Figure 1.
FACS analysis of UMSCs and UHSCs.
A. UMSCs isolated from human umbilical cord were characterized by flow cytometry using CD markers; and these plastic adherent cells were positive to CD105/SH, CD13, CD29, CD44, CD73/SH3, and CD90, and negative to CD34, CD14, CD45, CD31, and HLA-DR (MHC-II). B. There were large amounts of lipid droplets (red) in UMSCs shown by Oil-red O staining after adipogenesis induction. C. Under osteogenic culture conditions, UMSCs expressed alkaline phosphatase (blue). D. Toluidine blue staining revealed that UMSCs synthesized chondroitin sulfate after the induction of chondrogenesis. E. UHSCs were positive to CD34, CD45, CD59, CD133, and BCRP1, which were identified by flow cytometry. F. Immunofluorescent staining also revealed that UMSCs were positive to CD90, CD44, and negative to CD34, CD45; UHSCs were positive to CD34. Neither UMSCs nor UHSCs expressed keratocan and lumican before the transplantation. Scale bars: 10 µm; Blue: DAPI.
Figure 2.
Corneal stromal thickness and transparency in Lum-/- mice was improved by the transplantation of UMSCs.
A. Before UMSC transplantation, there were similar levels of corneal stromal light scattering and thickness in the right (OD, a) and left (OS, b) eye. Panel c contains histograms of light scattering and stromal thickness. B. Panel a and b represented the 3-dimensional images of HRT examination 12 weeks after UMSCs transplantation; and clearly, lower light scattering and increased thickness displayed in the transplanted corneal stroma (b) than that of an untransplanted cornea (a). The histograms showed thicker and less light scattering (more transparent) in the stroma of transplanted corneas than those of untransplanted corneas after 12 weeks. C. Corneal stromal thickness was significantly increased in the transplanted cornea than that of the untransplanted cornea after 8 weeks. D. Compared to the untransplanted cornea the average light scattering significantly decreased in the transplanted cornea 8 and 12 weeks after UMSC transplantation. In untransplanted corneas, there is an increase of light scattering with age. E. The average pixel intensity per µm of light scattering in the anterior corneal stroma significantly decreased after 8 and 12 weeks. F. The improvement of light scattering as measured by average pixel intensity in the posterior stroma was not significantly improved until 12 weeks after transplantation. (n = 11; * p<0.05; ** p<0.01; *** p<0.001).
Figure 3.
Improved collagen fiber organization in the corneal stroma of Lum-/- mice 12 weeks after UMSC transplantation.
A. The SHG forward scattering images revealed that collagen fibers (cyan) were more regularly organized in the transplanted cornea (n = 5) than that of the untransplanted cornea (n = 2), especially in the middle and posterior stroma; moreover, the size of collagen fibers was larger in the transplanted cornea than that of the untransplanted cornea. B. The back scattering images showed more flattened images in the transplanted cornea and uneven lamellae (magenta) in the untransplanted cornea. Left panels, wild type mouse cornea; middle panels, Lum-/- cornea transplanted with UMSCs; right panels, untransplanted Lum-/- cornea. White arrows: UMSCs; Red: Syto59; Scale bars: 50 µm.
Figure 4.
Transplanted UMSCs undergoing morphological changes resembling that of host dendritic keratocytes.
A. The images of in vivo fluorescent stereoscopy showed that DiI-labeled UMSCs (red) gathered around the area of the injection tunnel and displayed a round cell shape within the first week following transplantation; later, the cells migrated out and became dendritic in shape. At 8 weeks, the cells were homogeneously distributed in the whole cornea. (magnification, 200×). B. Confocal microscopy revealed that transplanted UMSCs displayed a round-like cell shape by phalloidin staining with the whole mount cornea in the first week and afterwards the cells extended their protrusions and displayed a flat and dendritic cell shape (Scale bars: 50 µm; Blue: nuclear stain by DAPI). C. Following Phalloidin (green) staining with the whole mount Lum-/- mouse corneas 5 weeks after UMSCs transplantation, confocal images revealed that DiI-labeled UMSCs displayed a dendritic and flat cell shape and formed a three dimensional network between the host stromal cells and the donor cells via their extensive dendritic processes, which is similar to those of host keratocytes. Scale bar: 20 µm; Blue: nuclear stain by DAPI.
Figure 5.
The apoptosis of transplanted UMSCs and UHSCs in the recipient Lum-/- corneal stroma.
A. Two weeks after transplantation, the number of UHSCs and UMSCs decreased; afterwards, UHSCs continuously diminished and almost disappeared from the cornea after 5 weeks; however, UMSC numbers maintained at a relative constant level and more than half of UMSCs survived in the corneal stroma up to 12 weeks (n≥6 in each time point). B and C. TUNEL assay revealed that a large number of UMSCs was labeled (green) in 48 hours after transplantation, thereafter, very few apoptotic UMSCs could be detected after one week. In contrast very few apoptotic cells were seen in the UHSCs within the first 48 hours after the transplantation and the number of apoptotic UHSCs increased after 1 week and maintained at high levels thereafter (n≥4 in each time point). Scale bars: 50 µm; Blue: DAPI.
Figure 6.
The recipient inflammatory response after UMSCs and UHSCs transplantation into Lum-/- cornea.
A. Immunostaining with antibodies against inflammatory cells, e.g., CD45 (leukocyte), CD90 (T-Cell) and F4/80 (macrophage) demonstrated the presence of large numbers of immune and inflammatory cells in UHSC transplanted corneas but not in UMSC transplanted corneas, indicating that UMSCs might suppress the host immune response. B. More CD45+ leukocytes invaded UHSC-recipient corneal stroma after the transplantation and the peak of leukocyte infiltration occurred one week after transplantation (n≥5 in each time point). C. The recipient F4/80+ macrophages were present in corneas transplanted with UMSCs and UHSCs. In contrast, more macrophages were found in corneas with UHSC transplantation than that of UMSC transplantation (n≥5 in each time point). D. The infiltration of recipient CD90+ T-cells appeared at the first week and maintained at high levels after UHSC transplantation; very few CD90+ cells were seen after UMSCs transplantation (n≥5 in each time point). Scale bars: 10 µm; Blue: DAPI.
Figure 7.
UMSCs-derived cells changed to be quiescent in Lum-/- cornea after the transplantation.
A. BrdU labeled cells were identified by whole mount immunostaining with anti-BrdU antibody (green) and the results revealed that BrdU-labeled UMSCs (red) presented at 1 and 3 weeks after transplantation, no BrdU-labeled cells were found in corneas 8 weeks after transplantation. B. The numbers of BrdU-labeled UMSCs decreased 2 weeks after transplantation; by eight weeks, there were no BrdU-labeled cells found in the stroma, suggesting that transplanted UMSCs became quiescent. n = 6 for each time point and condition. Scale bars: 10 µm; Blue: DAPI.
Figure 8.
The synthesis of KS-keratocan, KS-lumican, and expression of CD34 by transplanted UMSCs.
A. Immunostaining with anti-human keratocan antibody displayed that keratocan (red) was distributed surrounding the transplanted UMSCs (green) in the anterior stroma of the Kera-/- mouse. B. Western blot revealed that human keratocan (45 kD) was detected after enzymatic digestion to remove keratan sulfate glycosaminoglycan (KS-GAG) in the Kera-/- cornea transplanted with UMSCs, but not in untransplanted corneas of Kera-/-, Lum-/- and wild type mice. n = 5. C. Immunostaining with anti-mouse lumican antibodies (cross reacts to both human and mouse lumican) displayed that lumican (red) presented in Lum-/- mouse cornea after UMSC (green) transplantation. D. Western blot revealed that lumican (45 kD) was detected after enzymatic removal of KS-GAG in Lum-/-, Kera-/-, and wild type mouse corneas transplanted with UMSCs, but not in the Lum-/- mouse cornea (n = 4). E. Immunostaining revealed that transplanted human UMSCs (red) were stained by anti-CD34 (green) in Lum-/- mouse corneas six weeks after transplantation. Scale bars: 50 µm; Blue: DAPI.
Figure 9.
Up-regulation of keratocan and ALDH3A1 was observed in the Lum-/- mouse cornea after UMSC transplantation.
Semi-quantitative Western blot analysis was performed to determine increased synthesis of keratocan and aldehyde dehydrogenase by recipient keratocytes in corneas transplanted with UMSCs. Panels A and B indicated that the keratocan expression level significantly increased after UMSC transplantation compared to untransplanted corneas. Panels C and D showed that the expression of ALDH3A1 was significantly increased in Lum-/- mice after UMSC transplantation (n = 4).