Box 1. Evidence demonstrating the absence of transformation of v-myc
perpetuated hNSCs
• No reported transforming activity of v-myc on human neural tissue in the scientific
literature.
• Absence of growth in soft agar assays (Villa eat al., 2000).
• Their immortalization is conditional, since they are EGF/bFGF dependent. In the
absence of these factors, and even in the presence of fetal bovine serum, the cells do
not grow. After differentiation, the cells do not proliferate, as assessed by 3Hthymidine incorporation in vitro or PCNA immunocytochemistry. The cell cycle
length increases from 40 hours to over 30 days (Villa eat al., 2000).
• After in vitro differentiation, the cells:
o
o
o
o
o
o
achieved by growth factor withdrawal (Villa et al., 2000; Flax et al., 1998;
Rubio et al., 2000).
stopped expressing nestin and vimentin, both in vitro and in vivo (Villa et al.,
2000; Flax et al., 1998; Rubio et al., 2000).
reduced expression of v-myc, as demonstrated by RT-PCR (Villa et al., 2000;
Flax et al., 1998; Rubio et al., 2000; unpublished data).
stopped expressing telomerase protein, and show no detectable telomerase
activity (unpublished data) in contrast to transformed cells.
changed morphology and expressed neuronal and glial differentiation markers
proper of terminally differentiated (and not just quiescent) cells (Villa et al.,
2000; Flax et al., 1998; Rubio et al., 2000).
pool of cells in G2/S/M phases of the cell cycle profoundly decreased. This
change was more clear-cut in genetically expanded (GE) cells than in human
neurospheres (unpublished data).
• In vivo:
o
o
o
o
o
GE-hNSCs do not form tumors neither in fetal (Ourednik et al., 2001), neonatal
(Flax et al., 1998) nor in adult immune-suppressed animals (Rubio et al., 2000;
Aboody et al., 2000).
Transplanted GE-hNSCs downregulate nestin as soon as one week following
implantation (Rubio et al., 2000).
Transplanted GE-hNSCs do not express PCNA, a marker of cycling cells
(Rubio et al., 2000).
Transplanted GE-hNSCs differentiate to generate cells expressing mature
neuronal and glial markers (Flax et al., 1998; Rubio et al., 2000; Ourednik et
al., 2001).
Cell cycle duration, colony formation potential and neuron generation
capability is unchanged over years in culture (unpublished data).
Reviews: Villa et al., 2001; Martinez-Serrano et al., 2001; Villa et al., 2002.