Fig. 6
Knockdown of uPAR in iMSCs reduced the efficiency of cell transplantation. A Outline of the experiments. iMSCs were transfected with huPAR shRNA plasmids or control shRNA plasmids. After puromycin treatment for 24 h, iMSCs were co-cultured with Hu5/KD3 cells in 10% FBS/DMEM in a Transwell plate for 3 d. The TA muscles of NOD/Scid mice were injured with 1.2% BaCl2 solution 24 h before cell transplantation and injected with 5 × 105 myogenic cells (Hu5/KD3 cells). Cells co-cultured with iMSCs transfected with control shRNA plasmids were injected into right TAs (control), and cells co-cultured with iMSCs transfected with uPAR shRNA plasmids were injected left TAs. Ten days after transplantation, the mice were killed, and the TA muscles were dissected for immunohistochemical analysis using an anti-human lamin B1 antibody and human spectrin antibody as described in Fig. 5. B RT-qPCR analysis performed on RNAs extracted from iMSCs transfected with shRNA plasmids. Prep. 1 and Prep. 2 showed 60% reduction of uPAR mRNA. C Western blotting was performed using proteins extracted from iMSCs transfected with uPAR shRNA plasmids or control shRNA plasmids. Prep. 1 and Prep. 2 showed 40–70% reduction of uPAR protein. D Numbers of human lamin B1-positive, human spectrin-positive myofibers in TA muscles transplanted with Hu5/KD3 cells after co-culture with iMSCs (summary of two independent experiments). n = 5 mice. Paired t-test. E Recombinant PAI-1 (100 ng/ml) was added to the coculture and three days later Hu5/KD3 cells were transplanted into left TA muscles of NOD/Scid mice (n = 4) or to NSG-mdx4Cv mice (n = 4). Right TA muscles were injected with Hu5/KD3 cells that had been co-cultured with iMSCs without PAI-1 (control). Data are shown as the mean ± SEM