Reprogramming somatic nuclei to a pluripotent state with cell-free extracts

Abstract

The live birth of cloned animals has shown that somatic cells are not irrevesibly committed to their fate. A functional reprogramming of differentiated cells to pluripotency may present beneficial applications in regenerative medicine. A differentiated cell can be reprogrammed to pluripotency by transfer into enucleated oocytes, fusion with a pluripotent cell or forced expression of pluripotency genes. Some of these genes can be substituted by proteins, suggesting that reprogramming cells by non-genetic means is possible. The main hypothesis of this thesis is whether the introduction of factors derived from pluripotent cells into somatic target cells epigenetically and functionally could reprogram the target cell to pluripotency. This thesis reports the reprogramming of function of human kidney epithelial 293T cells treated with extracts from human embryonal carcinoma cells and of mouse NIH3T3 cells treated with extract from mouse embryonal stem cells (ESC). Morphological observations, genome-wide gene expression analysis, RT-qPCR analysis, immunolabeling, in vitro differentiation assays, locus-specific assessments in DNA methylation and post-translational histone modifications, as well as changes in surface markers and intracellular protein expression analysed by mass spectrometry are consistent with long-lasting alterations in somatic cell fate as a result of treatment with pluripotent cell extracts. Extract-mediated reprogramming is non-genetic and thus may be more easily ethically amd medically acceptable than methods using viruses or human eggs.