Male C57BL/6 mice (CLEA Japan, Tokyo, Japan) were used. Male and female Aldh1l1-EGFP mice (B6;FVB-Tg(Aldh1l1-EGFP/Rpl10a)JD130Htz/J) [17] were purchased from The Jackson laboratory. All mice used were 8–10 weeks of age at the start of each experiment and were housed at 22 ± 1 °C with a 12-h light-dark cycle. All animals were fed food and water ad libitum. All animals were housed in standard polycarbonate cages in groups of same-sex littermates. All animal experiments were conducted according to relevant national and international guidelines contained in the ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan) and ‘Regulation of Laboratory Animals’ (Kyushu University) and under the protocols approved by the Institutional Animal Care and Use committee review panels at Kyushu University.

Mice were deeply anesthetized with intraperitoneal (i.p.) injection of pentobarbital and transcardially perfused with phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde (PFA)/PBS. The transverse fourth lumbar (L4) segments of spinal cord was removed, postfixed in the same fixative for 3 h at 4°C. According to a method of our previous paper [12], transverse sections of L4 spinal cord (30 µm) were incubated for 48 h at 4°C with primary antibodies for ACSA-2-PE (1:50, 130-116-244, Miltenyi Biotec, Bergisch-Gladbach, Germany), IgG-PE (1:50, 130-113-450, Miltenyi Biotec, Bergisch-Gladbach, Germany), polyclonal goat anti-SOX9 (1:2000, R&D Systems, MN, USA), polyclonal goat anti-SOX10 (1:500, AF2864, R&D Systems, MN, USA), monoclonal rabbit anti-NeuN (1:2000, ab17748, abcam, Cambridg, UK), polyclonal guinea pig anti-IBA1 (1:2000, 234 00, Synaptic Systems, Goettingen, Germany) and monoclonal rat anti-galectin-3 (1:500, CL8942AP, Cedarlane Labs, Burlington, Canada). Tissue sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 (1:1000, A-21208, Thermo Fisher), Alexa Fluor 546 (1:1000, A-11056, Thermo Fisher) or DyLight 405 (1:1000, 711-475-152 or 706-475-148, Jackson ImmunoResearch) and mounted with or without 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). Three sections from one tissue were randomly selected and images were taken using an LSM700 Imaging System (Zen 2012, Carl Zeiss) and BZ-X800 fluorescence microscope (Keyence Corporation, Osaka, Japan).

Mice were deeply anesthetized by i.p. injection of pentobarbital and perfused transcardially with ice-cold PBS. The L3/4 spinal cord was rapidly removed from the vertebral column and placed into ice-cold Hanks’ balanced salt solution (HBSS) without Ca2+, Mg2+ (HBSS (-)). The dura and pia matter and dorsal/ventral roots were carefully removed from the spinal cord, and spinal cord tissue was cut into 8 pieces. The spinal tissue pieces were treated with ice-cold 1 mL enzymatic solution [10 mg/mL of protease from Bacillus licheniformis enzyme (Sigma-Aldrich, P5380) with 125 units/mL of DNaseI (Worthington Biochemical Corporation) and 5 mM CaCl2] in HBSS (-) for 10 or 30 min at 4 °C. The tissues were homogenized by passing through a 23G needle attached with 1 mL syringe and were further incubated for 10 or 30 min at 4 °C. After that, the tissues were homogenized by passing through a 26G needle, and the enzymatic reaction was stopped by HBSS with Ca2+, Mg2+. After centrifugation (1000 × g, 5 min, 4 °C), the cells were resuspended in HBSS (-) with 0.5% BSA, 2mM EDTA and 10 or 20 µL Myelin Removal Beads II (Milteny Biotec) for 15 min at 4 °C to remove myelin debris and oligodendrocytes from the suspension. In the case of the hemi-section of the L3/4 spinal cord, the tissue pieces were treated with ice-cold 0.5 mL enzymatic solution for 20, 40 or 60 min at 4 °C and the cell suspension was incubated with 5, 7.5 or 10 µL Myelin Removal Beads II. After centrifugation (300 × g, 10 min, 4 °C), the cells were resuspended in HBSS (-) with 0.5% BSA and 2mM EDTA, and passed through a magnetic cell sorting (MACS) LS column (Milteny Biotec) according to the manufacturer’s protocol at 4 °C. After centrifugation (300 × g, 10 min, 4 °C), the cell suspension was blocked by incubating with Fc Block (1:200, 553142, BD Biosciences) for 5 min and immunostained with IgG-PE (1:200, 130-113-450, Miltenyi Biotec) or ACSA2-PE (1:200, 130-116-244, Miltenyi Biotec) for 10 min at 4 °C in the dark. After washing, the cell suspension was treated with 7-aminoactinomycin D (7-AAD; 1:40, 00-6993-50, eBioscience) and DRAQ5 (1:666, 130-117-343, Milteny Biotec) and incubated for at least 15 min on ice for viability and targeting cells with nucleus staining prior to cell sorting. 7-AADneg DRAQ5+ ACSA-2+ cells were gated as live cells and sorted depending on ACSA-2 fluorescent intensity using cytoFLEX SRT (Beckman coulter, CA, USA).

The sorted cells were subjected total RNA extraction using the Quick-RNA Micro-Prep kit according to the manufacturer’s protocol (Zymo Research, CA, USA). The extracted RNA from the sorted cells was transferred to reverse transcriptional reaction with Prime Script reverse transcriptase (Takara, Japan). qPCR was performed with Premix Ex Taq (Takara, Japan) using QuantStudio3 (Applied Biosystems, MA, USA). Expression levels were normalized to the value for Gapdh and values below detection level were regarded as zero. The following primers and probes were obtained from Integrated DNA Technologies (IA, USA): Sox9 (NM_011448; SRY (sex determining region Y)-box 9), Sox10 (NM_011437; SRY (sex determining region Y)-box 10), Mog (NM_010814; myelin oligodendrocyte glycoprotein), Itgam (NM_001082960; integrin subunit alpha M), Rbfox3 (NM_001039168; RNA binding protein, fox-1 homolog (C. elegans) 3), Pecam1 (NM_001032378; platelet/endothelial cell adhesion molecule 1), Cspg4 (NM_139001; chondroitin sulfate proteoglycan 4), Aqp4 (NM_009700; aquaporin 4), Slc7a10 (NM_017394; solute carrier family 7 (cationic amino acid transporter, y+ system), member 10), Hes5 (NM_010419; hairy and enhancer of split 5) and Dynlrb2 (NM_029297; dynein light chain roadblock-type 2). The sequences of TaqMan primer pairs and probe were described below.

Gapdh: 5′-FAM-ACCACCAACTGCTTAGCCCCCCTG-TAMRA-3′ (probe), 5′-TGCCCCCATGTTTGTGATG-3′ (forward primer), 5′-GGCATGGACTGTGGTCATGA-3′ (reverse primer).

Lgals3: 5’-FAM-CCCGCTGGACCACTGACGGTG-TAMRA-3’ (probe), 5’-TCCTGCTGCTGGCCCTTAT-3’ (forward primer), 5’-TTCACTGTGCCCATGATTGTG-3’ (reverse primer).

Quantitative data were expressed as the means ± SEM. Data were analyzed by one-way analysis of variance (ANOVA) with the post hoc Tukey’s multiple comparisons test (Fig. 4a, b and Additional file 1: Supplementary Fig. 3a), Kruskal–Wallis test with the post hoc Dunn’s multiple comparisons test (Additional file 1: Supplementary Fig. 3b), as appropriate, after determining the normality (Shapiro–Wilk test) and variance (F-test or Brown–Forsythe test) of the experimental data. Statistical analyses were performed using Prism 7 (GraphPad, CA, USA). Values were considered significantly different at P < 0.05.