Title

The Generation of Induced Pluripotent Stem Cell by Recombinant Proteins, and The Essential Epigenetic Role of Jak/Stat3 in Promoting Murine Somatic Cell Reprogramming

Date of Completion

January 2012

Keywords

Biology, Molecular|Biology, Genetics|Biology, Cell

Degree

Ph.D.

Abstract

The generation of induced pluripotent stem cells (iPSCs) directly from somatic cells by ectopic expression of reprogramming factors opened unprecedented opportunities for regenerative medicine. However, the genomic integration of cells by transgenes, the low efficiency of iPSC induction, and the limited understanding of reprogramming hindered its clinical advance. The generation of integration-free iPSCs by recombinant proteins (piPSCs) is inefficient and time consuming, which may result from the lack of proper protein activity, as no functional assay was conducted to test each protein individually prior to the reported piPSC induction. In the first section of this dissertation, we used iPSC induction assay from mouse embryonic fibroblasts (MEFs) to screen the recombinant Klf4 constructs linked to cell-penetrating peptides (CPPs) at the N- or C- terminus. We found distinct potency among these Klf4 constructs for iPSC induction, with the fusion protein harboring Discosoma red fluorescent protein (DsRed) between the CPP and Klf4 exhibiting the most significant reprogramming activity. We further optimized the iPSC induction conditions using this recombinant Klf4 and showed that four consecutive protein transductions at nano-molar level are sufficient for effective iPSC induction. ^ In the second section of this dissertation, we aimed to better understand the mechanism of reprogramming and to improve its efficiency, by studying the role of Janus kinase/signal transducer and transactivator 3 (Jak/Stat3), which is activated by leukemia inhibitory factor (LIF) and maintains the self-renewal of mouse pluripotent stem cells with ill-defined mechanism(s). We discovered that enhanced Stat3 activation promotes MEF reprogramming, and that Stat3 activity is essential for both terminally differentiated and partially reprogrammed cells to acquire pluripotency. Inhibiting Jak/Stat3 blocked the expression of de novo DNA methyltransferase 3L (Dnmt 3L), with the failure of cells to silence retroviral transgenes during reprogramming. Furthermore, Jak/Stat3 inhibition leads to hyper-methylation of Oct4 and Nanog regulatory regions, which is correlated with the elevated expression of DNA methyltransferase 1 and histone deacetylases (HDACs). Finally, we demonstrated that the Dnmt or HDAC inhibition but not Nanog expression rescues the arrested MEF reprogramming resulted from either Jak/Stat3 inhibition or LIF deprivation. We propose that Jak/Stat3 signaling promotes pluripotency by epigenetic regulations during reprogramming. ^