Background:
Cornea is continues renewed by the limbal epithelial stem cells (LESCs), but due to damage of the limbus, this function can be lost and can lead to limbal stem cell deficiency (LSCD). Treatments of LSCD are either by cultivated limbal epithelial transplantation or collateral transplantation of LESCs. Induced pluripotent stem cell (iPSCs) shows great potential in treating single cell defects like Parkinsons, Diabetes and LSCD.
Objective:
The current work will focus on the reprogramming of limbal fibroblasts to iPSCs by the Epi5TMEpisomal iPSC Reprogramming Kit (Invitrogen), and both episomal and viral plasmids obtained from Addgene.org
Methods/Results:
Results from previous experiments using the Vivid ColorTM pLenti6.2-GW/EmGFP Expression Control Vector was used for optimization experiments by electroporation it into human limbal fibroblasts, resulting in two experiments, concluding that previously using a narrower cuvette could halve electroporation voltage. Optimal parameters was 125 V, 0% modulation and 10 pulses.
In addition, these results were used for the purpose of reprogramming limbal fibroblast to iPSCs, by the Epi5TM Episomal iPSC Reprogramming Kit (Invitrogen) and the episomal reprogramming vector from Addgene.org. Futhermore viral production and viral transfection of limbal fibroblasts were also tried.
Conclusion:
The current project has further optimization the electroporation setup for electroporation of limbal fibroblasts for the usage in formation of iPSCs. Plasmids obtained from Addgene.org were verified by restriction digests and electrophoreses. Viral production was done by transfecting HeLa cells with a viral plasmid and a viral packaging system and harvesting the medium. Even though cell morphology changes during the experiments, it was not succeed to form iPSCs