Establishment of a β1-tubulin–Venus imMKCL reporter line to fluorescently monitor MK maturation. (A) Schematic representation of MK maturation. The increased expression level of TUBB1 is induced in the terminal stage of MK maturation. (B) The mRNA expression of several markers of MK maturation. Samples were obtained from imMKCLs (Cl-7 clone; 2 × 105 cells) incubated in the absence or presence of SR1 (0.75 μM) on day 4 (DOX-Off stage). Gene expression levels were measured using quantitative real-time reverse transcription polymerase chain reaction and normalized to GAPDH expression. (C) Schematic illustration of the establishment of a β1-tubulin–Venus reporter line from secondary imMKCL-derived iPSCs. The imMKCL-derived secondary iPSCs were used to establish the reporter cell line with a green (Venus) fluorescent transgene, followed by redifferentiation into the imMKCL state. (D) CRISPR/Cas9-mediated knock-in strategy to generate β1-tubulin–Venus imMKCLs. A self-cleaving 2A peptide with a green (Venus) transgene was inserted downstream of exon 4. (E) The mRNA expression of TUBB1 increased upon MK maturation in β1-tubulin–Venus imMKCLs. Samples were obtained from β1-tubulin–Venus imMKCLs (2 × 105 cells) on the indicated days. (F) Venus intensity and CD42b+ population were increased upon the maturation of β1-tubulin–Venus imMKCLs. The Venus fluorescence intensity was assessed using an ArrayScan over 7 days. The CD42b+ population was detected using flow cytometry. (G) Effect of SR1 on PLP yield from β1-tubulin–Venus imMKCLs. Cells (2 × 105) were incubated in the absence or presence of SR1 (0.75 μM) for 7 days, and CD41+CD42b+ PLPs were detected and counted using flow cytometry. Data are expressed as the mean ± SD from 3 independent experiments. **P < .01 vs Vehicle, Student t test.
