Thesis

Algorithm selected transcription factors used to initiate reprogramming and enhanced differentiation in somatic and pluripotent stem cells

Direct reprogramming offers a promising alternative method in producing cells for regenerative medical applications. However, a major obstacle in this process is the selection of transcription factors best suited to initiate reprogramming. By comparing differential gene transcription levels across numerous tissue samples, the Core Expression Module Analysis (CEMA) developed by the Lowry lab identified and compiled compact sets of genes unique to a given cell lineage. With additional gene ontology analysis, a list of 3-10 transcription factor (TF) were generated for each cell type. We hypothesized that ectopic expression of these selected TF can induce direct reprogramming in terminally differentiated somatic cells and direct differentiation and maturation in pluripotent stem cells. To test this, we incorporated these lineage conversion factors into a YFP marker gene containing lentiviral polycistronic cassette that uses an rtTA Inducible Tet-On promoter system. For the direct reprogramming project, two plasmids were generated to produce two distinct cell lineages; tissue endothelial cells and mature neural progenitor cells. Fibroblast were infected with rtTA and TF cassette lentivirus and using puromycin and neomycin selection, with YFP expression, we were able to positively identify infected cells. Treatment with doxycycline over an extended period resulted in morphological changes appearing in a small subset of infected cells, with ectopic and endogenous up regulation of key genes measured via IF staining and qRT-PCR. Using a similar protocol with ESCs/iPSC, infected YFP positive colonies ectopically expressing early neural progenitor cell transcription factors were selected and differentiated towards the neural fate using lab established methods. After sorting and analyzing cells with FACS and qRT-PCR, cells that had received continuous doxycycline treatment while remaining YFP positive demonstrated a decrease in Lin28B expression, a microRNA preventing cell maturation, with cell and colony morphology comparative to fetal NPC samples. With additional refinement to CEMA and the reprogramming protocols, mature cells from all lineages may be generated, with applications ranging from disease modeling to cell replacement therapy.

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