Quiescence of adult oligodendrocyte precursor cells requires thyroid hormone and hypoxia to activate Runx1

The adult mammalian central nervous system (CNS) contains a population of slowly dividing oligodendrocyte precursor cells (OPCs), i.e., adult OPCs, which supply new oligodendrocytes throughout the life of animal. While adult OPCs develop from rapidly dividing perinatal OPCs, the mechanisms underlying their quiescence remain unknown. Here, we show that perinatal rodent OPCs cultured with thyroid hormone (TH) under hypoxia become quiescent and acquire adult OPCs-like characteristics. The cyclin-dependent kinase inhibitor p15/INK4b plays crucial roles in the TH-dependent cell cycle deceleration in OPCs under hypoxia. Klf9 is a direct target of TH-dependent signaling. Under hypoxic conditions, hypoxia-inducible factors mediates runt-related transcription factor 1 activity to induce G1 arrest in OPCs through enhancing TH-dependent p15/INK4b expression. As adult OPCs display phenotypes of adult somatic stem cells in the CNS, the current results shed light on environmental requirements for the quiescence of adult somatic stem cells during their development from actively proliferating stem/progenitor cells.

The forward primer for Hif2α was 5'-GTAGCGGCCGCACGGGGTTAAGGAACCC -3', and the reverse primer for Hif2α was 5'-CCATCGATCCCTGGCTCAGGTGG-3'.   Gene symbols are shown in italic. Average raw data of the P7 OPCs cultured without TH in 1.5% O 2 for 15 days are shown (n = 3). 129 Genes up-regulated with TH treatment are shown in (+) fold, and 60 genes down-regulated are shown in (-) hold.
The moderate genes described in the text (11 genes) are also shown. For calculating probability (P value), Student's t test was carried out. The P value of every gene is P < 0.05 (t-test, n=3).
The TH-dependent up-regulations of the major genes described in the text were confirmed by qRT-PCR (Supplementary Figure 3a). And the TH-dependent up-regulations of protein expression of Runx1 and p15/INK4b were confirmed by western blot analysis (Supplementary Figure 3b).  Table 2 The nucleotide sequences of RT-PCR primers.
The nucleotide sequences of PCR primers are shown from 5' (left) to 3' (right) direction.
The numbers shown below each gene name are the length of predicted PCR products (bp). FW; forward primer, REV; reverse primer.  Table 3 The nucleotide sequences of qRT-PCR primers.

Primers for qRT-PCR
The nucleotide sequences of PCR primers are shown from 5' (left) to 3' (right) direction.
The numbers shown below each gene name are the length of predicted PCR products (bp). FW; forward primer, REV; reverse primer.  Table 4 The sequences of siRNA target sites.
The symbol of gene is shown in (Italic). For the gene silencing experiments, a mixture of four siRNAs (1 µM each) specific to each target gene was used.

Legend of videos
Video 1. Perinatal OPCs without TH in 1.5% O 2 condition (as control).
OPCs purified from P7 rat optic nerves were cultured in the hypoxic condition without TH. These cells passaged once on day 14. On day 21, time-lapse differential interference microscopic images of cells were recoded every 15 minutes for 24 hours.

Video 2. Adult-like OPC with TH in 1.5% O 2 condition.
OPCs purified from P7 rat optic nerves were cultured in the hypoxic condition with TH.
On day 21, time-lapse differential interference microscopic images of cells were recoded every 15 minutes for 24 hours.

Video 3. Adult-like OPCs differentiated into OLs in 1.5% O 2 condition.
300 of OPCs purified from P7 rat optic nerve were plated in BS medium containing PDGF with TH in 1.5% O 2 on PDL/fibronectin coated 12 mm glass-bottom dishes and pre-cultured at 37℃. On the culture day 10, the culture medium was replaced to PDGF free BS medium, after which (recording time 0) they were followed by time-lapse video microscopy. Images of cells were captured at every 15 minutes for 96 hours.      Note that the cells over expressing p15/INK4b proliferated much less than control. *P < 0.001 (unpaired Student's t-test, n = 3). The p15INK4b-IRES-zsGreen expressing cells were also labeled with A2B5 antibody (red) and DAPI (blue); as shown in the panels on right, the ZsGreen + OPCs (green) are bipolar and A2B5 + . Scale bar: 100 µm.  (b) 2,000 of OPCs purified from P7 rat optic nerve were inoculated in PDL-coated T25

Supplementary
flasks. Cells were pre-cultured in BS medium containing PDGF without TH in 1.5% O 2 for 10 days at 37℃. To investigate the immediate early responses of the genes of the deceleration of cell cycle related transcription factors, these cells were pre-treated with cycloheximide (50 µg/ml) for 6 hours, after then TH was added to the medium. Cells were harvested at 0, 0.5, 1 and 2 hours later. Total RNA was prepared from these cells and a RT-PCR assay was carried out. Gapdh (GAPDH) was used as a negative control.
The PCR products were detected by 2% agarose gel electrophoresis. (f) OPCs overexpressing Runx1 (green) in (d) were stained with the nuclear stain DAPI (blue) and rabbit anti-p15/INK4b antibodies (red). Scale bar: 100 µm. By using the BZ-Ⅱ Analyzer software (Keyence), the fluorescent intensities of the staining of anti-p15/INK4b antibodies in each cells were estimated. The average staining intensity of Runx1-overexpressing cells was 22,743 ± 11,425 pxl (n = 29), that was 11-folds higher than that of the non-transformed cells (2,032 ± 3,862 pxl; n = 49). Figure 6 (a) 2,000 of OPCs purified from P7 rat optic nerve were inoculated in PDL-coated T25

Supplementary Figure Legend for
flasks. Cells were cultured in BS medium containing PDGF without TH (-TH) or with were presented as the relative amount of transcripts to that of the DMOG free culture using comparative ∆∆Ct method. Actb is an endogenous negative control. Ldha, Pgk1 and Vegfa are HIFs-inducible positive control. The P values of these genes are P < 0.001 (ANOVA with Fisher's LSD test, n = 3).
(g) 1,000 of OPCs purified from P7 rat optic nerve were inoculated in PDL-coated slide flasks. Cells were pre-cultured in BS medium containing PDGF with TH in 3% O 2 for 10 days at 37℃. Then, some cells were treated with DMOG (1 mM) for 24 hours. After then, cells were stained with rabbit anti-Runx1 antibodies (green) and PI (red). Scale bars; 50 µm. Note; in the DMOG treated cells (3% O 2 + DMOG), Runx1 protein increased and colocalized with nucleus (yellow).