Figure 7. In vivo differentiation potential of re-isolated Sk-MSC-3d after “2^nd transplantation” into damaged skeletal muscle and sciatic nerve.

To confirm the differentiation potential of once engrafted Sk-MSC-3d, GFP⁺ cells were re-isolated from transplanted crushed nerves (using same method as described in previous RT-PCR analysis) from 1^st transplanted mice (n = 10), and re-transplanted into both the damaged skeletal muscle and sciatic nerve models (2^nd transplantation). Re-isolation and 2^nd transplantation were performed at 7, 12, 17 days and 4 weeks after 1^st transplantation (n = 3 in each stages in both the damaged skeletal muscle and sciatic nerve), and final sampling was performed at 4 weeks after 2^nd transplantation. The left column shows transplantation into damaged skeletal muscle, and the right column shows transplantation into damaged sciatic nerve. (A) Some GFP⁺ muscle fibers were detected in limited areas (arrows in A). (B, C) Incorporation of GFP⁺ cells to the vascular (arrows in B) and peripheral nerve (arrows in C) tissues were also evident. There were no GFP⁺ muscle fibers detected thereafter, with a lower rate of cellular engraftment (at 12 and 17 days, data not shown). (D) Few GFP⁺ cells related to nerves were seen in the case of re-isolated-4w (arrows in D). (E) There was a large number of GFP⁺ muscle fibers near the N-200⁺ nerve bundles when Sk-MSC-3d were directly transplanted into damaged muscle (as 1^st transplantation), thus indicating vigorous skeletal myogenic potential. These data indicate that diminished engraftment capacity of Sk-MSC-3d is associated with reduced myogenic potential in the 2^nd transplantation. (F) When original SNDC-D was directly transplanted into damaged skeletal muscle niche, they were unable to differentiate into muscle fibers, and their engraftment capacity was quite low. (G–I) 2^nd transplantation of re-isolated Sk-MSC-3d cells at -7d (G), -12d (H) and -4 w (I) into damaged sciatic nerve. Number of engrafted cells increased gradually with time after 2^nd transplantation. (J–L) Similar trends in cell differentiation which was observed in 1^st transplantation were seen for 2^nd transplantation (refer to Figs. 2 and 3). Scale bars represent 50 µm (A–F, and J–L), 20 µm (inset of F) and 200 µm (G–I).