Combinatorial Modulation of Signal Transduction for Accelerated Induced Pluripotency

Shafer-Sull, Ben-Ami

A pluripotent stem cell is a type of cell that is undifferentiated, meaning it can develop into any type of cell. Until recently, embryonic stem cells, which are derived from early stage mammalian embryos and hence controversial, were the only available pluripotent stem cells. However, we now know of another type of pluripotent stem cell: the induced pluripotent stem cell (iPS cell). An iPS cell is an adult cell that has gained pluripotency through the implementation of defined sets of transcription factors. Inducing pluripotency is a slow and inefficient process, but we have previously shown that a combination of activating Wnt signaling and inhibiting transforming growth factor β (TGF-β) would allow for the more rapid achievement of induced pluripotency. We achieved this combination of activation and inhibition by using a mixture of ascorbic acid (AA), ALK5 inhibitor II (iAlk5), and CHIR99021 (CHIR). To better understand the effects of these compounds on the process of iPS cell formation, we tested mouse embryonic fibroblasts (MEFs) in an intermediate step in the induction of pluripotency for expression of the transcription factors c-Myc, Klf4, Oct4, and Sox2, which are known indicators of pluripotency. As comparison we used MEFs before the induction of pluripotency, which are known to express c-Myc only; we used gel electrophoresis and chemiluminescence for these tests. The results of our experiment provide insight into methods for the acceleration of induced pluripotency.