PROM1 is expressed in human fetal neural stem cells and in two distinct cell populations in adult human brain.

<p>A. PROM1 RNA and protein are highly expressed in ependymal/subependymal cells of the ventricular zone (arrow) particularly in ciliated cells. Images are from the anterior-lateral germinal matrix/SVZ in the developing cortex. A more heterogeneous expression of PROM1 is detected in neurospheres derived from human fetal brain (15 gw). Immunofluorescence lineage analysis shows co-localization of PROM1 with SOX2 and GFAP (arrows) but only rare co-localization with OLIG2 (arrowhead). Co-localization was not detected with proliferating Ki67 positive cells (arrowhead). B. RISH and IHC analysis, on human adult normal brain tissue shows high PROM1 expression in ependymal cells (arrows) and in cells with glial morphology with complex processes dispersed within the white and grey matter regions (arrows). PROM1 is present in ependymal cells (ep) and cells within the subependymal astrocyte ribbon (ar) also positive for GFAPδ and SOX2. White and grey matter regions exhibit PROM1 cells with astrocytic morphology that co-localize with markers SOX2 and GFAP (arrows). PROM1 and OLIG2 co-localization was not detected (arrowhead).</p

<i>Prom1^hi</i> cells are slow dividing distributed progenitor cells distinct from adult NG2 progenitors.

<p>A. Two examples of long term BrdU labeling (7 days, daily dosing) identify rare Prom1+ (RISH; blue) BrdU+ (IHC; brown) cells in white and grey matter (c and f are magnified insets of the small marked area in a and d). High magnification of the SVZ (b, e) shows numerous BrdU+ stem/progenitor cells that do not express high <i>Prom1</i>. Arrows indicate rare Prom1^hi+ cells that do not express BrdU (b, e). B. In contrast to Prom1, double IHC for NG2 (brown) and BrdU (red) readily identifies numerous co-localized cells in SVZ and brain parenchyma. C. Manual quantification of the percentage of BrdU positive cells co-labeled with <i>Prom1</i> or NG2 in the white matter, grey matter and SVZ. svz: subventricular zone; lv: lateral ventricle; gm: grey matter; wm: white matter.</p

<i>Prom1^hi</i> cells are glial cells with novel characteristics.

<p>A. Combined RISH:IHC shows single cells with high <i>Prom1</i> RNA (blue, ISH) co-localize with oligodendroglial marker Olig2 and astroglial marker Sox2 (brown, IHC) in both white and grey matter. No co-localization was detected with Gfap, NG2 or NeuN. B. Olig1/2^−/− knock-out mice (Olig1/2 KO) show <i>Prom1</i> staining in ventricular zones (arrows) and fimbria white matter where <i>Prom1^hi</i> cells first emerge (arrows) in E18.5 mouse brain. Olig2+ cells retain protein in Olig1/2 controls (brown, IHC). C. Quantification of the <i>Prom1</i> RISH cells co-localizing with lineage markers. cc: corpus callosum; gm: grey matter, wm: white matter; vz: ventricular zone.</p

High PROM1 expression is associated with IDH1 wild-type, proneural GBM patients with poor survival.

<p>A. PROM1 RISH and IHC show similar patterns of expression <i>in vitro</i> (PDCL; BT216) and <i>in vivo</i> (PDX; BT216). B. Immunofluorescence on BT216 PDCL shows PROM1 co-localization with GFAP and SOX2 but rarely with proliferative marker Ki67 (arrow). C. <i>PROM1</i> RNA expression is highest in TCGA proneural subclass, not statistically significant. <i>PROM1</i>^high cases are found in all 4 groups. D. <i>PROM1</i> high expression correlated with shorter overall survival in GBM patients from TCGA. P values using Mantle-Cox. E. cBioPortal analysis of the proneural TCGA subclass shows that <i>PROM1</i>^low correlates with IDH mutation, while <i>PROM1</i>^high is more associated with PDGFRA amplification. F. Kaplan-Meier analysis of <i>PROM1</i>^high and <i>PROM1</i>^low cases from the TCGA proneural subclass.</p

<i>Prom1</i> expression in developing and adult mouse CNS.

<p>A. RNA in situ hybridization showing high <i>Prom1</i> expression in the ventricular zone (vz, arrows) of the prenatal brain and spinal cord (a). Levels decrease over time except in the floor plate (fp). In the cerebellum (b) <i>Prom1</i> is highly expressed in the caudal and rostral rhombic lips (rl, cRL, rRL) while postnatal expression is most intense in rare cells of the white matter (hi, arrows) and the internal granule layer (igl). In postnatal brain (c) expression is conserved at low levels in the stem cell niches (svz and sgz) but is highest in single cells within white matter (wm, red arrows). B. In adult brain <i>Prom1</i> RNA is highest in single, widely distributed cells (arrowheads) in the corpus callosum (a, d), cerebellum (f), gray matter (e) and only rarely in SVZ (b) and the SGZ (c) stem cell niches. Semi-quantitative RT-PCR of microdissected white and grey matter regions from 3 different mice (g) validate high <i>Prom1</i> levels in white matter relative to gray matter and whole brain (p = 0.05) (h). Mouse liver acts as a low level reference tissue. The differential level of <i>Prom1</i> is similar to the oligodendroglial marker <i>Olig2</i> (i). ge: ganglionic eminence; ml: molecular layer; gm: grey matter; hp: hippocampus; gcl: granule cell layer; sgz: subgranular zone; cc: corpus callosum; lv: lateral ventricle; ep: ependymal layer.</p