Intramuscular transplantation of human neonatal ADSC into Col6a1−/−Rag1−/−mice. (A) Representative in vivo images showing the hindlimbs that received a single intramuscular injection of ADSC. Cells were cultured in the presence of a red fluorescent lipophilic dye (DiOC18), and 0.5 × 106 DiOC18-ADSC were injected into the left GCM. The right GCM served as the control. IVIS imaging was performed at one, two, three, four and six weeks post-transplantation. (B) Morphometric analysis of DiOC18-ADSC engraftment into the GCM was performed at one, two, three, four and six weeks post-transplantation. The percentage of engrafted cells was determined by staining of transplanted GCM with lamin A/C antibody as a marker for donor cells and by counting lamin A/C positive-cells on at least fifty 7 μm sections. As an additional approach, the total number of DiOC18-positive ADSC was calculated using FACS analysis of a single-cell suspension of the GCM containing ADSC transplant. The vertical axis shows the percentage (%) of engrafted ADSC per injected muscle. The horizontal axis shows the time point after transplantation in weeks (w). (C) Morphometric analysis of α1(VI)-positive myofibers in the GCM transplanted with ADSC. The number of α1(VI)-positive myofibers was determined by counting a minimum of fifty 7 μm sections per animal. The vertical axis shows the number of α1(VI)-positive myofibers per section. The horizontal axis shows the time point after transplantation in weeks (w). In all studies, at least five animals were analyzed per time point. Error bars represent means ± SEM. ADSC, adipose-derived stem cells; FACS, fluorescence-activated cell sorting; GCM, gastrocnemius muscle; SEM, standard error of the mean.