Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

3.3 Glioma Culture Systems Introduction
GFAP, and performing flow cytometry for CD133. Furthermore, transplanta-
tion of these cells after in vitro exposure to differentiation-promoting conditions
delayed tumour formation. In most transplants a striking infiltration of the
brain reminiscent of the human disease was observable with the exception of
cell line G166, which generated a more defined tumour mass. To determine
the tumour-initiating potential of the GNS cell lines, transplants were carried
using 10-fold dilutions starting with 100 cells, which were sufficient for cell en-
graftment of all GNS cells and for G144 and G174 were capable of generating
an aggressive tumour mass, which required 1,000 cells in the case of G166 and
G179. Unlike GNS cells, normal fetal NS cells never generated tumours at
any dilution. Finally, to determine whether the tumour-initiating cells could
self-renew within the xenograft, serial transplantations from the tumour mass
into secondary and tertiary recipients was carried out using G144, G144ED
and G179, showing in each case the generation of a tumour and thus demon-
strating that long-term expanded GNS cell lines remain highly tumourigenic
and are capable of forming tumours that appear to recapitulate the human
disease [404].
Since the important defining property of stem cells is their ability to gen-
erate differentiated progeny, the differentiation programs of the GNS cells
were evaluated keeping in mind that the prevalent form of glioma is the
GFAP + astrocyte-like cell-containing astrocytoma, which can also contain
anaplastic cell populations and, in some cases, an oligodendrocyte compo-
nent [301]. For all GNS cell lines, the differentiation to Octamer-binding pro-
tein 4 (Oct4) + oligodendrocytes or TuJ1 + neurons was fully suppressed in the
presence of EGF and FGF2, in contrast to what is observed in glioma neuro-
spheres. Upon growth factor withdrawal, NS cells differentiate into neurons
(see Fig 2.9), but in contrast to that, G144 and G179 GNS cells differentiated
into Oct4 + or CNPase +38 oligodendrocyte-like cells, and TuJ1 + cells, respec-
tively. Neuronal-like cells or oligodendrocytes were not apparent in G166 cul-
tures, which continued to proliferate in the absence of EGF and FGF2 without
clear differentiation. The tendency of G144 cells to differentiate into oligo-
dendrocytes was surprising because efficient oligodendrocyte differentiation of
mouse and human fetal NS cells requires exposure to thyroid hormone, ascor-
38 CNPase is a 2Õ, 3Õ-cyclic nucleotide 3Õ-phosphodiesterase that catalyses the in vitro
hydrolysis of 2Õ, 3Õ-cyclic nucleotides to produce 2Õ-nucleotides and has an in vivo function
that remains to be elucidated. High CNPase expression is seen in oligodendrocytes and
Schwann cells, accounting for roughly 4% of the total myelin protein in the CNS [137]
80