Duchenne muscular dystrophy (DMD) is an X-linked, recessive and lethal genetic disease, which usually caused by gene mutations and the underlying mechanisms are complicated and diverse. The causal gene of DMD is the largest one in human that locates in the region of Xp21.2, encoding dystrophin. Currently there is no effective treatment for DMD patients. The treatment of DMD depends on gene mutation and molecular mechanism study of the disease, which requires reliable disease models such as mdx mouse model. Recently, researchers have increasingly discovered gene therapy strategies for DMD, and the efficacy has been demonstrated in DMD animal models. In addition, induced pluripotent stem cell technology can provide patient-specific cell source, offering a new platform for mechanism and therapy study of DMD.
Animals ; Disease Models, Animal ; Dystrophin ; genetics ; Genetic Therapy ; trends ; Humans ; Induced Pluripotent Stem Cells ; Mice ; Mice, Inbred mdx ; genetics ; Muscular Dystrophy, Duchenne ; genetics ; therapy

OBJECTIVETo investigate the expressions of myogenic markers MyoD, myogenin,and desmin in skeletal muscle differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

METHODSMyogenic markers MyoD, myogenin,and desmin of hBM-MSCs cultured in vitro were detected by immunofluorescence and RT-PCR. A total of 21 8-to-10 week-old immunosuppressed mdx mice were transplanted with 1x107 passage 5 of hBM-MSCs. The mice were euthanized 2-24 weeks after transplantation,and gastrocnemius muscle were analyzed for human MyoD, myogenin,desmin,and dystrophin (Dys) expressions by immunohistochemistry and RT-PCR.

RESULTSThe numbers of MyoD-,myogenin-,and desmin-positive cells per 100 hBM-MSCs were 23.5∓5.3, 30.7∓6.2, and 28.4∓5.7, respectively. MyoD, myogenin, and desmin mRNA was observed in passage 5 of hBM-MSCs. After two weeks of hBM-MSCs transplantation,a small number of MyoD-and myogenin-positive cells were observed in skeletal muscle of mdx mice,and desmin-positive cells were observed 4 weeks after transplantation. Expressions of MyoD and myogenin were detected in the muscle of mdx mice 2-4 weeks after hBM-MSCs transplantation, which reached a peak 12-16 weeks later. Desmin was expressed in the muscle of mdx mice 4-8 weeks after transplantation,with much more expression after 16 weeks of transplantation. A small number of Dys-positive cell and Dys mRNA expression were presented in the muscle of mdx mice 4 and 8 weeks after hBM-MSCs transplantation,respectively. The expression of Dys in the muscle of mdx mice increased gradually after transplantation.

CONCLUSIONhBM-MSCs have the potential of myogenic differentiation in vitro and contribute to myogenic conversion in xenogeneic animal,during which the up-regulation of MyoD and myogenin expressions may play an important role.

Animals ; Biomarkers ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Desmin ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal ; cytology ; metabolism ; MyoD Protein ; metabolism ; Myogenin ; metabolism ; Up-Regulation

OBJECTIVETo investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice.

METHODSBrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively.

RESULTSFour weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation.

CONCLUSIONThe myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Dystrophin ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; metabolism ; Muscle Fibers, Skeletal ; cytology ; metabolism ; Muscular Dystrophy, Animal ; metabolism ; therapy ; Rats ; Utrophin ; metabolism

OBJECTIVE: To investigate the effect of steroid administration on the apoptosis and heat shock protein 70 (HSP70) expression after exercise in the animal model of Duchenne muscular dystrophy. METHOD: We measured Bcl-2, BAX and HSP70 expression by western blotting. 20 control and 20 mdx mice were divided into free-living (n=10) and exercise (n=10) groups. Free-living and exercise groups were further divided into steroid-treated and sham-treated groups to evaluate the effect of steroid administration. RESULTS: Apoptosis was most prominent in the sham-treated exercise group, while apoptosis was significantly reduced in the steroid-treated exercise group. HSP70 expression was maximized in sham-treated exercise group, whereas steroid administration inhibited HSP70 expression after exercise in muscular dystrophy animal model. Exercise loading was found to cause severe apoptosis but steroid administration alleviated apoptotic damage in mdx mice. CONCLUSION: HSP70 expression was suppressed in the steroid-treated exercise group, which suggests steroid might have major preventive effect in exercise-induced apoptosis of muscular dystrophy animal model.
Animals ; Apoptosis ; Blotting, Western ; Heat-Shock Proteins ; Hot Temperature ; HSP70 Heat-Shock Proteins ; Mice ; Mice, Inbred mdx ; Models, Animal ; Muscular Dystrophies

OBJECTIVETo investigate the dynamic distribution of human bone marrow mesenchymal stem cells (hBM-MSCs) in mdx mice.

METHODSTwenty-four 8-10-week-old immunocompromised mdx mice were transplanted with 1 x 10(7) passage 5 hBM-MSCs labeled with bromodeoxyuridine (BrdU) by means of injection into the tail vein. The mice were euthanized 48 hours and 2, 4, 8, 12, 16, 20, and 24 weeks after transplantation. BrdU-positive cells in tissue and organs of the mice were detected by immunofluorescence analysis. Skeletal muscle was stained for anti-human nuclei mouse monoclonal antibody (anti-Hu) and analyzed for human dystrophin (Dys) expression by immunohistochemistry and reverse transcription-polymerase chain reaction.

RESULTSAfter transplantation, BrdU-positive cells were found in most organs (especially in bone marrow, liver, and lung) within 4 weeks, and these cells in liver and lung decreased gradually after 4 weeks. At 48 hours after transplantation, BrdU-positive cells were found in bone marrow, which reached a peak level after 2 weeks and were still detectable after 16 weeks. BrdU-positive cells in skeletal muscle increased gradually over time of transplantation. A small number of anti-Hu positive cells were detected in skeletal muscle 2 weeks after transplantation. A small number of Dys positive cell were seldom found at 4 weeks and small Dys mRNA expression detected 4 weeks after transplantation. The proportion of anti-Hu in parallel with Dys positive cells and Dys mRNA in skeletal muscle of mdx mice increased gradually over time of transplantation.

CONCLUSIONAfter being transplanted into mdx mice, hBM-MSCs are mainly distributed in bone marrow, liver, and lung during the early time (2-4 weeks) , and then in bone marrow and skeletal muscle (after 4 weeks).

Animals ; Bone Marrow Cells ; cytology ; Dystrophin ; genetics ; metabolism ; Humans ; Immunocompromised Host ; Immunohistochemistry ; Mesenchymal Stem Cell Transplantation ; methods ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. This study was purposed to investigate the feasibility to use adult adipose-derived mesenchymal stem cells (AD-MSCs) in the therapy of DMD. The Flk-1(+) MSCs were isolated from adipose tissue of adult GFP mice; the phenotype and cell cycle of MSCs were analyzed by flow cytometry; the AD-MSCs were directionally differentiated by myoblast and endotheliablast induction system in vitro and were identified by immumofluorecence staining and RT-PCR; the AD-MSCs were transplanted into CTX-injured mice model or mdx mice (DMD animal model) through tail vein; the distribution and differentiation of AD-MSCs were detected by immunofluorescence staining and RT-PCR respectively, and statistic analysis was performed. The results showed that the Flk-1(+) AD-MSCs could be induced to differentiate into myoblasts and endothelial cells in vitro. After transplanted into CTX-injured mice model or mdx mice, GFP-positive cells could be detected in damaged muscle, and these donor-derived cells were also positive for MHC, vWF, or Pax7. Flk-1(+) AD-MSC transplantation also partly reconstituted the expression of dystrophin, and reduced the percentage of centronucleated myofibers in mdx mice. It is concluded that Flk-1(+) AD-MSCs represent a possible tool for future cell therapy applications in DMD disease, as they can be delivered through the circulation for their potential of muscle homing. And Flk-1(+) AD-MSCs also show the ability to contribute to muscle repair, improvement of blood supply and long term reconstitution of dystrophy muscle.
Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; Cells, Cultured ; Dystrophin ; biosynthesis ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Mice, Transgenic ; Muscle Cells ; cytology ; Muscular Dystrophy, Duchenne ; pathology ; therapy ; Myoblasts ; cytology

Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy( DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme I , the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58 x 10(12) vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.
Adenoviridae ; genetics ; Animals ; Cells, Cultured ; Dystrophin ; genetics ; metabolism ; Gene Expression ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Immunohistochemistry ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; genetics ; pathology ; therapy ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transduction, Genetic

OBJECTIVETo investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation.

METHODSThe bone marrow stem cells of C57 BL/6 mice (aged 6 to 8 weeks) were injected intravenously into the mdx mice (aged 7 to 9 weeks), which were preconditioned with 7Gy gamma ray. The amount of dystrophin;expression in gastrocnemius was detected by immunofluorescence, reverse transcription-polymerase chain reaction and Western blot at week 5, 8, 12 and 16 after transplantation.

RESULTSAt week 5 after bone marrow stem cells transplantation, the dystrophin expression detected in mdx mice were very low; however, its expression increased along with time. At week 16 week, about 12% muscle cells of all transplanted mice expressed dystrophin. There were less centrally placed myonuclei than the control mdx mice, whereas peripheral myonuclei increased.

CONCLUSIONSAfter having been injected into mdx mice, the allogenic bone marrow stem cells have a trend to reach the injured muscle tissues and differentiate to fibers that can express dystrophin and the expression increased with time. The bone marrow stem cells participates in the repair and regeneration of the injured tissues permanently and constantly.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Disease Models, Animal ; Dystrophin ; biosynthesis ; Hematopoietic Stem Cell Transplantation ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; metabolism ; surgery ; Transplantation, Homologous

OBJECTIVETo investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD).

METHODSThe bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls.

RESULTSThe proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice.

CONCLUSIONBMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.

Animals ; Bone Marrow Transplantation ; methods ; Diaphragm ; metabolism ; pathology ; Dystrophin ; biosynthesis ; genetics ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; metabolism ; pathology ; surgery ; Rats ; Rats, Sprague-Dawley ; Transplantation, Heterologous

OBJECTIVETo construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dystrophin in mdx mice bone marrow-derived mesenchymal stem cells (MSCs) after retrovirus infection.

METHODSRetroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by Lipofectamine 2000. The retroviral supernatant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction.

RESULTSMicro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. CONCLUSION mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Dystrophin ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Animal ; metabolism ; Retroviridae Infections ; Transfection