Figure 2.
Ectopic Atoh1 expression drives neuronal conversion in induced pluripotent stem cells (iPSCs). (A): Diagram of the lentiviral vector for Dox-inducible Atoh1 expression. (B): Dox controls the on/off switch of Atoh1 expression. Human iPSCs were infected with lentivirus harboring Dox-inducible Atoh1. Stable Atoh1-iPSCs after puromycin selection were treated with or without Dox for 48 hours and transferred to Dox-free medium. Whole cell lysates collected on each indicated time point were subjected to immunoblot using anti-FLAG antibody. (C): Atoh1-iPSCs were treated with Dox (+Dox) for 3 days and changed to Dox-free medium (Dox withdrawal) for 3 days. Cells were immunostained with FLAG antibody. (D): Diagram of Atoh1-induced neuron differentiation protocol. Atoh1 is induced by Dox from days 1 to 5. (E): Immunostaining from cell cultures at differentiation day 6 shows TUJ1 expression in Atoh1-induced cells but not in control cells. (F): Bright-field microscope images show cell adhesion and neuronal process formation in Atoh1-induced cells on differentiation day 10. (G): Immunostaining shows the coexpression of TUJ1 and Synapsin in Atoh1-induced neurons on differentiation day 36. (H, I): During a 5-day time period, an equal number of Atoh1-iPSCs received different lengths of Dox treatment (from 1 to 5 days). After being matured for 30 days, cells were immunostained against neuronal marker TUJ1 and MAP2. The percentage of TUJ1+/MAP2+ cells over DAPI+ cells and the total number of TUJ1+/MAP2+ cells were quantified in 10 random-selected microscopic fields (∗, p < .01 compared with cells that had 4- and 5-day Atoh1 induction). (J, K): Atoh1-iPSCs were treated with Dox for 2 days and returned to Dox-free medium for 3 days (J). The expression of Atoh1, NEUROD1, and Ngn2 was measured by quantitative real-time polymerase chain reaction in control, Atoh1 induction, and Atoh1 silencing samples (∗, p < .01 compared with control). In (C) and (E–G), cell nuclei were counterstained with DAPI. Scale bars = 20 μm. The data represent means ± SEM. Abbreviations: Con, control; DAPI, 4′,6-diamidino-2-phenylindole; Dox, doxycycline; FLAG-Atoh1, FLAG-tagged Atoh1; IRES, internal ribosome entry site; Puror, puromycin selection marker; rtTA3, reverse tet-transactivator; TRE, tet-inducible promoter; UBC, human ubiquitin C promoter.