Aria 1 flow cytometer

Neurospheres consisting of ∼30% CD133+ NSCs and ∼70% CD133- neural progenitor cells (NPCs) [23] (link), [26] (link), were dissociated using a trypsin-based neural tissue dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were centrifuged (400×g) and resuspended in FACS buffer (PBS supplemented with 0.5% bovine serum albumin (BSA) (Sigma) and 2 mM ethylene diamine triacetic acid (EDTA) (Sigma). Cells were blocked with fragment crystallisable region (FcR) (Miltenyi Biotec) for three min at room temperature and incubated with AC133/1-Phycoerythrin (PE) and CD133/2(C293)-PE antibodies (Miltenyi Biotec) on ice for 20 min. As negative controls, cells were incubated with IgG_2b-PE (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at the same concentration, or antibodies were omitted. Excess antibody was removed by washing in FACS buffer, and samples were isolated using an ARIA-I flow cytometer (BD Biosciences, San Jose, CA, USA). CD133⁺ NSCs were isolated from neurosphere cell lines MEL1, hES3, and hES4, at 90% or greater purity (Table S1). The purity of CD133^- NPCs derived from the same cell lines was consistently greater than 99%. The expression of PROM1 (p<0.05) and NES (p<0.01) was significantly higher in the CD133+ compared to the CD133-depleted samples consistent with successful enrichment of CD133⁺ NSCs (data not shown).

Cells were seeded in 10 cm dishes at 2 × 10⁵ cells/mL, followed by drug treatment. At the end of the drug incubation period, cell cultures were transferred to 15-mL conical tubes and gently centrifuged for 5 min. The cell pellets were resuspended in 1 mL of fresh complete medium and incubated for 40 min at 37 °C with either an APC-labeled human CD11b (clone ICRF44) antibody or an APC-labeled IgG1, κ (clone MOPC-21) isotype control (BD Biosciences, Franklin Lakes, NJ, USA) per manufacturer’s recommendations. At the end of the incubation time, the cells were pelleted again and resuspended in ice-cold PBS supplemented with 10% FBS. FACS analysis was performed on an Aria I flow cytometer (BD Biosciences) equipped with four laser lines, including a red (633 nm) laser.

Sox9⁺ cells were isolated from digits of E13.5 Sox9-GFP embryos (Nakamura et al., 2011 (link)). Lgr5⁺ and surrounding non-Lgr5 (Lgr5⁻) cells were isolated from forelimb digit interzones of E14.5 Lgr5^GFP/+ mice (Figures S3A–S3C). Cells were released with a mixture of TrypLE Express (Gibco) and 0.1% DNase I for 20 min, filtered through a 40 μm cell strainer, and sorted by FACS using an Aria I flow cytometer (BD Biosciences). Total RNA was extracted using a mirVana miRNA Isolation Kit (Ambion) and cDNA libraries were constructed with 10 ng of RNA using a SMARTer Ultra Low Input Kit (v3, Illumina), and sequenced using the Illumina HiSeq 1500 platform (Center for Genomic Sciences, The University of Hong Kong). cDNA fragment sequences were aligned to mouse genome (mm10) using the HISAT program (Kim et al., 2015 (link)). FPKM values were generated for comparison (Figure S3D). Genes with FPKM ≥ 5 were considered as expressing genes. Pathway and expression analyses were performed with DAVID (Huang da et al., 2009 (link)) and Eurexpress (Diez-Roux et al., 2011 (link)) databases, respectively. Datasets have been uploaded to the Gene Expression Omnibus for public access (GEO: GSE110281).