Construction and characterization of inducible cell lines
(A) Schematic of PiggyBac transposon-based inducible overexpression vector.
(B) Schematic of cell line generation and validation process.
(C) Inducible NEUROD1 (iN) cell line validation at 3 weeks post induction via (1) qRT-PCR analysis of signature neuronal markers MAP2, TUBB3, VGLUT2, and VGAT; data represent the mean ± SD (n = 4 independent experiments); (2) immunofluorescence micrograph of MAP2+ cells (scale bars, 50 μm); and (3) representative spike plots from MEA measurements of spontaneously firing iN cells.
(D) Inducible ASCL1+DLX2 (iAD) cell line validation at 3 weeks post induction via (1) qRT-PCR analysis of signature neuronal markers MAP2, TUBB3, VGLUT2, and VGAT; data represent the mean ± SD (n = 4 independent experiments); (2) immunofluorescence micrograph of MAP2+ cells (scale bars, 50 μm); and (3) representative spike plots from MEA measurements of spontaneously firing iAD cells.
(E) Inducible MYOD1+BAF60C (iMB) cell line validation at 2 weeks post induction via (1) qRT-PCR analysis of signature skeletal muscle markers MYH8, TNNC1, and RYR; data represent the mean ± SD (n = 3 independent experiments); and (2) immunofluorescence micrograph of MYH+-, MYOG+-, and SAA+-labeled cells (scale bars, 50 μm). (C–E) ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001; ns, not significant.