Poster #039, Cedars Sinai Medical Center
Investigating Tumor Development Risk in Diabetic and Non-Diabetic iPSC-Derived Limbal Epithelial Stem Cells
Mentors: Vanessa Borges, PhD and Alexander Ljubimov, PhD
Up to 70% of diabetes mellitus (DM) patients face ocular surface complications, many resulting from corneal issues. These complications often manifest as corneal epithelial degeneration, slow wound healing, neuropathy, tear film abnormalities, and may eventually result in blindness. In efforts to mitigate this, limbal epithelial stem cells (LESCs) have been a recurrent topic of focus due to their dysfunction in DM patients. To address LESC dysfunction in DM patients, this project uses prior protocols to convert defective LESCs from DM patients into induced pluripotent stem cells (iPSCs) using genes known as Yamanaka factors and then differentiate the iPSCs into LESC-like cells. However, to understand the drawbacks of such a procedure in terms of patient outcomes, potential risks must be investigated, specifically increased potential for tumor development. To investigate whether the resulting cells were functional LESC-like cells, immunofluorescence was used to check for presence of putative LESC markers. A flow cytometry protocol was developed on differentiated LESC-like cells to see whether pluripotency markers (SOX2 and OCT4) were still present, whether putative LESC markers (ΔNp63 and cytokeratin 17) could be found, and whether these cells contained concerning levels of markers (CD133 and Nestin) known to be highly expressed in cancer stem cells. Continued presence of pluripotency markers would highlight potential problems in differentiation while putative LESC/epithelial markers would indicate successful differentiation of iPSCs into functional LESC-like cells. It was found that limbal-derived iPSCs differentiated into LESC-like cells expressed putative LESC markers at non-diabetic levels and none to minimal expression of cancer stem cell markers and pluripotency markers. These findings demonstrate that the differentiated cells are molecularly similar to primary non-diabetic LESCs without elevated potential to create tumor cells. This affirms procedure viability and demonstrates possibility for its future safe implementation for autologous cell therapy in DM patients.