Triiodothyronine t3

Primary Neuron Basal Medium (PNBM) and all supplements were purchased from Lonza (Walkersville, MD, USA). Triiodothyronine (T3) and bicuculline were purchased from Sigma Aldrich (St. Louis, MO, USA).

Derivation of NPCs was performed as described [30 (link), 60 (link)] (also described in Supplementary Methods). Briefly, NPCs were cultured in suspension as neurospheres in Advanced DMEM/F12 containing: 1% N-2 supplement, 1% B27 supplement, 1% Anti-anti, 1% GlutaMAX (all from Life Technologies), FGF2 20 ng/ml (PeproTech), PDGFα 20 ng/ml (PeproTech), purmorphamine 1 μM (Calbiochem), SAG 1 μM (Calbiochem), IGF-1 10 ng/ml (PeproTech), and Tri-iodothyronine (T3) 30 ng/ml (Sigma) for 6–12 weeks. Following papain dissociation of neurospheres (Worthington), cells were plated on 1/100 diluted Matrigel (BD Biosciences), 20 µg/ml Fibronectin (Sigma), and 10 µg/ml Laminin (Sigma) coated coverslips or plates at a density of 20,000–30,000 cells per 0.3 cm². Differentiation of oligodendrocytes was achieved by mitogen withdrawal (except for T3 and IGF-1).

Preadipocytes were cultured and differentiated as reported before [22 (link)]. Preadipocytes from passage 1 were thawed at 37°C and expanded into a T-75 flask using preadipocytes medium. Upon reaching 70% confluence, the cells were trypsinized and seeded again into a 12 well plate at density 15,000 cells/cm (passage 2) using preadipocytes media. After reaching confluence, adipogenesis was induced by adding a differentiation cocktail DMEM-F12, 1% PEST, 100 nM insulin, 17 µM pantothenate (Sigma), 33 µM biotin (Sigma), 1 µM dexamethasone (Sigma), 1 µM rosiglitazone (Sigma), 250 µM 3-isobutyl-1-methylxanthine (IBMX, Sigma), 10 µg/ml transferrin (Sigma), 2 nM triiodothyronine (T3, Sigma) for 5 days, changing the medium on day 3. The differentiation continued using maintenance media (composition as that of differentiation cocktail except for omitting IBMX) for 14 days to obtain mature adipocytes. Medium was replenished every 2–3 days. CABLES1 gene expression was measured in cells collected upon confluence (day 0) and days 2, 4, 8 and 14 post induction for temporal profiling, or, for gene editing experiments (see below), on days 0, 7 and 14 for gene edited cells.

HSE was constructed according to our previous study with some modification [16 (link)]. Briefly, adult fibroblasts and keratinocytes were isolated from discarded skin (under patients’ consent and approved by the Monash Ethics Committee). Fibroblasts were expanded in single layer dermal templates, Integra^® and BTM (with no sealing film), in DMEM with bovine calf serum (10%, Sigma, St. Louis, MI, USA) and gentamicin (50 µg/mL, Life Technologies, Carlsbad, CA, USA) 4–7 days. Dermal templates were soaked in human plasma (20–25 mg/mL) and CaCl₂ (1%) for 30 min at 37 °C prior to seeding keratinocytes. The HSEs were allowed to expand and stratify in Green’s media: DMEM: F12 media (3:1) (Life Technologies, Carlsbad, CA, USA) supplemented with L-glutamine (4 mM, Life Technologies, Carlsbad, CA, USA), adenine (0.18 mM, Calbiochem, San Diego, CA, USA), hydrocortisone (0.4 ug/mL, Calbiochem, San Diego, CA, USA), triiodothyronine (T3)( 2 × 10⁻⁹ M, Sigma), insulin (5 ug/mL, Sigma, St. Louis, MI, USA), transferrin (TRF) (5 ug/mL, Sigma, St. Louis, MI, USA), Epidermal Growth Factor (EGF) (10 ng/mL, R&D Systems, Minneapolis, MI, USA), foetal calf serum (10%, Thermo Fisher Scientific, Waltham, MA, USA), and gentamicin (50 µg/mL, Life Technologies, Carlsbad, CA, USA) 10 to 14 days prior to grafting (n = 5–6 mice per group).

Media (DMEM/F12, MEM, Neurobasal medium), GlutaMAX^TM, StemPro^® NSC SFM, B27 supplement, N2 supplement, poly-L-ornithine (PLO), and Lipofectamine 2000 were purchased from Invitrogen. Horse serum (HS) was obtained from HyClone Laboratories. Apotransferrin, biotin, bovine serum albumin (BSA), 5-bromo-2′-deoxyuridine (BrdU), cytosine arabinoside (Ara-C), diethylpyrocarbonate (DEPC), hydrocortisone, insulin, N-acetyl-cysteine (NAC), poly-D-lysine (PDL), sodium ampicillin, sodium pyruvate, sodium selenite, triiodothyronine (T3), and lysolecithin were obtained from Sigma. Ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and platelet derived growth factor-AA (PDGF-AA) were obtained from ProSpec. The antibodies used in this study are listed in Table 1.