School of Medicine

Human somatic cells are capable of being reprogrammed to induced pluripotent stem (iPS) cells through the introduction of several transcription factors (Oct4, Sox2 and either Klf4, and c-Myc or Lin28 and Nanog). While iPS cells hold therapeutic potential for the future of regenerative medicine, current methods of induction and maintenance of the pluripotent state are extremely inefficient (with reported efficiencies of <0.01%) and pose significant oncogenic risk. Several studies have demonstrated that the efficiency of reprogramming can be significantly enhanced by modifying culture conditions through the addition of small molecules or growth factors. The goal of this project is to study the impact of growth factors encoded by the Wnt gene family on the reprogramming and the regulation of the induced pluripotent state. This work will address the utility of Wnt proteins as reprogramming agents and potentially establish a safer method to generate iPS cells than is currently available. Here we propose a set of experiments to determine whether Wnt proteins, a class of signaling molecules with potent stem cell activities, and their signaling pathways regulate the acquisition of the pluripotent state. WT83 human foreskin fibroblasts were infected with retroviruses containing Oct-4, Sox-2, Klf-4 and c-Myc (Yamanaka factors), and assessed for iPS reprogramming efficiency while activating or inhibiting Wnt signaling. Wnt signaling was activated by adding either canonical Wnt3a or non-canonical Wnt5a proteins, or concurrently transducing lentiviruses encoding [Beta]-catenin. Wnt signaling was inhibited by adding small molecule antagonists of Wnt signaling IWP and IWR, or concurrently transducing with lentiviruses encoding Axin. 11 days after gene transduction, fibroblasts were assessed for iPS cell reprogramming efficiency by staining for alkaline phosphatase, a marker for pluripotency, and quantifying the number of AP-positive colonies. In comparison to untreated cells, we observed a significant increase in AP-positive c.