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1126. Further Characterization of Msx1 De-Differentiated Cells
MUSCLE AND CONNECTIVE TISSUE: GENE THERAPY FOR CONNECTIVE TISSUE 1125. AAV-Mediated Myostatin Propeptide Gene Delivery Results in Growth and Hypertrophy of Skeletal but Not Cardiac Muscles Chunping Qiao,1 Jianbin Li,1 Tong Zhu,1 Chunlian Chen,1 Terry O’Day,2 Jon Watchko,2 Juan Li,1 Xiao Xiao.3,1 1 Department of MGB, University of Pittsburgh, Pittsburgh, PA; 2 Dept. of Pediatrics, University of Pittsburgh, Pittsburgh, PA; 3 Dept. of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. Muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness. Because of the lack of effective treatment with traditional pharmaceutical agents, gene therapy and stem-cell therapy have been vigorously explored. Besides gene replacement therapy, a different form of gene therapy, i.e., expression of “booster genes”, has been investigated, aiming at alleviating the secondary deficiencies in muscular dystrophies rather than the primary ones. Blockade of myostatin is one of such strategies. Myostatin is a member of the TGF-beta family and a negative regulator of skeletal muscle growth. Studies using transgenic mice or antagonist antibody against myostatin showed promotion of muscle growth and amelioration of muscular dystrophy. However, gene delivery of myostatin inhibitors has not been reported. In this study we examined whether myostatin propeptide gene transfer into normal mice by AAV vectors could increase muscle mass and strength. AAV vector carrying myostatin propeptide (MPRO) gene was delivered into neonate and adult BL10 mice respectively. For neonates study, AAV serotype 8 carrying MPRO gene was delivered into 3 to 5 days old mice by intraperitoneal injection. Two months after vector injection we observed very significant hypertrophy of the skeletal muscles. The treated mice gained body weight and had less fat when compared to the controls. The skeletal muscle mass including TA, GAS, Quadriceps, and Diaphragm from the treated mice were significantly larger (p<0.01). Myofiber diameters of the treated mice were larger than the untreated ones. The hypertrophy was not observed in the hearts of the treated mice despite the gene expression there. However, the larger muscle did not yield stronger force. The peak twitch and peak tetanic force of the treated muscle did not increase significantly, and the treated mice also did not increase performance on the treadmill. For the adult study, AAV-MPRO gene was delivered by local and systemically. Local intramuscular injection study indicated that the treated muscle mass also increased significantly. However, the treated muscle showed increased central nucleation, suggesting muscle regeneration. The study of systemically delivery of AAV-MPRO gene to the adult mice is under way. In conclusion, these data have shown for the first time that muscle mass can be increased by gene delivery of myostatin propeptide in normal mice. Further studies on muscular dystrophy animal models are warranted.
1126. Further Characterization of Msx1 DeDifferentiated Cells Qiang Liu, Wen Liu, Yaming Wang. 1 Anesthesia, Brigham Women’s Hospital, Harvard. Medical School, Boston, MA. Msx1 is a homeobox-containing transcription repressor that has been demonstrated to play an important role in cellular generation and regeneration processes. Our group, as well one other, has demonstrated that ectopic expression of msx1 in murine multinucleated myotubes could lead to de-differentiation of myotubes into mononucleated cells that are capable of proliferation and which possess properties of pluripotent stem cells. Previously, we have demonstrated that msx1-dedifferentiated cells (MDCs) differed from myoblasts by exhibiting reduction in the expression S434
levels of myogenic transcription factors, such as MyoD, Myf5 and Myogenin. These cells also differed in their ability to differentiate into other mesoderm-derived cells, such as adipocytes and osteocytes. We have also demonstrated, that MDCs transcribed oct4 (expressed in undifferentiated ES and germ cells) and nucleostemin (NST), which is expressed in early multipotent stem cells and several cancer cell lines. Current study has confirmed the expression of oct4 and NST in MDCs with Western Blot analysis. Further characterization of MDCs, by RT-PCR has demonstrated that MDCs exhibit the exact transcription profile of ES cells and not the transcription profile of primary myoblasts. Comparable to ES cells, MDCs were positive for C-met, Bcl-2 and peroxisome proliferative activated receptor and were negative for runt-related transcription factor 2 (an essential transcription factor for regulation of osteoblast), Wnt5a, BMP-2, CD44 (a marker for mesenchymal stem cells), and FLK-1 (a marker for endothelial cell progenitor). In contrast, the primary myoblasts were positive for all but one of above described marker genes, the CD44. These data provide additional evidence that MDCs bear a larger resemblance to ES cells than to myoblasts. To assess if MDCs are more advantageous for muscle engraftment over primary myoblasts, 3x105 of MDCs were injected into right tibialis anterior (TA) muscles of mdx mice. To suppress the expression of Tet-Off regulated msx1 gene, mice were given doxycyclinesupplemented water before and after the injections. As a control, the left TA muscles of the same mice were injected with the same number of primary myoblasts. One group of mdx mice received 18Gy radiation treatment in their legs before the injection and another group remained un-irradiated. Two months after the injection, TA muscles were examined for the presence of donor-derived cells by immunohistochemical detection of dystrophin expression. In irradiated MDC-injected TA muscles we observed an abundant number of dysdrophin-positive myofibers. These dystrophin positive myofibers were highly variable in size and tended to aggregate into groups. The TA muscles that received the unmanipulated myoblasts showed only few dystrophin-positive myofibers that were mostly scattered. Non-irradiated TA muscles from right legs exhibited a similar but significantly lower number of donor-derived myofibers. Current results indicate that MDCs survive and divide better than wt myoblasts after transplantation.
1127. Perivascular CD45-:Sca-1+:CD34- Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration Sheng Li,1 Morayma Reyes,1 En Kimura,1 Jessica Foraker,1 Miki Hagakura,1 Leonard Meuse,1 Brent Fall,1 Jeffrey S. Chamberlain.1,2 1 Department of Neurology, University of Washington School of Medicine, Seattle, WA; 2Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA. Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that mouse CD45-:Sca-1+:CD34- cells may play a role in the maturation of skeletal muscles and regeneration of mdx4cv dystrophic skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45-:Sca-1+:CD34- cells in mouse skeletal muscle, we reconstituted lethally irradiated wild type or mdx4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found BM-derived (GFP+) CD45-:Sca-1+:CD34- cells in the skeletal muscles of these GFP+ BM transplant recipients. We also demonstrated that Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
1126. Further Characterization of Msx1 DeDifferentiated Cells Qiang Liu, Wen Liu, Yaming Wang. 1 Anesthesia, Brigham Women’s Hospital, Harvard. Medical School, Boston, MA. Msx1 is a homeobox-containing transcription repressor that has been demonstrated to play an important role in cellular generation and regeneration processes. Our group, as well one other, has demonstrated that ectopic expression of msx1 in murine multinucleated myotubes could lead to de-differentiation of myotubes into mononucleated cells that are capable of proliferation and which possess properties of pluripotent stem cells. Previously, we have demonstrated that msx1-dedifferentiated cells (MDCs) differed from myoblasts by exhibiting reduction in the expression S434
levels of myogenic transcription factors, such as MyoD, Myf5 and Myogenin. These cells also differed in their ability to differentiate into other mesoderm-derived cells, such as adipocytes and osteocytes. We have also demonstrated, that MDCs transcribed oct4 (expressed in undifferentiated ES and germ cells) and nucleostemin (NST), which is expressed in early multipotent stem cells and several cancer cell lines. Current study has confirmed the expression of oct4 and NST in MDCs with Western Blot analysis. Further characterization of MDCs, by RT-PCR has demonstrated that MDCs exhibit the exact transcription profile of ES cells and not the transcription profile of primary myoblasts. Comparable to ES cells, MDCs were positive for C-met, Bcl-2 and peroxisome proliferative activated receptor and were negative for runt-related transcription factor 2 (an essential transcription factor for regulation of osteoblast), Wnt5a, BMP-2, CD44 (a marker for mesenchymal stem cells), and FLK-1 (a marker for endothelial cell progenitor). In contrast, the primary myoblasts were positive for all but one of above described marker genes, the CD44. These data provide additional evidence that MDCs bear a larger resemblance to ES cells than to myoblasts. To assess if MDCs are more advantageous for muscle engraftment over primary myoblasts, 3x105 of MDCs were injected into right tibialis anterior (TA) muscles of mdx mice. To suppress the expression of Tet-Off regulated msx1 gene, mice were given doxycyclinesupplemented water before and after the injections. As a control, the left TA muscles of the same mice were injected with the same number of primary myoblasts. One group of mdx mice received 18Gy radiation treatment in their legs before the injection and another group remained un-irradiated. Two months after the injection, TA muscles were examined for the presence of donor-derived cells by immunohistochemical detection of dystrophin expression. In irradiated MDC-injected TA muscles we observed an abundant number of dysdrophin-positive myofibers. These dystrophin positive myofibers were highly variable in size and tended to aggregate into groups. The TA muscles that received the unmanipulated myoblasts showed only few dystrophin-positive myofibers that were mostly scattered. Non-irradiated TA muscles from right legs exhibited a similar but significantly lower number of donor-derived myofibers. Current results indicate that MDCs survive and divide better than wt myoblasts after transplantation.
1127. Perivascular CD45-:Sca-1+:CD34- Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration Sheng Li,1 Morayma Reyes,1 En Kimura,1 Jessica Foraker,1 Miki Hagakura,1 Leonard Meuse,1 Brent Fall,1 Jeffrey S. Chamberlain.1,2 1 Department of Neurology, University of Washington School of Medicine, Seattle, WA; 2Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA. Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that mouse CD45-:Sca-1+:CD34- cells may play a role in the maturation of skeletal muscles and regeneration of mdx4cv dystrophic skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45-:Sca-1+:CD34- cells in mouse skeletal muscle, we reconstituted lethally irradiated wild type or mdx4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found BM-derived (GFP+) CD45-:Sca-1+:CD34- cells in the skeletal muscles of these GFP+ BM transplant recipients. We also demonstrated that Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy