Abstract 14806: Generation of Tumor Free Induced Pluripotent Stem Cells for Safe Cell Based Therapy
Background The use of induced pluripotent stem cells (IPS) for patient-specific regenerative therapy necessitates the generation of tumor free IPS. Previously we showed that predifferentiation of IPS reduced the risk of tumor formation and improved cardiac function in vivo. However, exact mechanism of tumor genesis by IPS cells need to be explored.
Methods and Results: Glycogen synthase kinase 3 (GSK3) is an important component of diverse signaling pathways involved in the regulation of cell fate, proliferation and survival. It binds and phosphorylates several proteins in the Wnt pathway which is critical for tumor development. We established a serum free (SF) culture system to investigate the tumorigenic role of Wnt/GSK3β signaling. Previously we have reported IPS generation by using a small molecule without viral vectors.IPS were grouped in Serum or SF conditions with or without GSK3B inhibitor(3uM);with or without Wnt agonist (2uM); with or without Frizzled protein (0.5ug/ml) and with GSK3β/ Frizzled protein together. RTPCR analysis showed that IPS cells retained the pluripotency markers Oct4, Sox2, Nanog under serum free conditions. However the expression of tumorogenic regulators, c-Myc and Eras was down regulated under serum free conditions as compared to IPS cultured under standard culture media containing serum. The cell growth and proliferation was measured by cell doubling time and Ki67 staining. To assess the tumor formation in vivo, IPS (1×106) treated under various groups were injected into nude mice. IPS which expressed GSK3β grew slow and did not generate teratoma which was reversed by GSK3β inhibitor. IPS cells cultured with Wnt agonist grew normal but generated teratoma. No teratoma was observed with frizzled protein treatment of IPS cells which expressed increased total Akt activity by western blot analysis. Teratomas were developed after 2-3weeks in nude mice that received IPS treated with frizzled protein and GSK3β inhibitors.
Conclusion: We report here for the first time that Akt/Wnt/GSK3β signaling pathway is closely associated with tumor formation by IPS cells. It is feasible to generate tumor free IPS cells by regulating the Wnt signaling pathway and its downstream targets.
- © 2012 by American Heart Association, Inc.