Fig 1.
Healing of vaginal incision in sham, local and systemically transplanted rats.
On day one post injury, disruption of the epithelial layer and the adjacent lamina propria was noted in all groups (A-C). On day three post injury, the epithelial cut was bridged but the layer was still thick in the sham (D) and I.V. (F) groups while it was disrupted in the local group (E). On day seven post injury, in the sham (G) and I.V. (I) treated rats the epithelial layer had a normal appearance while there was still partial epithelial disruption and disorganization of the sub-epithelial fibromuscular tissue in local transplanted rats (H). Complete re-epithelization was observed on day 30 in the sham (J), local (K), and I.V. (L) treated rats. n = 5 rats in each treatment group at each time point. The location of the incision is marked with the black arrow. Abbreviation: V = vagina; Ep = epithelium; Lp = lamina propria; Scale bar = 200μm.
Fig 2.
Survival of MSCs following systemic or local transplantation.
On day three post injury, GFP- labeled cells homed to the injury site in the I.V. treated group (A). GFP positive cells were also evident at day three in the locally treated group (B). On day seven, GFP transplanted cells could be detected in the I.V. treated group (C). A higher magnification of the GFP labeled cells occupying the white square in C, is shown in D. In the locally treated rats, GFP transplanted cells could not be detected on day 7 (E). A group of rats (n = four rats) were transplanted systemically with MSCs, without making a vaginal incision. GFP-expressing cells were not detected in the vagina in this group (F). Scale bar A-B; D-E = 50 μm; C = 100 μm; F = 200 μm.
Fig 3.
Systemically transplanted MSCs differentiate into endothelial cells.
(A) DAPI stained section at seven days post injury and transplantation. Adjacent to the incision site a capillary-like structure is observed (red square). Fluorescence image of this capillary-like structure shows that it mostly made of GFP-expressing cells (B). A higher magnification of B is demonstrated in (C). Sections were stained with anti- GFP (D) and the endothelial marker CD31 (E). The co-localization of CD31 and GFP is shown in (F). PKH-26 transplanted cells (G), expressed laminin (H) and the co-localization of PKH-26 and laminin is shown in (I). FACS analysis prior to transplantation showed that the MSC population did not include CD31- expressing cells (J). Jurkat T cells decorated with anti-CD31, were used as positive controls (K). Scale bar: A = 500 μm; B, D, E, F = 100 μm; C, G, H, I = 50 μm.
Fig 4.
Systemically transplanted MSCs form blood vessel structures on day 30 after transplantation.
Confocal microscopy showed that at thirty days after transplantation, PKH-26 transplanted cells (red) were organized in blood vessels-like structures (A) and were expressing CD31 (B). Nuclei are counterstained with DAPI (C). A merged image showing the co- localization of PKH-26 and CD31 is shown in (D). PKH-26 transplanted cells, organized in a capillary like structure (E), decorated with anti-vWF are demonstrated in (F). Nuclei are counterstained with DAPI (G). A merged image showing co-localization of PKH-26 and vWF (H). An example of a blood vessel composed of PKH-26 labeled cells is seen in (I). H&E staining of an adjacent section demonstrates the existence of enucleated erythrocytes in the vessel’s lumen (J). Proliferating cells expressing Ki67 (white arrows) were observed in the inner cell layer of blood vessel composed of PKH-26 transplanted cells. (K). Double staining with GFP (L) and anti trimethyl- Histone H3 (M) shows only one red focus in each cell. A merged picture is demonstrated in (N) and a higher magnification of N is shown in (O). Staining with anti trimethyl- Histone H3 in vitro of a hiPSC line with 93XXXXY karyotype that was formed by fusion between 46XX and 47XXY lines shows two red foci per cell (P). Scale bar: (A-D, F) 100 μm; (E) 500 μm; (G-L) 50 μm; (M-N) 5 μm.
Fig 5.
The effect of MSCs transplantation on tissue vascularization.
To assess the effect of MSCs transplantation on tissue vascularization, vaginal sections from sham, local and I.V. transplanted rats were stained with anti-muscle actin and GFP antibodies. Representative images from day 7 of sham (A) and local (B) transplanted rats and of day 30 I.V. transplanted animals (C) are shown. The number of blood vessels without any staining to GFP (indicated with white arrows) per field at 10x magnification were analyzed (each group included five rats; three slides per rat; three fields were counted per slide). In addition, the number of blood vessels which had positive staining to GFP in the inner cell layer (green arrows) was analyzed. At days 7 and 30, MSCs transplantation induced angiogenesis (blood vessels without GFP) in both the local and systemically transplanted groups (D). Only in the systemically transplanted group, blood vessels composed of GFP-expressing cells, probably representing neovascularization involving the transplanted MSCs, were observed at 7 and 30 days post transplantation (C and D).*p<0.05. Scale bar: A-B = 500 μm; C = 50 μm.