Complete dmem f12 medium

Choosing the cerebral cortex of mice (1-3 days old) to extract primary astrocytes, the cortex was digested by 0.125% trypsin-EDTA (Gibco, Grand Island, NY, USA), then centrifuged and incubated in DMEM/F12 complete medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 9-11 days, the cells were shaken at 260 rpm at 37° C for 16 h to purify. Finally, purified astrocytes were obtained.
Choosing the cortex of fetal mice (16–18 days old) to isolate primary neurons, the culture flasks were pretreated with poly-d-lysine (PDL) (Sigma-Aldrich, St. Louis, MO, USA). The fragment was digested with 0.125% trypsin and grown with DMEM (Gibco, Grand Island, NY, USA) for 5 h, then replaced with neurobasal medium containing 2% B27 (Gibco, Grand Island, NY, USA) and 0.5 mmol/L glutamine (Sigma-Aldrich, St. Louis, MO, USA).
Choosing Adult Brain Dissociation Kit and ACSA-2 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for magnetic isolation of astrocytes from adult mice. The former was used to digest the cortex. Then, purified astrocytes were obtained. Astrocytes were incubated for 15 min at 4° C with ACSA-2 MicroBeads and separated from single-cell suspension in a magnetic field using MS columns, MACS MultiStand and QuadroMACS (Miltenyi Biotec, Bergisch Gladbach, Germany).

BMSCs were cultured with Ficoll Hypaque density gradient centrifugation. Resuspended bone marrow mononuclear cells were placed into T25 flasks, the cell number was adjusted to 10⁶ cells/mL, and cells were incubated at 37 °C and 5% carbon dioxide. The culture medium for BMSCs comprised Dulbecco's modified eagle medium (DMEM) with 15% fetal bovine serum (FBS) (Gibco), 100 U/mL of penicillin and streptomycin (Solarbio) in DMEM/F-12 complete medium (Gibco). BMSCs were incubated in the culture medium for 72 h, followed by a half volume change to remove non-adherent cells, after which the medium was changed every 3–4 days until the appearance of adherent cells arranged in a fusiform radial or swirling pattern and the medium change was completed. When the cell confluence reached 80–90%, the cells were digested with 0.25% trypsin (Gibco) and passaged. P3 and fourth generation (P4) BMSCs were used in the follow-up experiments.

Constructs were created using the Gateway® recombination technology (Life Technologies). The open reading frame of YARS1 was amplified by PCR using specific primers to allow insertion of the product in a pcDNA5/FRT (Invitrogen) vector. YARS construct was tagged at its C-terminal end with ProtA-TEV-Flag tags. The YARS1^CMT mutations (p.E196K and p.G41R) were generated by site-directed mutagenesis. All constructs were validated by Sanger sequencing and transferred by recombination to a pLenti6 destination vector (Life Technologies). Stable cell lines were generated by lentiviral transduction of SH-SY5Y human neuroblastoma cells (purchased from ATCC, CRL 2266) as described in ref. ⁶⁴ . SH-SY5Y cell lines were cultured at 37 °C and 5% CO₂ in a humidified atmosphere in DMEM/F-12 complete medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco).