Subconjunctival Injection of Transdifferentiated Oral Mucosal Epithelial Cells for Limbal Stem Cell Deficiency in Rats

Abstract

Rat limbal niche cells (LNCs) have been proven to induce transdifferentiation of oral mucosal epithelial cells (OMECs) into corneal epithelial-like cells termed transdifferentiated oral mucosal epithelial cells (T-OMECs). This investigation aimed to evaluate the effect of subconjunctival T-OMEC injections on alkali-induced limbal stem cell deficiency (LSCD) in rats. LNCs were cocultured with OMECs in the Transwell system to obtain T-OMECs, with NIH-3T3 cells serving as a control. Subconjunctival injection of single T-OMEC or OMEC suspension was performed immediately after corneal alkali injury. T-OMECs were prelabeled with the fluorescent dye CM-DiI in vitro and tracked in vivo. Corneal epithelial defect, opacity, and neovascularization were quantitatively analyzed. The degree of corneal epithelial defect (from day 1 onward), opacity (from day 5 onward), and neovascularization (from day 2 onward) was significantly less in the T-OMEC group than in the OMEC group. Cytokeratin 12 (CK12), pigment epithelium–derived factor, and soluble fms-like tyrosine kinase-1 were expressed at a higher rate following T-OMEC injection. Some CM-DiI-labeled cells were found to be coexpressed with CK12, Pax6, and ΔNp63α in the corneal epithelium after subconjunctival injection. Subconjunctival injection of T-OMECs prevents conjunctival invasion and maintains a normal corneal phenotype, which might be a novel strategy in the treatment of LSCD:

Pathogenesis of Alkali Injury-Induced Limbal Stem Cell Deficiency: A Literature Survey of Animal Models

“…In LAB models, phenotypic assessment of the re-epithelialized cornea confirms induction of LSCD. This is validated by the loss of a well-established corneal epithelial differentiation marker cytokeratin (K)12 [73,[76][77][78], whilst biomarkers of conjunctival squamous epithelia (K13 and K19 [73,79]) as well as conjunctival MUC5AC + goblet cells (GCs) are present (Figure 1h-j). GCs appear as early as 7 days post-injury [58] and persist throughout the early reparative phase [76,77,79].…”

“…This is validated by the loss of a well-established corneal epithelial differentiation marker cytokeratin (K)12 [73,[76][77][78], whilst biomarkers of conjunctival squamous epithelia (K13 and K19 [73,79]) as well as conjunctival MUC5AC + goblet cells (GCs) are present (Figure 1h-j). GCs appear as early as 7 days post-injury [58] and persist throughout the early reparative phase [76,77,79]. Notably, the onset of CNV precedes the appearance of GCs, which are often associated with vascularized areas [58], although the significance of this correlation is not fully understood.…”

Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up

“…Several studies assume that the presence of corneal cells observed in patients after COMET is due to a change in the phenotype of oral cells into corneal one, a phenomenon called transdifferentiation [22,52]. However, it has been reported that epithelial cells transplanted in ectopic districts maintain their original phenotype, making the transdifferentiation hypothesis unlikely [53][54][55].…”

Effects of Exosomal microRNAs on Oral Mucosal Epithelial Cells Cocultured with Limbal Niche Cells

“…Currently, autologous limbal tissue/stem cell transplantation is the most established treatment for LSCD, which shows a favorable and stable therapeutic effect; however, this procedure cannot be used in patients with binocular LSCD. Clinical observations revealed that the most severe LSCD often involved both eyes, forcing ophthalmologists to choose allogeneic sources of corneal tissue or cells for treatment [ 22 ]. Nonetheless, this option is accompanied by a high risk of immune rejection and requires long-term adjuvant therapy using immunosuppressants, thus limiting the clinical benefits of the treatment.…”