Immunophenotypic characterization of human pluripotent stem cell-derived limbal stem cells

Abstract

Limbal stem cells are responsible for maintaining vision by renewing the corneal epithelium and preventing the conjunctival cells and surrounding vasculature from invading onto the cornea. If these functions of the limbal stem cells are disturbed, humans are in danger of losing their sight. One of the most severe disorders associated with limbal stem cells is the limbal stem cell deficiency. It may develop due to acute trauma, chronic inflammation or genetic cause. The only suitable treatment for limbal stem cell deficiency is the transplantation of healthy limbal cells, but the lack of suitable cell material creates a drastic issue. Human pluripotent stem cell-derived limbal stem cells may offer a solution to the problem and serve as an alternative treatment. Further knowledge on the immunogenicity of human pluripotent stem cell-derived cells is needed to enable safe cell replacement therapies in the future. Immunophenotype of the cells plays a huge role in immunogenicity. Most significant surface proteins contributing to the immunogenicity are polymorphic human leukocyte antigens, i.e. HLA-molecules, which are needed to activate T cell-mediated immune reaction. In addition to HLA-molecules, co-stimulatory molecules contribute to the activation of T cells. The immunogenicity of human pluripotent stem cell-derived limbal stem cells depends largely on the expression of these molecules. Thus, for the future clinical applications it is critical to investigate the expression of these molecules determining the immunophenotype of the cells. In this study, the expressions of these molecules contributing to the T cell activation were studied with cryopreserved human embryonic stem cell-derived limbal stem cells that were differentiated using the specific method developed by the Skottman laboratory. Expressions of the five relevant antigens were studied with immunofluorescent antibody labeling followed by flow cytometric analysis. The experiments were repeated three times. The results showed that human embryonic stem cell-derived limbal stem cells typically express HLA class I molecules, whereas the expressions of other molecules in the studied cells were rather insignificant. However, the expression of a co-stimulatory molecule CD28 varied between individual repetitions. Therefore, the immunopheno-type of the human pluripotent stem cell-derived limbal stem cells regarding the CD28-molecule should be restudied. These results on the immunophenotype of human pluripotent stem cell-derived limbal stem cells serve as a basis for the understanding of their immunogenicity. Nevertheless, further analysis should be conducted to understand the overall immunological properties that these cells possess. To make cell replacement therapy possible, studying how inflammation affects the immunopheno-type of these cells would be of interest. Also, it would be important to understand, if the particular method used to differentiate the limbal stem cells of this study changes the immunogenicity of the cells.