Effect of oocyte factors on epigenetic memory in induced pluripotent stem cells for reduction of cell of origin dependency

Abstract

The derivation of induced pluripotent stem cells (iPSCs) has opened doors for numerous applications for which limited human cell sources were available. However, their use has limitations due to epigenetic memory; histone and DNA modifications that iPSCs retain from their cells of origin. This affects chromatin structure and therefore activation or inhibition of specific genes. Some inhibitors against these modifications have been assessed to enhance reprogramming efficiency, but little has been reported about their influence on differentiation potential. What has been discovered, however, is that somatic cell nuclear transfer (SCNT) creates stem cells with DNA methylation levels more similar to embryonic stem cells (ESCs) compared to iPSCs, likely due to involvement of oocyte factors. Therefore, the aim of this proposed research is to determine the effect of a selection of these factors on epigenetic memory and differentiation potential of iPSCs generated from human dermal fibroblasts and neutrophils. Expression of TH2A, TH2B, ASF1A, GDF9, GLIS1, H3.3B, Wave1 and Gata3 will be transiently induced during reprogramming, while BMP7 will be supplemented to the culture medium and DJ-1 will be knocked down. Additionally, they will be investigated in combination of aforementioned inhibitors. Consequently, changes in modifications and differentiation potential of resulting iPSCs will be analysed and compared. This will provide the possibility of utilizing non- invasively acquired cells for creating a neutral iPSC line without limitations in differential potential, allowing for non-confounding comparisons across studies and between in vitro and in vivo circumstances due to no dependence on cells of origin.