title: In vitro and in vivo analysis of human fibroblast reprogramming and multipotency.
abstract: Multipotent stem cells have potential therapeutic roles in the treatment of Duchenne muscular dystrophy (DMD). However, the limited access to stem cell sources restricts their clinical application. To address this issue, we established a simple in vitro epigenetic reprogramming technique in which skin fibroblasts are induced to dedifferentiate into multipotent cells. In this study, human fibroblasts were isolated from circumcised adult foreskin and were reprogrammed by co-culture for 72 h with fish oocyte extract (FOE) in serum-free medium. The cells were then observed and analyzed by immunofluorescence staining, flow cytometry and in vitro differentiation assays. Then FOE-treated human fibroblasts were transplanted by tail vein injection into irradiated mdx mice, an animal model of DMD. Two months after injection, the therapeutic effects of FOE-treated fibroblasts on mdx skeletal muscle were evaluated by serum creatine kinase (CK) activity measurements and by immunostaining and RT-PCR of human dystrophin expression. The results indicated that the reprogrammed fibroblasts expressed higher levels of the pluripotent antigen markers SSEA-4, Nanog and Oct-4, and were able to differentiate in vitro into adipogenic cells, osteoblastic cells, and myotube-like cells. Tail vein injection of FOE-treated fibroblasts into irradiated mdx mice slightly reduced serum CK activity and the percentage of centrally nucleated myofibers two months after cell transplantation. Furthermore, we confirmed human dystrophin protein and mRNA expression in mdx mouse skeletal muscle. These data demonstrated that FOE-treated fibroblasts were multipotent and could integrate into mdx mouse myofibers through the vasculature.
authors.forename: Rongqing
authors.initials: R
authors.lastname: Pang
authors.forename: Xiangqing
authors.initials: X
authors.lastname: Zhu
authors.forename: Jia
authors.initials: J
authors.lastname: Geng
authors.forename: Yongyun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.forename: Jie
authors.initials: J
authors.lastname: He
authors.forename: Jinxiang
authors.initials: J
authors.lastname: Wang
authors.forename: Guangxu
authors.initials: G
authors.lastname: Zhu
authors.forename: Fu
authors.initials: F
authors.lastname: Xiong
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Guangping
authors.initials: G
authors.lastname: Ruan
authors.forename: Xinghua
authors.initials: X
authors.lastname: Pan
mesh_terms: D000328:Adult; D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation; D005399:Fishes / Q000378:metabolism; D052816:Foreskin / Q000166:cytology; D006801:Humans; D007275:Injections, Intravenous; D008297:Male; D018101:Mice, Inbred mdx; D008856:Microscopy, Fluorescence; D039902:Multipotent Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation; D009137:Muscular Dystrophy, Animal / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D009210:Myofibrils / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous
chemical_list: D002457:Cell Extracts; D016895:Culture Media, Serum-Free; D016189:Dystrophin