Abstract We001: Doxorubicin-Induced hiPSC Hormetic Model

Abstract

Doxorubicin (Dox), an anticancer anthracycline drug, is an important tool to understand how cells develop resistance to anthracyclines. Hormesis is a biological phenomenon where low doses of drugs can have beneficial effects on cell proliferation and differentiation. hiPSCs are becoming the model of choice for Dox studies yet the low dose hormetic effect of Dox on hiPSC proliferation and differentiation is not well understood. We assess the hypothesis that low-dose Dox preconditioning of hiPSCs enhances pluripotency gene expression while maintaining pluripotency state. To determine the low-dose effect (50 nM), we treated the JHU001 hiPSC line with Dox (hiPSC-D50) for two hours and a vehicle control without Dox (hiPSC-D0). Both hiPSC-D50 and hiPSC-D0 were maintained in E8 flex medium culture until reaching 70% confluency and were passaged five times and frozen for experimental studies. hiPSC-D50 and -D0 were thawed at passage 5 and cultured on coverslips for pluripotent gene expression (Oct-4, Nanog, and SSEA-4) analysis by immunocytochemistry. The cells were imaged using epifluorescence Nikon Eclipse Ti2 microscopy and the images were analyzed using NIS-Elements BR software. Phase-contrast microscopy shows significant cell death in hiPSC-D50 24 hours post-treatment compared to hiPSC-D0. However, 72 hours post-Dox-treatment, several colonies with pluripotent morphology were observed in hiPSC-D50 that were similar to hiPSC-D0. While some differentiated cell morphologies were observed in hiPSC-D50, these were significantly reduced after five passages. Immunofluorescent staining of Oct-4 and Nanog showed nuclear localization whereas SSEA-4 was localized on the cell surface, in both hiPSC-D0 and hiPSC-D50 cells at passage five. However, immunocytochemistry analysis of pluripotent markers revealed significantly higher expression of Oct-4, Nanog and SSEA-4 in hiPSC-D50 when compared to hiPSC-D0 (1.3-fold,1.9-fold and 2.9-fold increase with p-values: 0.00057, 0.018, and 0.0000026, respectively; n= 5) suggesting a more robust pluripotent state and self-renewal capacity. Interestingly, higher SSEA-4 expression might also be associated with resistance to Dox. Our data suggest that low-dose Dox preconditioning enhances pluripotency state. This Dox-induced hormesis model of hiPSCs is a unique platform to help understand how Dox upregulate pluripotency markers and the potential link between SSEA-4 and drug resistance.