Figure 1.
Morphology and surface markers of rabbit MSCs.
(A–C): Bright field images of rabbit ASCs (A), BM-MSCs (B), and DFs (C). Cells were grown in culture dishes with growth medium and photos were taken. Scale bar; 100 µm. (D): Western blot analysis. Whole cell extract of rabbit ASCs from passage 1 (P1) to P9 was prepared and loaded 20 µg per well. The expression of CD29, CD44, CD90, and CD105 were detected with their specific antibodies as indicated. β-actin was detected as a loading control.
Figure 2.
Rabbit MSCs differentiate to mesodermal lineages in vitro.
Passage 2 ASCs (A, D, G), BM-MSCs (B, E, H), and DFs (C, F, I) were used for differentiation. (A–C): Adipogenic differentiation. Cells were cultured in adipogenesis differentiation medium for 8 days. Oil Red O staining was performed to detect lipid accumulation. Nuclei were stained with Hematoxylin. (D–F): Chondrogenic differentiation. Cells were cultured in chondrogenesis differentiation medium for 21 days. Alcian blue staining was performed. (G–I): Osteogenic differentiation. Cells were cultured in osteogenesis differentiation medium for 35 days. Alizarin Red S staining was performed to detect calcium accumulation. Abbreviation: MSCs, mesenchymal stem cells; ASCs, adipose derived stem cells; DFs, dermal fibroblasts; BM-MSCs, bone marrow derived mesenchymal stem cell. Scale bar; (A–F) 50 µm, (G–I) 100 µm.
Figure 3.
Histological quantification of MSCs treated wounds.
(A–C): A total of 1×105 ASCs (A), BM-MSCs (B), and DFs (C) in PBS were delivered to 7 mm wounds on one ear. In the contralateral ear, PBS alone was delivered as a control. Wounds were harvested at POD7 and granulation tissue area was measured. Number of wounds analyzed; (A, n = 35 for saline & n = 36 for ASCs; B, n = 17 for saline & n = 20 for BM-MSCs, C, n = 17 for saline & n = 24 for DFs). N represents the total number of wounds from six (A) or four (B, C) rabbits. Data shown as mean + SEM. ***p<0.001, ns = not significant.
Figure 4.
Transplanted ASCs express α-SMA in wounds.
GFP-expressing ASCs were analyzed 7 days after transplantation in wounds. Chicken anti-GFP and mouse anti-α-SMA antibodies were used to detect GFP and α-SMA. Nuclei were stained with DAPI. (A): Low magnification of wounds. The areas analyzed in Figure 6 were indicated by ‘a’ and ‘b’. (B–D): Higher magnifications of the indicated regions in A (white squares; labeled as i, ii, iii). (E–G): Higher magnifications of the indicated regions in B–D (white squares; labeled as iv, v, vi). Merged images of α-SMA (red) and GFP (green) indicate that α-SMA is expressed in ASCs. Scale bars: 500 µm (A), 50 µm (B–G).
Figure 5.
Transplanted ASCs proliferate in wounds.
GFP-expressing ASCs were analyzed 7 days after transplantation in wounds. Chicken anti-GFP (A) and mouse anti-Ki67 (C) antibodies were used. Nuclei were stained with DAPI (B). Merged image was shown in D. Scale bars: 50 µm.
Figure 6.
Analysis of protein expression in ASCs treated wounds.
Saline control (A, B, C, D) and ASCs (E, F, G, H) were delivered to wounds and harvested as described in Figure 3. α-SMA (A, E) and CD3 (B, F) were visualized by DAB after staining with their specific antibodies. CD31 (C, D, G, H) was stained with its specific antibody and visualized using fluorescence conjugated secondary antibody. (A, B, C, E, F, G): images were taken from the area labeled as ‘a’ in Figure 4. (D, H): Immunostaining for CD31 in the area labeled as ‘b’ in Figure 4. The junction area between cartilage and wound beds was demarcated by white dot lines. CD31 positive signals were indicated by arrows in H. Scale bars: 50 µm.
Figure 7.
Higher infiltration of macrophages was found in ASCs treated wounds.
Saline control (A, C) and ASCs (B, D) were delivered to wounds and harvested as described in Figure 3. Neutrophils (A, B) and macrophages (C, D) were visualized by DAB after staining with their specific antibodies. Scale bars: 100 µm. (E): Number of macrophages per high-power microscopic fields (HPF) at 400 X magnification. Macrophages were counted and averaged from four HPF. Data are from four independent wounds and presented as mean + SEM. ***p<0.001.