Supplementary Material
Materials and Methods
Generation of astrocytes from mouse neural stem cells. Passage 2-5 neurospheres generated from E14
mouse neural stem cells (ganglionic eminences)(Louis et al., 2008) were dissociated into single cell and
plated at a high density (5105 cells/ml) in NSA medium with no growth factor and containing 5% FCS
(Gibco) to induce differentiation process. After one week, a monolayer of astrocytes containing neuronal
and oligodendroglial cells formed. Trypsinization and passaging of this monolayer depleted nonastrocytic cells. This step may be repeated to further eliminate other cell types. Under these conditions the
culture is comprised primarily of GFAP immunoreactive cells (1:500, Dako, supplementary figure 2),
with very little contamination by neuronal (TUJ1+) and oligodendrocytic (O4+) cells (data not shown) and
no proliferating cells (supplemental figure 2C). Therefore this procedure is appropriate to generate nonproliferative astrocytes used to measure the decay of CFSE fluorescence intensity unrelated to cell
proliferation. CFSE labeling of the astrocytic monolayer was performed according to the manufacturer’s
instructions.
qPCR analyses. After isolation of RNAs from the individual subpopulation, cDNA synthesis, using
High-Capacity cDNA reverse transcription kit (Applied Biosystems, #4374966) on a 7900HT Thermal
Cycler (Applied Biosystems), was performed. cDNA (50 ng) was used in relative quantitative PCR
containing Taqman Gene Expression Master Mix (Applied Biosystems), probe, and primers for CD133
(#Hs01009257_m1) and MCM2 (Hs01091564_m1). qPCR was performed using the SDS 2.3 software
(Applied Biosystems) and the data were analyzed with RQ manager 1.2 software (Applied Biosystems).
PCR data were normalized with 18S rRNA.
References
Louis SA, Rietze RL, Deleyrolle L, Wagey RE, Thomas TE, Eaves AC, et al. Enumeration of neural stem
and progenitor cells in the neural colony-forming cell assay. Stem Cells 2008; 26: 988-96.
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Figure Legend
Figure S1. Identification of label-retaining cells in hGBM and human grade III glioma derived cell
cultures.
21 gliomasphere cultures were generated from 20 different hGBM patients and one grade III glioma
patient. Flow cytometric histograms show the presence of CFSE-retaining cells in the individual culture.
The name of the lines is indicated on the top right corner of the histograms.
Figure S2. CFSE loaded mouse astrocytes.
In order to attribute the CFSE decay, observed in hGBM cells, to cell proliferation we generated a culture
of mouse astrocytes GFAP immunoreactive (A), which we loaded with CFSE (B) so as to follow the
CFSE intensity overtime (fig. 1D). (C) The lack of proliferation in the astrocyte culture was confirmed by
the absence of EdU incorporation after 45 minutes or 48H of pulse. (D) However active cell division was
observed in hGBM cell culture where approximately 30% of the cells incorporated EdU after a 45
minute-pulse.
Figure S3. Limiting dilution assay.
A log-fraction plot of the limiting dilution model fitted to the data in figure 4C. The slope of the line is
the log-active cell fraction (Slow cycling cells, red line; and overall population, black line). The dotted
lines give the 95% confidence interval. Limiting dilution analyses were performed using the limdil
function of the ‘StatMod’ package (Hu et al. 2009) (http://bioinf.wehi.edu.au/software/limdil/), part of the
R statistical software project (http://www.r-project.org).
Figure S4. Detection of hGBM cells.
(A) hGBM cells (black) can be separated from murine striatum cells (red) using a human specific antiCD56 antibody. Virtually all the hGBM cells showed positive staining whereas mouse cells were
negative. (B) Fluorescence picture of CD56 (red)/ DAPI (blue) double stained hGBM cells.
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Figure S5. CFSE-retaining cell distribution.
Undifferentiated gliomasphere fluorescence picture revealing a stochastic distribution of the CFSE
retaining cells (yellow arrows).
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