Three to four-year-old nonpassaged EGF-Responsive neural progenitor cells:Proliferation, apoptosis, and DNA repair

Epidermal growth factor responsive (EGFr) neural progenitor (NP) cells have been shown to be a potential alternative tissue source for neural transpla ntation and for developmental study. We have shown that nonpassaged EGFr NP cells can self-renew for 2 years in neurospheres and can robustly differen tiate into glia and a number of neuronal cell types. We are now attempting to investigate if the EGFr NP cells will die or continue to live beyond the life span of the donor. In addition, we and other investigators have also found that EGFr NP cells, after transplant, retain only a small number of c ells in the transplant site. In this study, we investigate the plasticity a nd fate of the EGFr NP cells. Using the nonpassaged method, we found EGFr N P cells live in the EGF supplement medium for over 4 years-the longest-live d EGFr NP cells ever reported. The 4-year-old striatal or cortical EGFr neu rospheres, when subplated with substrate coating, migrate out of neurospher es and have robust growth with many processes. Furthermore, when nucleotide marker bromodeoxyuridine (BrdU) was added 3 days prior to the subplating, the EGFr NP cells were labeled positively with BrdU in the nucleus, indicat ing active proliferation activity. Meanwhile two other events were also fou nd in the long-term EGFr NP cells. In the midst of the proliferation, apopt osis occurred. A subpopulation of EGFr NP cells are undergoing programmed c ell death as indicated by the cell morphology and the TUNEL staining for DN A strand breaks. The TUNEL fluorescein-staining indicates that over 50% of EGFr NP cells are positive in the nuclei. On the other hand, we have also f ound that the major base excision repair enzyme, APE/ref-1, which is respon sible for recognizing and repairing baseless sites in DNA, was present in t he progenitor cells. However, in those cells undergoing apoptosis, APE/ref- 1 levels were dramatically reduced or missing, and only a small percentage of cells were TUNEL and APE/ref-1 positive. These observations indicate tha t EGFr neural progenitor cells can live beyond the life span of the donor a nimal. The longevity of these cells in culture may be enhanced due to decre ased apoptosis and the retention of normal DNA repair capacity. (C) 2000 Ac ademic Press.