Analysis was performed with the statistical software package Prism Origin (GraphPad Software, La Jolla, USA). Statistical analysis of iMN survival experiments was performed using a two-sided log-rank test to account for events that did not occur (i.e. iMNs that did not degenerate before the end of the experiment). For each line, the survival data from 50 iMNs were selected randomly using Microsoft Excel, and these data were used to generate the survival curve. If all iMNs degenerated in a given experiment, statistical significance was calculated using a two-tailed Student’s t-test. For all other experiments, differences between two groups were analyzed using a two-tailed Student’s t-test, unless the data was non-normally distributed for which two-sided Mann-Whitney testing was used. Differences between more than two groups were analyzed by one way-ANOVA with Tukey correction for multiple testing. Significance was assumed at p < 0.05. Error bars represent the standard deviation unless otherwise stated.
To determine if the survival difference between C9ORF72 patient iMNs and controls was specific to our transcription factor-based reprogramming approach, we also measured the survival of Hb9::RFP+ control and C9ORF72 patient motor neurons derived from iPSCs by small molecule activation of the Sonic Hedgehog and retinoic acid signaling pathways 28 (Supplementary Fig. 3g, h). Similarly to iMNs, morphogen-generated motor neurons showed a significant survival difference between C9ORF72 patients and controls (Supplementary Fig. 3i-l).
Yingxiao Shi,#1,2,3 Shaoyu Lin,#1,2,3 Kim A. Staats,1,2,3 Yichen Li,1,2,3 Wen-Hsuan Chang,1,2,3 Shu-Ting Hung,1,2,3 Eric Hendricks,1,2,3 Gabriel R. Linares,1,2,3 Yaoming Wang,3,4 Esther Y. Son,5 Xinmei Wen,6 Kassandra Kisler,3,4 Brent Wilkinson,3 Louise Menendez,1,2,3 Tohru Sugawara,1,2,3 Phillip Woolwine,1,2,3 Mickey Huang,1,2,3 Michael J. Cowan,1,2,3 Brandon Ge,1,2,3 Nicole Koutsodendris,1,2,3 Kaitlin P. Sandor,1,2,3 Jacob Komberg,1,2,3 Vamshidhar R. Vangoor,7 Ketharini Senthilkumar,7 Valerie Hennes,1,2,3 Carina Seah,1,2,3 Amy R. Nelson,3,4 Tze-Yuan Cheng,8 Shih-Jong J. Lee,8 Paul R. August,9 Jason A. Chen,10 Nicholas Wisniewski,10 Hanson-Smith Victor,10 T. Grant Belgard,10 Alice Zhang,10 Marcelo Coba,3,11 Chris Grunseich,12 Michael E. Ward,12 Leonard H. van den Berg,13 R. Jeroen Pasterkamp,7 Davide Trotti,6 Berislav V. Zlokovic,3,4 and Justin K. Ichida1,2,3,†
Samples were first fixed in 4% PFA (1x PBS) overnight at 4°C and were subsequently washed three times with 1x PBS. Next, cells were permeabilized with 0.3% Triton X-100 (1x PBS) for 10 min at room temperature, followed by three washes with 1x PBS for 10 min each. After permeabilization, the samples were equilibrated in 1x SSC buffer for 10 min at room temperature and then transferred into 50% formamide (2x SSC) for 10 min at 60°C. The repeat expansion-targeting probe and the negative control probe were ordered from Exiqon 58. During this step, the probe mixture (1µl salmon sperm (10 µg/µl), 0.5 µl E. coli tRNA (20 µg/µl), 0.4 µl probe (25 µM), 25 µl 80% formamide/per sample) was made and placed at 95°C for at least 10 min. The samples were submerged in 200 µl of hybridization buffer (4ml 100% formamide, 0.5 ml 20x SSC, 1 ml BSA fraction V, 0.5ml RVC (20 mM), 1ml NaPO4 (0.1 M), 3 ml nuclease-free water) and 27 µl of the probe mixture was added to each sample and incubated for 1 hour at 60°C. After probe hybridization, the samples were washed twice with 50% formamide (2x SSC) for 20 min each at 65°C and once more with 40% formamide (1x SSC) for 10 min at 60°C. The remaining formamide was removed by briefly washing with 1x SSC three times. A final crosslinking step was performed by first incubating the samples with 1x Tris-Glycine for 5 minutes followed by a 5 min incubation in 4% PFA. Samples were washed three times with 1x PBS, stained with DAPI, and imaged using a Zeiss LSM 800 confocal microscope.
A 241-bp digoxigenin (DIG)-labeled probe was generated from 100 ng control genomic DNA (gDNA) by PCR reaction using Q5® High-Fidelity DNA Polymerase (NEB) with primers shown in Supplementary Data Table 4. Genomic DNA was harvested from control and patient iPSCs using cell lysis buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% w/v sodium dodecyl sulfate (SDS)) at 55ºC overnight and performing phenol:chloroform extraction. A total of 25 µg of gDNA was digested with XbaI at 37 ºC overnight, run on a 0.8% agarose gel, then transferred to a positive charged nylon membrane (Roche) using suction by vacuum and UV-crosslinked at 120 mJ. The membrane was pre-hybridized in 25 ml DIG EasyHyb solution (Roche) for 3 h at 47 ºC then hybridized at 47 ºC overnight in a shaking incubator, followed by two 5-min washes each in 2X Standard Sodium Citrate (SSC) and in 0.1% SDS at room temperature, and two 15-min washes in 0.1x SSC and in 0.1% SDS at 68 ºC. Detection of the hybridized probe DNA was carried out as described in DIG System User’s Guide. CDP-Star® Chemilumnescent Substrate (Sigma-Aldrich) was used for detection and the signal was developed on X-ray film (Genesee Scientific) after 20 to 40 min.
Biotinylation of plasma membrane localized glutamate receptors was performed using the Piece™ Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Dox-NIL iMNs were incubated with 0.25mg/ml Sulfo-NHS-SS-Biotin in cold room for 1~2 hrs with end-to-end shaking. After quenching, cells were harvested by scraping and lysed with lysis buffer from the Piece™ Cell Surface Protein Isolation Kit or the M-PER™ mammilian protein extraction buffer (Thermo Fisher Scientific). Cell lysate was incubated with High Capacity NeutrAvidin™ agorase beads (Thermo Fisher Scientific), and the bound protein was eluted in 2X SDS-PAGE sample buffer supplemented with 50mM DTT for 1 hr at room temperature with end-to-end rotation, and further analyzed by western blot.
To generate Dox-NIL iMNs, the Dox-NIL construct was integrated into the AAVS1 safe harbor locus of the control, C9+/−, and C9-ALS patient iPSC lines using CRISPR/Cas9 editing (gRNA sequence shown in Supplementary Table 4). Dox-NIL iMNs were generated by plating at ~25% confluency on matrigel coated plates and adding 1 μg/ml of doxycylin in N3 media +7.5 μM RepSox 1 day after plating. Mouse primary mixed glia were added to the cultures at day 6, and doxycyline was maintained throughout conversion. iMN cultures were harvested at day 17.
GCaMP6 was cloned into the pMXs-Dest-WRE retroviral vector and transduced into reprogramming cultures concurrently with the motor neuron factors. To assess GCaMP6 activity, 1.5 μm glutamate was added to iMN cultures and cells were imaged continuously for 2 minutes at 24 frames per second. GFP flashes were scored manually using the video recording. At least 3 different fields of view from three independent cultures, totalling 50–100 iMNs, were scored per condition.