Cytotune 2

For iPS induction, MEFs were seeded per well of a six-well plate and 12 h later overnight infections were performed using equal amounts of each indicated retrovirus in the presence of 8 μg ml⁻¹ polybrene (Sigma, 107689). The day after, wells were rinsed twice and cells were cultivated in medium supplemented or not with 150 or 750 ng ml⁻¹ recombinant Netrin-1 (rNetrin-1). Forty-eight hours after infection, MEFs were reseeded onto irradiated feeders in iPS media. Media were replaced every day with medium alone or freshly supplemented with rNetrin-1. Emerging iPS colonies were monitored until days 12–14 when cells were harvested or individual colonies picked for further analysis. Similar protocol was employed with RNA sendai virus (cytotune 2.0, Life Technologies). The dox-inducible mice were purchased from the Jackson Laboratories (stock number 011001). MEFs, adult fibroblasts from ear and tail tip and intestinal epithelium were plated on irradiated feeders and treated with doxycycline at 2 μg ml⁻¹. Recombinant Netrin-1 was purchased from Adipogen (AB40B-0075). Because of the poor stability of the recombinant Netrin-1 molecule and variability between batches, batch activity was systematically tested in cell death assays using UNC5b-transfected cells. The blocking Netrin-1 antibody was obtained from the Netris Pharma company.

All cultures were at 37°C at 5% CO₂ in a humidified atmosphere. Unless otherwise stated, all reagents were from ThermoFisher. HUES7 hESCs were gifted by Chad Cowan and Doug Melton at the Harvard Stem Cell Institute. Fibroblasts were derived under ethical consent from individual with the genotypes RYR2^6739C/T (NRES Committee East Midlands–Nottingham 2 approval 09/H0408/74) and ACTC1^301G/G (Biomedical Institute of A Coruna, INIBIC). Reprogramming to hiPSCs was via CytoTune 2.0 (ThermoFisher), according to the manufacturer's instructions. Culture was in E8 medium on Matrigel, although processes could also be completed in hESC medium conditioned using mouse embryonic fibroblasts [20 (link)]. In the first 4–5 passages after reprogramming, cell harvesting was done using 0.5 mM EDTA and thereafter with accutase.

Cell services were performed using the Harvard Stem Cell Core: whole blood was reprogrammed via Cytotune 2.0 (Thermo Fisher), and colonies were allowed to grow and were then assessed for the iPS markers SSEA, Oct4, Tra-1-60, and Nanog using immunocytochemistry and qPCR for trilineage. Successful screened colonies were then processed further for genetic editing. Guide RNA (gRNA) and single-stranded oligodeoxynucleotide (ssODN) sequences were determined (Supplementary Table 1) using the CRISPOR suite through the highest specificity score and lowest off-target homology, and then, colonies were karyotyped. Normal karyotype colonies were sequenced for their inclusion of the desired genetic mutation, and isogenic controls were also selected (Supplementary Figure 2).