Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

3.3 Glioma Culture Systems Introduction
NS cell markers and did not respond to differentiation cues such as RA.
· In clonogenicity assays in which cells were tested for neurosphere forma-
tion upon single cell plating, NBE cells showed clonogenic frequencies
reminiscent of NS cell-derived neurospheres, whereas serum cells failed
to form neurosphere-like cells when plated in NBE conditions.
· The telomerase activities of NBE and serum-cultured cells differed in
that, although both cells retained telomeres as determined by fluores-
cence in situ hybridization (FISH), NBE cells had consistent telomerase
activity regardless of passage number, whereas telomerase activity was
lost when these cells were cultured in serum-containing media, consis-
tent with what occurs in normal NS cells. Likewise, early passage serum
cells did not have appreciable telomerase activity, which, however, they
gained back in later passages of exponential growth phase.
Taken together, these data demonstrate that NBE cells, as observed by the
two NBE cell lines followed in the study, contain many of the self-renewal
and differentiation characteristics of NS cells, whereas serum cultured cells do
not [261]. Other important characteristics tested on these two cell types were:
· Tumourigenic potential in vivo. Upon injection into the brains of
neonatal immunodeficient mice, NBE cells demonstrated retention of a
tumourigenic potential independent of passage number with as low as
1,000 cells, whereas serum-cultured cells at early passages did not show
any tumourigenicity. When established NBE cell-derived tumours were
dissociated and cultured under NBE conditions to be injected again into
the brains of new recipient mice, there was no loss of tumourigenic po-
tential, unlike when the same xenograft-derived cells were grown under
serum conditions and all subsequent tumourigenic potential was lost.
This suggested that the loss of tumourigenicity was due to the serum-
culture conditions. Interestingly, although early passage serum-cultured
cells did not form tumours, the late passage, exponentially growing,
telomerase-positive ones did at an increasing rate with progressive pas-
sage number.
· Phenocopy of the original human glioblastoma tumour. While
the intracranial tumours generated by NBE cells demonstrated exten-
sive infiltration along white matter tracts as observed in glioblastoma
patients, all the tumours generated from late passage serum cells were
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