cytokines.Stem Cells. 2007; 25: 236-244Crossref PubMed Scopus (96) Google Scholar,20Laflamme MA Gold J Xu C Hassanipour M Rosler E Police S et al.Formation of human myocardium in the rat heart from human embryonic stem cells.Am J Pathol. 2005; 167: 663-671Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar,21Singla DK Hacker TA Ma L Douglas PS Sullivan R Lyons GE et al.Transplantation of embryonic stem cells into the infarcted mouse heart: formation of multiple cell types.J Mol Cell Cardiol. 2006; 40: 195-200Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar,22Blin G Nury D Stefanovic S Neri T Guillevic O Brinon B et al.A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.J Clin Invest. 2010; 120: 1125-1139Crossref PubMed Scopus (261) Google Scholar,23Behfar A Zingman LV Hodgson DM Rauzier JM Kane GC Terzic A et al.Stem cell differentiation requires a paracrine pathway in the heart.FASEB J. 2002; 16: 1558-1566Crossref PubMed Scopus (429) Google Scholar,24Behfar A Perez-Terzic C Faustino RS Arrell DK Hodgson DM Yamada S et al.Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.J Exp Med. 2007; 204: 405-420Crossref PubMed Scopus (203) Google Scholar,25Ménard C Hagège AA Agbulut O Barro M Morichetti MC Brasselet C et al.Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study.Lancet. 2005; 366: 1005-1012Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar,26Laflamme MA Chen KY Naumova AV Muskheli V Fugate JA Dupras SK et al.Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.Nat Biotechnol. 2007; 25: 1015-1024Crossref PubMed Scopus (1759) Google Scholar This confers the potential ability to rebuild true cardiac tissue, to replenish areas that have been depopulated following ischemic accidents or the progression of fibrosis.8Menasché P Embryonic stem cells for severe heart failure: why and how?.J Cardiovasc Transl Res. 2012; 5: 555-565Crossref PubMed Scopus (10) Google Scholar In contrast, this positive benefit is limited by the formation of teratomas27Nussbaum J Minami E Laflamme MA Virag JA Ware CB Masino A et al.Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response.FASEB J. 2007; 21: 1345-1357Crossref PubMed Scopus (503) Google Scholar or if too many cells are in uncommitted state at the time of injection.24Behfar A Perez-Terzic C Faustino RS Arrell DK Hodgson DM Yamada S et al.Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.J Exp Med. 2007; 204: 405-420Crossref PubMed Scopus (203) Google Scholar In the present study, we have compared the integration and functional efficacy of the CGR8 line of murine ESC with the D7LNB1 line of murine Mb, in the myocardium of LmnaH222P/H222P mice. This genetic model of laminopathy reproduces a human missense mutation in the Lamin A/C gene causing Emery-Dreifuss muscular dystrophy. This model exhibits a rapidly progressive and lethal DCM,28Arimura T Helbling-Leclerc A Massart C Varnous S Niel F Lacène E et al.Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies.Hum Mol Genet. 2005; 14: 155-169Crossref PubMed Scopus (249) Google Scholar showing pathophysiological evolution and conduction defects comparable to the human situation. Of note, these animals are immunocompetent. The CGR8 cell line of ESC was chosen because it can be grown feeder-free, and it is efficiently committed toward cardiogenic differentiation in vitro upon treatment with bone morphogenic protein 2 (BMP-2),23Behfar A Zingman LV Hodgson DM Rauzier JM Kane GC Terzic A et al.Stem cell differentiation requires a paracrine pathway in the heart.FASEB J. 2002; 16: 1558-1566Crossref PubMed Scopus (429) Google Scholar,24Behfar A Perez-Terzic C Faustino RS Arrell DK Hodgson DM Yamada S et al.Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.J Exp Med. 2007; 204: 405-420Crossref PubMed Scopus (203) Google Scholar,29Pucéat M Protocols for cardiac differentiation of embryonic stem cells.Methods. 2008; 45: 168-171Crossref PubMed Scopus (41) Google Scholar,30Pucéat M TGFbeta in the differentiation of embryonic stem cells.Cardiovasc Res. 2007; 74: 256-261Crossref PubMed Scopus (57) Google Scholar a treatment that indirectly lowers the risk of teratoma formation in vivo by decreasing the proportion of pluripotent cells.24Behfar A Perez-Terzic C Faustino RS Arrell DK Hodgson DM Yamada S et al.Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.J Exp Med. 2007; 204: 405-420Crossref PubMed Scopus (203) Google Scholar,27Nussbaum J Minami E Laflamme MA Virag JA Ware CB Masino A et al.Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response.FASEB J. 2007; 21: 1345-1357Crossref PubMed Scopus (503) Google Scholar The committed CGR8 cells, whether selected or not, have been previously shown to efficiently improve cardiac function following injection into the scar tissue in animal models of postischemic heart failure.7Liu J Zhang Z Liu Y Guo C Gong Y Yang S et al.Generation, characterization, and potential therapeutic applications of cardiomyocytes from various stem cells.Stem Cells Dev. 2012; 21: 2095-2110Crossref PubMed Scopus (23) Google Scholar,8Menasché P Embryonic stem cells for severe heart failure: why and how?.J Cardiovasc Transl Res. 2012; 5: 555-565Crossref PubMed Scopus (10) Google Scholar,23Behfar A Zingman LV Hodgson DM Rauzier JM Kane GC Terzic A et al.Stem cell differentiation requires a paracrine pathway in the heart.FASEB J. 2002; 16: 1558-1566Crossref PubMed Scopus (429) Google Scholar,24Behfar A Perez-Terzic C Faustino RS Arrell DK Hodgson DM Yamada S et al.Cardiopoietic programming of embryonic stem cells for tumor-free heart repair.J Exp Med. 2007; 204: 405-420Crossref PubMed Scopus (203) Google Scholar,25Ménard C Hagège AA Agbulut O Barro M Morichetti MC Brasselet C et al.Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study.Lancet. 2005; 366: 1005-1012Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar The time window for the addition of BMP-2 is of crucial importance,30Pucéat M TGFbeta in the differentiation of embryonic stem cells.Cardiovasc Res. 2007; 74: 256-261Crossref PubMed Scopus (57) Google Scholar therefore we pretreated CGR8 ESC for a short period of time, and we designed the experiments using limited amounts of cells to reach a compromise between myocardial differentiation and risk of teratoma formation (3 × 105 per heart, at four different sites). The Mb have been assayed, in the present study, as a gold standard for validating the injection procedure, the efficacy of the immunosuppression regimen, the natural evolution of the implanted cells, the immunohistological procedures. Comparisons between Mb and ESC in murine models of postischemic heart failure have pinpointed important intrinsic differences in the efficacies and persistence of these two cell types, which now deserve a comparison in a DCM model.11Ebelt H Jungblut M Zhang Y Kubin T Kostin S Technau A et al.Cellular cardiomyoplasty: improvement of left ventricular function correlates with the release of cardioactive cytokines.Stem Cells. 2007; 25: 236-244Crossref PubMed Scopus (96) Google Scholar The D7 Mb cell line was originally derived from the dy/dy mouse model of laminin-2 deficient congenital muscular dystrophy.31Yaffe D Saxel O Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.Nature. 1977; 270: 725-727Crossref PubMed Scopus (1561) Google Scholar It was engineered to express β-Galactosidase (β-Gal) constitutively and named D7LNB1. It showed no modification in its ability to form myotubes in vitro and in vivo. In addition, unlike C2 or G8 murine Mb, D7LNB1 Mb cell line did not produce carcinomas in vivo.32Vilquin JT Guérette B Puymirat J Yaffe D Tomé FM Fardeau M et al.Myoblast transplantations lead to the expression of the laminin alpha 2 chain in normal and dystrophic (dy/dy) mouse muscles.Gene Ther. 1999; 6: 792-800Crossref PubMed Scopus (26) Google Scholar CGR8 ESC and D7 Mb originate from close parental strains of 129 mice derived from the same animals in the 1970's (129P/Ola background for ESC, 129/ReJ background for D7), and they share identical markers.33Simpson EM Linder CC Sargent EE Davisson MT Mobraaten LE Sharp JJ Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice.Nat Genet. 1997; 16: 19-27Crossref PubMed Scopus (603) Google Scholar To avoid immune rejection triggered by murine ESC34Swijnenburg RJ Schrepfer S Cao F Pearl JI Xie X Connolly AJ et al.In vivo imaging of embryonic stem cells reveals patterns of survival and immune rejection following transplantation.Stem Cells Dev. 2008; 17: 1023-1029Crossref PubMed Scopus (101) Google Scholar or Mb35Huard J Roy R Guérette B Verreault S Tremblay G Tremblay JP Human myoblast transplantation in immunodeficient and immunosuppressed mice: evidence of rejection.Muscle Nerve. 1994; 17: 224-234Crossref PubMed Scopus (86) Google Scholar further expressing β-Gal in allogenic contexts, recipient mice were immunosuppressed using Tacrolimus.36Kinoshita I Vilquin JT Guérette B Asselin I Roy R Tremblay JP Very efficient myoblast allotransplantation in mice under FK506 immunosuppression.Muscle Nerve. 1994; 17: 1407-1415Crossref PubMed Scopus (162) Google Scholar We compared the engraftment of committed ESC and D7LNB1 Mb in terms of functional benefits and fate of engrafted cells in vivo. Our results suggest that cardiac-committed ESC failed to engraft into the dilated myocardium of the LmnaH222P/H222P mice model, whereas Mb have a higher transplantation efficiency and greater functional improvement of cardiac function in this genetic model of dilated heart failure. Murine ESC from CGR8 line and the murine D7LNB1 Mb line were engineered to express the reporter β-Gal transgene to follow their fate within recipient heart tissue. These ESC and derived embryoid bodies (EBs), and D7LNB1 Mb cell lines all expressed β-Gal activity in vitro (Figure 1a–c), as demonstrated by the blue staining following incubation in X-Gal reagent. ESC were committed to a cardiac fate using BMP-2 pretreatment. The BMP-2 incubation decreased the proportion of cells expressing the CD15 (stage-specific embryonic antigen 1, SSEA-1) marker, indicating the enrichment in cardiac-committed cells by 60 to 65% (Figure 1d). The pretreatment also promoted the differentiation of EBs in vitro. By 14 days (from day 7 to day 21), beating clusters appeared within all of the EBs prepared from BMP-2-primed ESC, while only 20% of EBs prepared from unprimed ESC contained such beating bodies (Figure 1e). The incubation also promoted several-fold increases in expression of cardiac markers as evaluated by reverse transcription PCR (Figure 1f). Female LmnaH222P/H222P mice develop abnormal phenotypes more progressively than males and present longer life spans, although reduced when compared with the wild-type mice. Then female mice have been used throughout this long-term study. Twenty-six animals of matched ages (177 ± 21 day old) and left ventricular fractional shortening (LVFS; 39 ± 4%) were randomized into three groups receiving no cell implantation (Lmna-Sham, n = 8), ESC (Lmna-ESC, n = 11) or D7LNB1 cells (Lmna-D7, n = 7) to evaluate cell-based therapy during a 2-month period. Upon small left thoracotomy, 3.105 cells or medium were injected at four sites on the anterior-lateral wall of the left ventricle. To avoid rejection of β-Gal-expressing CGR8 ESC and D7LNB1 cells, all animals were immunosuppressed using Tacrolimus. Survival analysis (Figure 2) indicated that all mice of Lmna-D7 group survived until the end of the study compared with the Lmna-ESC and Lmna-Sham groups, because 5 out of the 8 mice from the Lmna-Sham group and 6 out of the 11 mice from the Lmna-ESC group showed a severe deterioration and had to be killed prematurely. It should be noted that two mice in the Lmna-ESC group developed a lethal thoracic teratoma within the first month. Nevertheless, none of the animals in the Lmna-D7 group showed any deterioration during the same period (Figure 2a). All surviving animals were killed at the end of the 2-month period, however, because animals of Lmna-Sham and Lmna-ESC groups had to be killed prematurely, we also killed two of the mice in the Lmna-D7 group at 1 month to compare the fate of injected cells. X-Gal staining revealed positive engraftment in cell-injected groups at 1 and 2 months: β-Gal–positive cells were found in 2 out of 11 animals of the Lmna-ESC group (i.e., 18%) and in 6 out of 7 animals of the Lmna-D7 group (i.e., 86%). D7LNB1 cells were observed in all but one injected heart, thus validating the injection procedure, and the robustness of the immunosuppressive protocol. In the Lmna-D7 group, the surfaces occupied by the transplanted cells identified by X-Gal staining were estimated by morphometric analysis and reached 4.8 ± 0.8% of total myocardial surface in certain sections 1 month after transplantation. Interestingly, although the number of animals was small and did not allow statistical comparisons, we observed a trend towards a decrease in surface occupied by transplanted cells with time, because β-Gal–positive cells represented only 2.2 ± 1.4% of the total section surface 2 months after transplantation (Figure 2b, n = 4). These clusters were observed at variable localizations (apex, ventricular wall, septum) probably reflecting the multisite injection methodology. The most substantial engraftment was generally localized in the septum (Figure 2c). The injected D7LNB1 cells formed small β-Gal–positive structures resembling myotubes or small skeletal muscle fibers (Figure 3a,b) and expressing myosin heavy chains (MHC) isoforms (see below). In contrast, the structures which were observed in the two animals of the Lmna-ESC group presented a very different nature. Indeed, β-Gal–positive cells in this group corresponded to the development of thoracic and/or intracardiac teratomas. No β-Gal–positive cells could be found in 9 out of 11 of the Lmna-ESC group which did not develop teratomas and survived for the duration of the experiment, 1 or 2 months after implantation. Sirius Red staining illustrated the extent of fibrosis within the hearts of these models. Apart from interstitial fibrosis, which constitutes a hallmark of remodeling in such animal models with DCMs, larger and irregular tracks of collagen depositions were observed in all hearts and evoked the needle injection trajectories (Figure 4c). Such collagen depositions were also observed surrounding the areas occupied by D7LNB1 β-Gal–positive cells in the group of Lmna-D7 animals. As reported above, important differences exist between the fates of D7LNB1 Mb and ESC 1 and 2 months after transplantation. Also, the implantation yield of ESC in our mouse model is strikingly different from that reported in mouse models of postischemic heart failure. These observations may not be predictive of the final functional efficacy, because cells may exert their effects through different mechanisms and kinetics. However, such differences prompted the examinations of the short-term fate of ESC. LmnaH222P mice were injected with ESC or D7LNB1 Mb and killed 12, 24, and 72 hours later (ESC) or at 72 hours later (D7LNB1). While β-Gal activity associated to the presence of ESC can be readily observed 12 hours after injection (Figure 4a), very few cells, if any, were observed at 24 hours (Figure 4a) and 72 hours after injection (not shown). In parallel, D7LNB1 cells occupied much larger areas 72 hours after injection (Figure 4c). These results suggested that at short times after injection D7LNB1 cells showed a better survival and implantation than ESC. We then tried to determine the causes of this decrease in survival using caspase 3 staining to detect cellular necrosis, TUNEL methodology to visualize the extent of apoptosis, and macrophage staining to illustrate the extent of nonspecific inflammation. Both necrosis and apoptosis were activated upon injection of ESC (Figure 4b), and the tissue also became infiltrated by macrophages and neutrophils. These cells may participate to the removal of cellular debris, and to the regeneration of myocardial tissue harmed by the needle puncture. As indicated above, β-Gal–positive ESC were not observed in 9 out of 11 animals. In the two animals, where β-Gal–positive ESC were observed, the formation of cardio-thoracic teratomas precluded their survival. Within these cellular masses (Figure 5a), we observed small clusters of cells expressing cardiac-specific antigens such as connexin-43 (Cx43) and α-actinin (Figure 5b–c). This indicated that some ESC have acquired a cardiogenic phenotype, upon development within the teratoma tissue. As expected, D7LNB1 Mb did not promote teratoma formation upon implantation. They also did not express Cx43 and α-actinin, further confirming that Mb cells do not transdifferentiate into cardiac tissue upon integration (Figure 5d,g). By contrast, and as a positive control, Cx43 labeling was observed at the periphery of the implantation area surrounding this new myogenic tissue. The nature of structures formed by D7LNB1 cells was further investigated using antibodies directed against specific isoforms of MHC, that are classified by their velocity of shortening in this following order: MHC I slow twitch oxidative (SO-MHC I), MHC IIa fast-twitch oxidative glycolytic (FOG-MHC IIa) and the MHC IIx and IIb fast-twitch glycolytic (FG-MHC IIx/b). Hybrid fibers expressing FOG-MHC IIa and FG-MHC IIx/b represent a subpopul