| Human embryonic stem (hES) cells are pluripotency (multipotency) stem cells which have the potential to differentiate into all cell types of the body. It's a hot field to induce this stem cells to myogenic lineage to provide enough cells for the treatments of the muscle degenerative disorders, such as Duchenn's Muscular Dystrophy (DMD). In this study, we focused on this issue and induced the hES cells to appropriate myogenic precursor cells in vitro. For testing these cells, we established a mouse model. Using this model, we show that precursor cells derived from hES cells can incorporate effectively into the mouse skeletal muscle after transplantation, and differentiate to a range of cell types in the myogenic lineage, including satellite cells, muscle and satellite cells. The results showed as following:1. In order to establish a optimal mouse model for myogenesis, the tibialis anterior muscles of mice were pretreated with either 20ug of cardiotoxin alone or in combination with different doses of irridiation (18, 25, or 50Gy) 24h later. The results of the pathology confirmed that the cardiotoxin damaged muslce severely and the irridiation supressed proliferation of the endogenous cells. The combined treatments improved the incorporation with human cells effectively. Irradiation at 25Gy severely inhibited muscle regeneration(28.65%±13.69%, P<0.01). Irradiation at 18Gy mildly inhibited, the dose of 50Gy was not so good.2. Following the cross-backcross method, 8-week-old NOD-scid male mouse (scid-/-) was mated with 8-week-old mdx female mouse(mdx-/-), and then their 8-week-old son (scid+/-/mdx-/-) mated with daughter (scid+/-/mdx+/-). The genome DNA was extracted from the F2 generations and PCR method was employed to amplify the normal and aberrant fragments of dystrophin and scid gene. Among the 46 individuals of F2 generation, there were 13 homozygotes deficient in double gene of scid and dystrophin(scid-/-/mdx-/-), which was used for DMD immunodeficient mouse model.3. hES cells were cultured in suspention to induce the formation of embryoid bodies. After EBs attachment, changed to the medium sutiable for myogenic precursor cells growth. After the morphology of differentiated cells was like fibroblast, the cells were injected into animal models to test their ability in vivo. To analysis the optimal time course, 8-week-old NOD-scid mice were treated with 20ug of cardiotoxin followed by irradiation(25Gy) 24h later, cells were transplanted 0, 24, or 48h after irradiation. The results of the immunohistochemistry confirmed that transplantation at both 24 and 48 h led to a greater incorporation of hES cell-derived cells into the TA muscles, they were24.32% and 23.48% respectively.4. After the combined treatments with 20ug of cardiotoxin and irradiation(25Gy) 24h later, hES cell-derived cells were transplanted into the tibialis anterior(TA) muscles of NOD-scid mice. 2-16 weeks after the transplantation, TA muscles were collected and frozen sectioned. The results of the immunohistochemistry and in situ hybridization(ISH) confirmed that about 10%-20% precursors differentiated from hES cells can undergo myogenesis in NOD-SCID mouse and give rise to a range of cell types in the myogenic lineage including satellite cells expressing the related markers, such as Pax 7. This cells also can incorporate into scid/mdx mouse (the animal model for the Duchenn's muscular dystrophy) and express the dystrophin gene. 5. Among the 12 mice(26 transplantation sites), which were transplanted with hES cell-derived cells 14-128 days later, we can find all of the cells incorporation, but no terotoma formation.The results showed that it was feasible to use hES cells for the treatments of DMD. |
PDF Full Text Request