Generating Transgenic iPSC Lines With Inducible Transcription Factors

The iPSCs were engineered to contain a 13-kb vector containing the human transcription factors NGN2, ISL1, and LHX3 (hNIL) under the control of doxycycline (Fernandopulle et al., 2018). To knock-in the hNIL construct into safe harbor loci, 1 × 10⁶ cells were added to 100 μL P3 buffer with 6 μg of the hNIL donor plasmid, and 3 μg of each paired TALEN (Addgene, 59025/59026 or 62196/62197, respectively) targeting either AAVS1 or CLYBL (Cerbini et al., 2015). Cells were transfected using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, V4XP-3024) with pulse code DS138. After nucleofection, cells incubated for 5 min at room temperature and were then plated in Stemfit media with 10-μM Y-27632 in a serial dilution into five wells of a six-well plate. Non-transfected cells were plated in the sixth well. After 2–3 days post-nucleofection, cells were fed with Stemfit with Puromycin or G418 (depending on which donor vector was used) and 10-μM Y-27632. Cells were kept on antibiotic selection until all cells in the control wells were dead and only red fluorescent colonies remained in the experimental wells. Fluorescent, antibiotic-resistant colonies were then manually picked and transferred to individual wells of a 48-well plate.

DNA from each clone was extracted using the DNeasy Blood and Tissue Kit (Qiagen, 69506). PCRs were performed with primers spanning the 5′ and 3′ junctions of the integration, with one primer annealing within the construct and the other outside of the corresponding homology arm. A third PCR amplifying the intact safe harbor locus was performed to determine whether the clone harbored a heterozygous or homozygous insertion. PCR primer sequences are listed in Supplementary Table 1. For the CMT2E line, we inserted hNIL in the AAVS1 locus (Supplementary Figure 1) and obtained a previously published WTC line with hNIL inserted in the AAVS1 locus as a control (Shi et al., 2018). We also independently inserted hNIL in the CLYBL locus of the same parental WTC line (Supplementary Figure 1). When performing neuronal differentiation, we found equivalent results with the AAVS1 and CLYBL versions of the WT line. For comparison with the N98S-hNIL-AAVS1 line, we used WTC-hNIL-AAVS1 for all experiments, with the exception of manual image analysis shown in Figure 2, in which case we used the WTC-hNIL-CLYBL line.

Pathologic accumulation of NF-L protein in the cell body of NEFL-N98S motor neurons. (A,B) Representative images of day-7 WT (A) and N98S (B) i³LMNs stained with anti-NF-L (green) and anti-HB9 (blue) antibodies. Boxed regions are shown in larger scale to the right of each image with manual cell body segmentation superimposed. Scale bars = 25 μm. (C) Distribution of NF-L fluorescence intensity in individual HB9+ cell bodies after manual segmentation. Individual data points are shown with horizontal lines representing median and interquartile range. (D) Quantification of mean NF-L fluorescence intensity from four biological replicates per cell line, demonstrating significantly increased intensity in N98S neurons. Points represent mean of >160 neurons quantified in each sample population, bars represent mean of all replicates ± S.E.M., ***p < 0.001 by t-test.