Isolation and Culture of Cerebellar GNPs

For GNPs isolation, we followed the previously described protocol (Li et al., 2013 (link)), cerebella were harvested from C57BL/6J mice at P4-P7 then digested in a solution containing 10 units/ml papain (Worthington), 25 U/ml DNase I (Solarbio) and 2 mg L-cysteine (Solarbio) at 37°C for 30 min to obtain a single-cell suspension. After filtered with 70 μm strainer, cells were centrifuged through a 35%–65% Percoll gradient (GE Healthcare). Cells from the 35–65% interface were suspended in Neurobasal Medium with B27 supplement. Then GNPs were suspended in NB-B27 and plated on Poly-L-omithine hydrobromide (PLO) and Laminin (all from Sigma)-coated coverslips. GNPs was treated with 5 μM PTL, 50% sonic hedgehog conditional medium (Shh-CM) and Shh-CM adding 5 μM PTL for 24 h. EDU (Proteintech) was added 20 μM and incubated GNPs for 1 h. Then stained according to the instructions.