Abstract
We assessed the maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using tissue-engineered oral mucosal cell sheets in a rat model. Oral mucosal biopsy specimens were excised from green fluorescent protein (GFP) rats and enzymatically treated with Dispase II. These cells were cultured on inserts with mitomycin C-treated NIH/3T3 cells, and the resulting cell sheets were harvested. These tissue-engineered cell sheets from GFP rats were transplanted onto the eyes of a nude rat limbal stem cell deficiency model. Eight weeks after surgery, ocular surfaces were completely covered by the epithelium with GFP-positive cells. Transplanted corneas expressed p63 in the basal layers and K14 in all epithelial layers. Epithelial cells harvested from the central and peripheral areas of reconstructed corneas were isolated for a colony-forming assay, which showed that the colony-forming efficiency of the peripheral epithelial cells was significantly higher than that of the central epithelial cells 8 weeks after corneal reconstruction. Thus, in this rat model, the peripheral cornea could maintain more stem/progenitor cells than the central cornea after corneal reconstruction using oral mucosal epithelial cell sheets.
Highlights
Corneal epithelial stem cells are located in the basal layer of the limbus, which is the narrow transition zone between the cornea and the conjunctiva [1,2]
Corneal epithelial cell sources are exhausted, the peripheral conjunctival epithelium invades inwardly, and the corneal surface becomes enveloped by vascularized conjunctival scar tissue, which results in corneal opacification that leads to severe visual impairment [4,5]
Cell suspensions were cultured on temperature-responsive culture inserts (CellSeed Inc., Tokyo, Japan) at an initial density of 46105 cells/23-mm insert along with mitomycin C (MMC)treated NIH/3T3 cells that were separated by these cell culture inserts in the keratinocyte culture medium (KCM) (Dulbecco’s modified Eagle’s medium [DMEM]/F12 [3:1] supplemented with 10% fetal bovine serum [Japan Bio Serum, Hiroshima, Japan], 0.5% Insulin–Transferrin–Selenium [ITS; Invitrogen, Carlsbad, CA], 10 mM isoproterenol [Kowa, Tokyo, Japan], 2.061029 M triiodothyronine [MP Biomedicals, Aurora, OH], 0.4 mg/mL hydrocortisone succinate [Wako, Osaka, Japan], and 10 ng/mL EGF [R&D Systems, Minneapolis, MN]) [6]
Summary
Corneal epithelial stem cells are located in the basal layer of the limbus, which is the narrow transition zone between the cornea and the conjunctiva [1,2]. The limbal epithelium is a reservoir for replacing corneal epithelial cells that are normally continuously lost from the corneal surface [3] Severe corneal diseases, such as Stevens–Johnson syndrome, or chemical burns destroy the limbus and cause limbal stem cell deficiency (LSCD). In cases of severe LSCD, we and others recently demonstrated the successful application of constructs involving ex vivo expansion of autologous oral mucosal epithelium [6,7,8] This method averts the risks of immune rejection and long-term immunosuppression, and offers clinical advantages over conventional allogeneic corneal transplantation [9]. Corneal transparency was restored and postoperative visual acuity remained improved for 2–8 years, whereas abnormal corneas were successfully reconstructed using conventional allogeneic transplantation in only 20–30% of patients for 2–3 years [10,11]
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