2. The isolated hNSCs are normal and do not form tumors in normal nude rats
The self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic.