Restoration of muscle fibers and satellite cells after isogenic MSC transplantation with microdystrophin gene delivery.

Duchenne muscular dystrophy is the most prevalent inheritable muscle disease. Transplantation of autologous stem cells with gene direction is an ideal therapeutic approach for the disease. The current study aimed to investigate the restoration of myofibers in mdx mice after mdx bone marrow-derived mesenchymal stem cell (mMSC) transplantation with human microdystrophin delivery. Possible mechanisms of action were also studied. In our research, mMSCs were successfully transduced by retrovirus carrying a functional human microdystrophin gene. Transplantation of transduced mMSCs enabled persistent dystrophin restoration in the skeletal muscle of mdx mice up to the 12th week after transplantation. Simultaneous coexpression of human microdystrophin and desmin showed that implanted mMSCs are capable of long-term survival as muscle satellite cells.

Promoter analysis of mouse Scn3a gene and regulation of the promoter activity by GC box and CpG methylation.

Voltage-gated sodium channel α-subunit type III (Na(v)1.3) is mainly expressed in the central nervous system and is associated with neurological disorders. The expression of mouse Scn3a product (Na(v)1.3) mainly occurs in embryonic and early postnatal brain but not in adult brain. Here, we report for the first time the identification and characterization of the mouse Scn3a gene promoter region and regulation of the promoter activity by GC box and CpG methylation. Luciferase assay showed that the promoter region F1.2 (nt -1,049 to +157) had significantly higher activity in PC12 cells, comparing with that in SH-SY5Y cells and HEK293 cells. A stepwise 5' truncation of the promoter region found that the minimal functional promoter located within the region nt -168 to +157. Deletion of a GC box (nt -254 to -258) in the mouse Scn3a promoter decreased the promoter activity. CpG methylation of the F1.2 without the GC box completely repressed the promoter activity, suggesting that the GC box is a critical element in the CpG-methylated Scn3a promoter. These results suggest that the GC box and CpG methylation might play important roles in regulating mouse Scn3a gene expression.

[Mesenchymal stem cells transplanted in mdx mice differentiate into myocytes and express dystrophin/utrophin].

OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice. METHODS: BrdU-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. RESULTS: Four 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. CONCLUSION: The 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.

Comparative study of mesenchymal stem cells from C57BL/10 and mdx mice.

BACKGROUND: Human mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Since most human diseases models are murine, mouse MSCs should have been studied in detail. The mdx mouse - a Duchenne muscular dystrophy model - was produced by introducing a point mutation in the dystrophin gene. To understand the role of dystrophin in MSCs, we compared MSCs from mdx and C57BL/10 mice, focusing particularly on the aspects of light and electron microscopic morphology, immunophenotyping, and differentiation potential. RESULTS: Our study showed that at passage 10, mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not at the same passages. By passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, a significant difference in the expression of CD34, not Sca-1 and CD11b, was observed between the MSCs from the 2 mice. CONCLUSION: Our current study reveals that the MSCs from the 2 mice, namely, C57BL/10 and mdx, exhibit differences in proliferative and myogenic abilities. The results suggest that the changes in mouse MSC behavior may be influenced by lack of dystrophin protein in mdx mouse.

Signals in pathological CNS extracts of ALS mice promote hMSCs neurogenic differentiation in vitro.

The capability of MSCs to differentiate into neurons has been proven by many studies. Recently, other studies have cast doubt on MSCs neurogenic differentiation with non-physiological chemical inducing agents in vitro. This present study was designed to use conditioned medium to investigate whether signals from pathological condition of ALS were competent to induce a program of neurogenic differentiation in expanded cultures of hMSCs. Incubation of hMSCs with conditioned medium prepared from CNS extracts of ALS mice (SOD1-G93A ALS mice) resulted in a time-dependent morphological change from fibroblast-like into neuron-like, concomitant with increase in the expression of Nestin and subsequent beta-tubulin III, NSE and GAP43. Moreover, signals in pathological CNS extracts of ALS mice were more effective in promoting hMSCs neurogenic differentiation than those in physiological extracts of normal adult mice. These results show that pathological condition of ALS is endowed with capacity to induce hMSCs neurogenic differentiation and hMSCs have shown a potential candidate in cellular therapy for ALS.

[Construction of recombinant adenovirus including microdystrophin and expression in the mesenchymal cells of mdx mice].

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.

[Expression of human micro-dystrophin gene after retrovirus infection in mdx mice bone marrow-derived mesenchymal stem cells].

OBJECTIVE: To 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. METHODS: Retroviral 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. RESULTS: Micro-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.

[Dystrophin expression in mdx mice after bone marrow stem cells transplantation].

OBJECTIVE: To investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation. METHODS: The 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. RESULTS: At 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. CONCLUSIONS: After 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.

[Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells].

OBJECTIVE: To construct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells (rMSCs) in vitro. METHODS: The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1(+) to prepare the eukaryotic expression vector-pcDNA3.1(+)/ microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression of microdystrophin was detected by RT-PCR and indirect immunofluorescence assay. RESULTS: Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1(+), as conformed by sequencing and digestion with Not I and Hind III. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immunofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs. CONCLUSION: Eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.

[Dystrophin expression and pathology of diaphragm muscles of mdx mice after xenogenic bone marrow stem cell transplantation].

OBJECTIVE: To 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). METHODS: The 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. RESULTS: The 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. CONCLUSION: BMT 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.