Year 1

iPS cell lines display high variability with respect to their growth properties, differentiation ability and disease phenotype manifestation; this is a major challenge for both in vitro disease modeling and drug screening, as well as cell replacement therapy. The cause for this variability is currently unknown and heterogeneity of the starting fibroblast population, composition of reprogramming factors, and viral integration into the genome have all been proposed to contribute to variability among different iPSC clones derived from the same starting population. In particular, the use of DNA viral vectors to deliver the reprogramming factors was suggested to be the key obstacle for eventual use of iPSCs in cell replacement therapies. Recently, several methods have been developed to derive integration-free iPS cells, but they mostly suffer from unacceptably low reprogramming efficiency or labor-intensive delivery of reprogramming factors.

The goal of this work is to produce integration-free iPS cells with high efficiency. To date, we developed a high-throughput platform to screen small molecules that enhance reprogramming in the presence of 3 transcription factors required for reprogramming of fibroblast cells – KLF4, SOX2, and OCT4. These efforts included assay development, characterization of our engineered stable cell lines expressing reprogramming factors under control of an inducible promoter, and optimization for high-throughput screening. We screened over 100,000 compounds from our small molecule library, comprising compounds with broad chemical diversity and covering multiple cellular target classes. We identified 130 hit candidates in our primary screen, however, in follow-up assays none of these compounds showed an effect comparable with that of a positive control compound. Moving forward, we plan on detailed characterization of other integration-free reprogramming methods to determine the differences between integrating vs. non-integrating methods for reprogramming fibroblasts. We will assess the quality and variability of iPS cell lines, as well as their potential and variability of differentiation and disease phenotype manifestation in integration-free iPSCs, and compare them to DNA virus-derived iPSCs. Our results should contribute to the understanding of the source of variability between iPS cells and bring us closer to reaching the ultimate goal: production of integration-free human iPS cells with high efficiency.