Formation of Induced Pluripotent Stem Cells and Ocular Regenerative Medicine: By Episomal and Viral Reprogramming
Author
Rasmussen, Andreas
Term
4. term
Publication year
2014
Submitted on
2014-05-28
Pages
40
Abstract
Hornhinden fornyes løbende af limbale epiteliale stamceller (LESC) ved kanten af hornhinden (limbus). Hvis limbus beskadiges, kan denne funktion gå tabt og føre til limbal stem cell deficiency (LSCD). Aktuelle behandlinger omfatter transplantation af dyrkede limbale epitelceller eller overførsel af LESC. Inducerede pluripotente stamceller (iPSC) viser potentiale for at behandle cellerelaterede defekter som Parkinsons, diabetes og LSCD. Dette projekt havde til formål at omprogrammere menneskelige limbale fibroblaster til iPSC ved hjælp af en ikke-integrerende, episomal tilgang (Epi5 Episomal iPSC Reprogramming Kit, Invitrogen) samt både episomale og virale plasmider fra Addgene. Før reprogrammeringen optimerede vi DNA-levering til fibroblasterne ved elektroporation (korte elektriske pulser, der midlertidigt gør cellemembranen gennemtrængelig) med et grønt fluorescens-kontrolplasmid (Vivid Color pLenti6.2-GW/EmGFP). Brug af en smallere kuvet gjorde det muligt at halvere den nødvendige spænding. De bedste parametre var 125 V, 0% modulation og 10 pulser. Plasmider fra Addgene blev verificeret ved restriktionsfordøjelse og gelelektroforese. Vi fremstillede desuden virus ved at transfektere HeLa-celler med et viralt plasmid og et pakningssystem og høstede mediet, og vi forsøgte at levere de virale vektorer til limbale fibroblaster. Selvom vi så ændringer i cellernes morfologi under forsøgene, lykkedes det ikke at danne iPSC. Konklusion: Projektet forfinede elektroporationsbetingelserne for limbale fibroblaster og etablerede arbejdsgange til plasmidverifikation og viral produktion, men omprogrammering til iPSC blev ikke opnået.
The cornea is continually renewed by limbal epithelial stem cells (LESCs) located at its edge (the limbus). When the limbus is damaged, this renewal can fail and lead to limbal stem cell deficiency (LSCD). Current treatments include transplantation of cultured limbal epithelium or transfer of LESCs. Induced pluripotent stem cells (iPSCs) show promise for treating cell-related defects such as Parkinson’s, diabetes and LSCD. This project aimed to reprogram human limbal fibroblasts into iPSCs using a non-integrating, episomal approach (Epi5 Episomal iPSC Reprogramming Kit, Invitrogen) and both episomal and viral plasmids obtained from Addgene. Before reprogramming, we optimized DNA delivery into fibroblasts by electroporation (brief electric pulses that temporarily make the cell membrane permeable) using a green fluorescence control plasmid (Vivid Color pLenti6.2-GW/EmGFP). Using a narrower cuvette allowed us to halve the required voltage. The best settings were 125 V, 0% modulation and 10 pulses. Plasmids from Addgene were verified by restriction digest and gel electrophoresis. We also produced virus by transfecting HeLa cells with a viral plasmid and packaging system and harvesting the medium, and we attempted to deliver the viral vectors to limbal fibroblasts. Although we observed changes in cell morphology during the experiments, we did not succeed in generating iPSCs. Conclusion: This project refined electroporation conditions for limbal fibroblasts and established workflows for plasmid verification and viral production, but successful reprogramming to iPSCs was not achieved.
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