KLAL rejection is a newly recognized entity. Pathologic findings of rejected specimens indicate that this is a T-cell mediated rejection phenomenon. The pattern of cytokeratin staining provided little evidence that the epithelium covering KLALs had a corneal phenotype. The scarcity of vimentin-positive epithelial cells suggests that the stem-cell/transient-cell pool was probably depleted. Early recognition of clinical rejection is important, as treatment with immunosuppressive therapy may reverse the process.
Purpose The integrity of the corneal epithelium is essential for clarity of vision. Under normal circumstances, superficial epithelial cells are shed into the tear film and regenerated by epithelial stem/progenitor cells located at the limbus. Damage or depletion of the limbal cells leads to conditions such as limbal stem cell deficiency (LSCD). Current treatment involves transplantation of limbal epithelial cells expanded ex‐vivo; however cell numbers are limited. In this study, we identify a source of multipotent stem cells located in the corneal stroma that express CD34 upon isolation. These cells demonstrate an ability to transdifferentiate into corneal epithelial cells.
Methods CD34 expression diminishes during traditional tissue‐culture plastic propagation; in this study we optimised culture conditions for maintained and efficient expansion of CD34+ cells. We investigated the role of CD34 in epithelial transdifferentiation of stromal cells, using siRNA gene knockdown and analysis by immunocytochemistry, flow cytometry and qPCR.
Results Early results suggest a three‐dimensional environment and semi‐solid medium intended for hematopoietic culture demonstrated extended CD34 expression. When cells were transferred to epithelial differentiation medium they showed an epithelial morphology, significantly increased cytokeratin 3 and 19 expression, and considerable upregulation of genes related to corneal epithelial cells (ABCG2, DeltaN63, LEF1, HES1, FRZB1, KRT19, DTC and CDH1).
Conclusion This work will help produce methodologies to create cell banks for generation of corneal epithelium, from a corneal stromal stem cell source, leading to improved surgical and visual outcomes in LSCD patients.
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