Human circulating AC133⁺ stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle
J. Clin. Invest. Yvan Torrente, et al. 114:182 doi:10.1172/JCI20325 [Go to this article.]

Figure 3
Myogenic differentiation of blood-derived AC133⁺ cells in vitro. (A and B) In myogenic differentiation, AC133⁺ cells from the blood do not form myotubes but remain as mononucleated, quiescent, desmin-positive cells (A) and M-cadherin–positive cells (B). (C) The early myogenic commitment (at 24 h of culture) was demonstrated by specific RT-PCR for (C) human markers performed on mRNA extracted from AC133⁺ cells isolated from blood (lane 1), a C2C12 murine cell line (lane –), and a human cDNA library (lane +). (D) AC133⁺ and AC133– cells derived from the blood were transduced with a third-generation lentivirus vector expressing GFP cDNA under the transcriptional control of the human phosphoglycerate kinase (PGK) promoter. GFP expression of the transduced cells is shown both in phase contrast (PhCo) and in fluorescence (GFP). Fluorescence analysis of transduced human circulating AC133⁺ cells cocultured with uninfected C2C12 mouse myoblasts revealed several multinucleated, human slow MyHC–positive myotubes (arrows) that incorporated the GFP⁺ cells (lower panel, bottom row). Moreover, transduced human blood–derived AC133– cells failed to differentiate into myosin-positive muscle cells when plated on the C2C12 feeder cells (lower panel, top row). (E) Double immunocytochemistry with antibodies against MyHC (red) and human lamin A/C (green) demonstrated the presence of human (arrowheads) and mouse (arrows) nuclei within myotubes, confirming the fusion of AC133⁺ cells with the C2C12 murine myoblasts. Nuclei were stained blue with DAPI. (F) Immunoblot analysis of human MyHC (MyHC-s) and human M-cadherin (M-Cad) confirmed the myogenic differentiation of the circulating AC133⁺ stem cells (CSC) cocultured for 14 days with C2C12 mouse myoblasts in low-serum fusion-promoting conditions. The 3T3 fibroblast and G8 myoblast cell lines were used as controls. The utrophin immunoblot (Utr) indicates that the same total protein concentration was present in all specimens.