Initial events of differentiation of mdx mice striated muscle fibers after bone-marrow stem cells therapy

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Mikhailov V.M., Karmanova A.V., Kaminskaya E.V., Zenin V.V., Kuzovatov S.N., Baranov V.S., Serikov V.B.

(Institute of Cytology of RAS and Ott’s Institute of Obstetrics and Gynecology RAMS, St.Petersburg, Russia ; Vladislav Baranov, Institute of Cytology of RAS and Ott’s Institute of Obstetrics and Gynecology RAMS, St.Petersburg, Russia; e-mail: baranov@VB2475.spb.edu)

Mdx mice are a model of human Duchenne muscular dystrophy (DMD). Striated muscle fibers (StMF) of these mice do not express dystrophin. The absence of dystrophin is a cause of development of oxidative stress of the muscle cells. In turn the oxidative stress is reason of high level of death of mdx mice StMF. StMF of mdx mice also show a defects in the formation of nerve-muscle junctions (NMJs) and in the organization of acethyl-choline receptors (AchRs) complexes. Disturbance of mdx StMF NMJs is one of the causes of low level of mdx StMF survival too. It is known that gene therapy of mdx mice StMF with cDNA of dystrophin genes restores a dystrophin synthesis and induces formation of myofibers with normal morphology. But positive effect of gene therapy is not enough long because of self-renewal of StMF population. It is impossible to receive durable positive effect by gene therapy for survival of StMF in case of constant cell renewal.

To restore normal structure of striated muscle cell population of mdx mice we have to change mutant stem cells population for population of wild type stem cells.

Constructively the mdx mice mutant stem cells of StMF would be replaced by transplantation of wild type stem cells. Stem cells of muscle origin or of bone marrow origin can take part in mdx StMF differentiation by two manner. The first one is the direct inclusion of wild type stem cells nuclei into sarcoplasma of mutant StMF. In this case it is possible to wait a restoration of dystrophin and dystroglycan synthesis and formation of normal structure of NMJs and AchRs. At the second model the wild stem cells can take part in the formation of new StMF. The change of the mutant population of bone marrow stem cell to stem cells of wild type is used in case of treatment of some forms of leukemia.

There are two sets of experiments. The first one was carried out on the chimeric mdx mice. Chimeric mice were prepared by intravenously injection of bone marrow derived cells from healthy C57BL/6 mice to X-ray irradiated (7.5 Gy) mdx mice. After C57BL/6 BMSCs transplantation the positive therapeutic affect was appeared as decrease of StMf death and accumalation of StMf without central nuclei. (table 1 and 2). At 2 months after BMSCs transplantation there was not accumulation of dystrophin positive StMF. There is accumulation of StMF without central nuclei. In such a manner chimeric mdx mice are characterized by intensification of striated muscle differentiation.

In case of preparation of chimeric C57BL/6 mice by transplantation after X-ray irradiaition 5Gy of BMSCs from GFP(+)-transgenic C57BL/6 mice the GFP(+)StMF were observed in 0.3±0.5% и 0.2±0.3% between all StMF as for damaged as for lateral (control) muscle consequently. In case of chimeric mdx mice, transplantation of GFP(+) BMSCs induces a positive results in 1.7±0.4% and 0.5±0.3% for damaged and lateral (undamaged) muscle consequently. This results show that muscle damage increase consumption of stem cell by muscles first of all in the case of mdx mice but not C57BL/6 mice.

In the 3nd set of experiment the GFP(+) bone marrow cell were intramuscularly injected to C57BL/6 or mdx mice M. quadriceps femoris in dose 2-5x105 cells per mouse (table 3). Before injections a GFP(+) bone marrow cells were fractionated in the 63% Percoll solution and exhausted by magnetic manner with mixture of CD4, CD8, CD38, CD45R, CD119, Ly-6G and F4/80 antibody. After 2 weeks there were 0.15±0.4% и 0.1±0.2% of GFP(+) muscle fibers in injected and lateral muscle of C57BL/6 mice. In case of mdx mice the frequency of GFP(+)-positive StMF were higher and reached 2±0.8% и 1.2±0.6% for injected and lateral muscle consequently. There is conclusion what BMCSs take part in the differentiation and regeneration of mdx mice muscles after delivery by multiple intramuscularly injections (table 3) (Mikhailov et al., 2006).

After intramuscularly injections of GFP(+) BMSCs into mdx mice muscle tissue the survival of GFP(+)StMF is not longer than 2 weeks because of immune conflict of recipients with GFP-protein. In this situation we decided to use for cell therapy of mdx mice StMF the BMSCs from C57BL/6 mice. Through one month after intra-muscle injections of C57BL/6BMSCs we did not observe accumulation of dystrophin(+) StMF in the mdx mice M. quadriceps femoris. Result is not contradicted with our early dates and results of another authors. There is descriptions what dystrophin and of dystroglycans synthesis are not repaired through one month after transplantation of wild type stem cells to muscles of mdx mice.

At the last experiment BMSCs from normal C57BL/6 mice were injected to muscle of mdx mice. It is known what mdx mice has C57BL/10 origin. There is not genetic difference between C57BL/6 and C57BL/10 mice in main histocompatibility complex. To determine the possible influence of BMSCs therapy for muscle morphology of mdx mice during one month after transplantation we decided to analyse the structure of nerve muscle junctions (NMJs) by means of staining with Bungarotoxin-rhodamine. It is known what absence of dystrophin in the muscle of mdx mice correlates with morphological abnormalities of NMJs. The morphology of mdx mice NMJs resembles the structure of C57BL mice NMJs after denervation. We estimated what through one month after BMSCs intramuscular injections the size of NMJs return back to the size of C57BL NMJs. The intensity of effects depended from cell composition of BMSCs suspension (table 4). In such a manner after one month of cell therapy by BMSCs of mdx mice striated muscles results in increase of part of StMF without central nuclei and of reparation of normal size of NMJs. Our results allow to challenge in transcription silence of injected stem cells wild type nuclei after their entry to sacroplasma of mdx mice StMF. The work is partly supported by RFBI grant # 05-04-49609 and 06-04-08338.

Таble 1. Change of differentiation parameters of M. quadriceps femoris striated muscle fibers (StMF) of mdx mice after X-ray irradiation.

Table 2. Progress of differentiation of striated muscle fibers M. quadriceps femoris of chimeric mdx mice after X-ray irradiation. Parts (%) of striated muscle fibers without central nuclei