Valproic acid

Astrocytes were grown to confluence before reprogramming. Cells were cultured on poly-L-lysine (PLO)/laminin substrates and media was changed to high glucose Dulbecco's Modified Eagle Medium (DMEM) with 2 mM Glutamax (Gibco), 1% N2/B27 (ThermoFisher Scientific), 1% Human Serum Replacement 3 (Sigma Aldrich), and 1% penicillin/streptomycin (ThermoFisher Scientific) for cellular reprogramming experiments. 15 μg/mL ASCL-IPTD (iProgen Biotech) was supplemented for all 12 days, with 50% media changes every 2 days. For days 1-2, 5μM SB431542 (Stemcell Technologies) and 0.25 μM LDN193189 (Stemcell Technologies) were added to cultures. For days 3–12 the following small molecules were added: forskolin (F, 10 μM, Medchem Express), CHIR99021 (C, 1.5 μM, Medchem Express), ISX9 (I, 20 μM, Medchem Express), DAPT (D, 5 μM, Medchem Express). The combinations tested were C, F, D, I, CF, CI, CD, FI, FD, ID, FCI, FCD, CID, FID, CFID. For days 3–6, 0.5 μM valproic acid (Stemcell Technologies) was also added to improve chromatin accessibility.

Young (4-month-old) and old (20-month-old) male C57BL/6 mice were acquired from the National Institute on Aging (NIA) aged rodent colonies (Charles River Laboratories, Wilmington, MA) and were fed standard 5053 chow. HEK293T cells were acquired from ATCC (Manassas, VA) and were verified by PCR to be free of mycoplasma contamination. Cell culture reagents were acquired from Thermo Fisher Scientific (Waltham, MA), reagents for isolating DNA and RNA were from Qiagen (Hilden, Germany) and Zymo Research (Irvine, CA), respectively, and general preparatory chemicals and reagents were from Sigma-Aldrich (St. Louis, MO). Chemical reprogramming reagents were obtained from the following suppliers: Sigma-Aldrich (repsox, trans-2-Phenylcyclopropylamine, DZNep), Cayman Chemical Company (TTNPB, CHIR99021), Tocris Bioscience (forskolin), and STEMCELL Technologies (valproic acid). Tetramethylrhodamine, methyl ester perchlorate (TMRM) was obtained from MedChem Express (Monmouth Junction, NJ). Antibodies were obtained from Abcam (Cambridge, United Kingdom). All experiments using mice were performed in accordance with institutional guidelines for the use of laboratory animals and were approved by the Brigham and Women’s Hospital and Harvard Medical School Institutional Animal Care and Use Committees under Protocol #2016N000368.

Neural differentiation was performed using a modified version of the dual SMAD inhibition protocol [49 (link)]. Briefly, hi-PSCs in the presence of small molecules: ROCK inhibitor, SMAD inhibitors (SB431542 and DMH1) for 6 days, SB431542 (Tocris), DMH1 (Tocris), RA and Purmorphamine (Tocris) for another 6 days. At that point, all the hi-PSC lines generated more than 90% OLIG2 + Motor Neuron Progenitor Cells. Afterwards, the cells were expanded in the same media, supplemented with valproic acid (STEMCELL Technologies).
To induce MN differentiation, OLIG2 + MNPs were dissociated and cultured at a density of 1:6 in suspension in neural medium with RA and Purmorphamine. The medium was changed every other day for 6 days. Next, they were dissociated into single cells and plated on Matrigel-coated plates and cultured with RA, Purmorphamine and Compound E (γ-Secretase-IN-1, Sigma) for 10 days, after that, the media was replaced with neuronal media (neurobasal media supplemented with 1% of B27, BDNF 10ng/mL, CNTF 10ng/mL and IGF 10ng/mL). The cells were then fed on alternate days with neuronal medium until day 40 to mature into CHAT + MNs.
Cell lines relevant clinical information is described in Supplementary Table 1.