Animals with the corneal injury
Male Balb/c mice were purchased from BioLASCO (Taipei, Taiwan). Animals were maintained in the animal facility of Wan Fang Hospital, Taipei Medical University (WFH-TMU, Taipei, Taiwan). All experimental protocols were approved by the animal use and care committee of WFH-TMU. An eight-week-old mouse’s cornea was covered by round filter paper, which had been rinsed by 0.5 N NaOH before covering the mouse corneal surface for 30 s. Mice were separated into two groups: filter paper in group A covered 70% of the central corneal area (5.9 mm in diameter) and in group B, filter paper covered 100% of the corneal area (7 mm in diameter). Corneal epithelial cells were smoothly removed with a no. 15 Bard-Parker scalpel blade after alkali damage.
Isolation, expansion of OFSCs
Isolation and culture of OFSCs were carried out as described previously
. Briefly, during blepharoplastic surgery, 0.5 to approximately 1 ml of redundant orbital fat tissues was removed from the intraorbital cavity. All samples were removed with informed consent and followed regulations of the Institutional Review Board of WFH-TMU. Tissues were fragmented, digested and filtered. The suspension was centrifuged, cells from the pellet were plated at a very low density, and colony-forming cells were maintained in MesenPro medium (Invitrogen, Carlsbad, CA, USA). OFSCs were mesenchymal stem cells which were negative for CD34, CD133, CD31, CD106, CD146, CD45, CD14, CD117 and HLA-DR and positive for CD58, CD90, CD105, CD29, CD49b, CD49e, CD44, CD49d and HLA-ABC
. The tri-lineage differentiation capacity of these cells was checked before this study.
Topical and intra-limbal OFSC transplantation
Six mice were used in Group A. After 70% of corneal injury to both eyes, the right eye was applied with topical OFSCs (T) and the left eye with PBS. Night mice were used in Group B for the topical (T) administration of OFSCs. After 100% of corneal injury for both eyes took place, topical OFSCs were given to the right eyes while topical PBS (six mice) or no treatment (dry control, three mice) to the left eyes. In addition, three mice without corneal injury received topical OFSCs on the right eyes and topical PBS on the left eyes.
For evaluation of the intra-limbal (IL) injection, 100% corneal injury was created on three mouse corneas. IL injection of OFSCs was performed in the right eye and PBS in the left eye. One mouse without corneal injury received IL OFSCs in the right eye and IL PBS in the left eye.
Before treatment, OFSCs were detached and re-suspended in PBS(Gibco, Grand Island, NY, USA). For topical (T) administration, 2 × 105 human OFSCs with or without quantum dots (Invitrogen) labeling in 5 μl of PBS were applied to the right corneal surface twice a day until the day of sacrifice, while 5 μl of PBS applied to the left eye twice a day served as the control. For the IL injection, 2 × 105 quantum dots (Invitrogen)-labeled OFSCs in 5 μl of PBS were injected into the lateral side of the right limbal epithelium on the first day, and a repeat injection on the nasal side of the right limbal epithelium was given on Day 6, while 5 μl of PBS was injected into the left limbal epithelium at the same time.
Quantification the area of corneal injury
The cornea wound was examined on days 0, 1, 2, 3 and 7. Before being photographed, the epithelial defect was stained with a topical fluorescent strip (HAAG-STREIT, Koeniz, Switzerland), and images were captured with a digital camera (Canon, Tokyo, Japan) under a cobalt-blue light source from a direct ophthalmoscope (Welch Allyn, Skaneateles Falls, NY, USA). The injured area of the cornea was determined using the software Image Pro-Plus version 6.0 (Media Cybernetics, Rockville, MD, USA) and calculated as a percentage of the residual epithelial defect.
Histological and immunohistochemical (IHC) staining
Mice were sacrificed at the end of day 2, 3 or 7 after injury. The eyeball was removed en bloc and fixed in formalin, then prepared in paraffin-embedded blocks for sectioning at a thickness of 10 μm. Tissue sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO, USA). For IHC staining, tissue sections were incubated with rabbit antibody against human immunoglobulin G (hIgG) (1:800, Abcam, Cambridge, MA, USA), or rabbit antibody against human beta-2 microglobulin (hβ2M) (1:800, Abcam) at 4°C for 1 h, followed by goat antibodies against rabbit IgG (Dako Cytomation, Glostrup, Denmark) for another 40 to approximately 60 minutes. Tissue sections were assessed by microscopy (Leica Microsystem, Wetzlar, Germany). Images were acquired with MetaMorph version 4.6 (Molecular Devices, Sunnyvale, CA, USA).
For cytokeratin 19 (CK19) and CK3 staining, frozen section tissue slides were fixed in cold methanol for 30 minutes, followed by two PBS washes. After being blocked in 5% skim milk at room temperature for 1 h, slides were incubated with a mouse antibody against human/mouse CK19 (1:500, Millipore, Billerica, MA, USA), a mouse antibody against human/mouse CK3 (1:500, Millipore), rabbit antibody against human p63 (1:150, Abcam), or rabbit antibody against hIgG (1:800, Abcam) at room temperature for 1 h, followed by incubation with DyLight 488-conjugated goat anti-mouse IgG (1:500, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA), or DyLight 594-conjugated goat anti-rabbit IgG (1:500, Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 30 minutes. Nuclei were then stained with 4,6-diamidino-2-phenylindole (DAPI, 1:1,000), and samples were assessed under a fluorescence microscope (Leica Microsystem). Images were acquired using MetaMorph version 4.6 (Molecular Devices).
Western blot analysis
Total protein was obtained from mouse cornea with Cell Lysis Buffer (Sigma-Aldrich) containing freshly added protease inhibitors (Sigma-Aldrich). Protein concentrations were determined with a Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was performed using 30 μg of total protein by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride membranes (Millipore). Nonspecific binding was blocked by 5% skim milk in TBST buffer (50 mMTris–HCl, pH 7.4; 150 mMNaCl, and 0.1% Tween 20) at room temperature for 1 h. The membrane was then probed with a rabbit polyclonal antibody (pAb) to inducible nitrous oxide synthetase (iNOS, 1:2000, Abcam), a rabbit monoclonal antibody (mAb) to CD68 (1:1,000, Epitomics, Burlingame, CA, USA), a rabbit pAb to tissue growth factor-beta (TGF-β, 1:4,000, Abcam), a mouse mAb to tumor necrosis factor-alpha (TNF-α, 1:1,000, Abcam), a rat mAb to lymphocyte antigen 6 complex (Ly6G, 1:1,000, Abcam), a mouse mAb to vascular endothelial growth factor (VEGF, 1:1,000, Abcam), a rabbit pAb to hIgG (1:800, Abcam), a mouse mAb to mouse and human β-actin (1:200, Sigma-Aldrich), or a mouse mAb to α-tubulin antibodies (1:104, Sigma-Aldrich) at 4°C overnight. After three washes with TBST (for 15 minutes each), the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody against to rabbit IgG (1:5,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or against to mouse IgG (1:5,000, Abcam) at room temperature for 1 h. After three more TBST washes, protein signals were detected by enhanced chemiluminescence (ECL; NEN Life Science, Boston, MA, USA) and their intensities were measured by densitometry (Image Pro-Plus version 6.0, Media Cybernetics).
Values are shown as the mean ± standard error. Statistical analyses were performed using the Statistical Package for Social Science version 16 software (SPSS, Chicago, IL, USA). Results of comparisons of the area of corneal injury at each time point between the PBS and OFSCs groups or between the dry control and OFSCs groups, and corneal protein expression between the PBS and OFSCs groups were analyzed by Student’s t-test, and P <0.05 was considered a statistically significant difference.