Host: The Japan Radiation Research Society, Chairman of the 52nd Annual Meeting, Toshiteru Okubo (Radiation Effects Research Foundation)
Purpose: Stem cells are defined as cells that self-renew indefinitely and also give rise to differentiated cells by suitable stimulation. There is increasing evidence that malignant tumors contain high-hierarchy cells that maintain the characteristics of tissue-specific stem cells. Recent findings support an idea that malignant gliomas can be generated from either a neural stem cells (NSCs) or glial lineage cells. The hypothesis can be viewed as cancer stem cells. The stem cells also are gaining attention of the application in regenerative medicine. However, radiosensitivity of stem cells is not fully determined due to difficulty of in vitro culture. The development of NSCs in vitro culture has been accomplished by neurosphere method. In this study, we demonstrated the induction of telomerase activity in mouse neural stem cells infected by retrovirus containing an hTERT. Additionally, we investigated radiation sensitivity of hTERT introduced neurosphere cells.
Methodology : NSCs were harvested from corpus striatum in E14.5d ICR mouse embryo. For 10 days culture, cells were grown as self-adherent complexes of cells, forming clusters known as neurosphere. An hTERT gene was introduced into neurosphere cells with retrovirus vector. The introduction of hTERT gene was checked by RT-PCR and telomerase activity of neurosphere cells was evaluated by ELISA. The radiosensitivity of neurosphere cells was determined by colony forming assay in soft agar.
Result : Telomerase activity of hTERT introduced neurosphere cells was elevated as compared with those of primary neurosphere cells and mouse embryo fibroblasts. Moreover, we defined that serum stimulation allowed NSCs to be differentiated to glial cells. We determined the radiosensitivity of primary, p53 and ATM deficient neurosphere cells, and hTERT introduced neurosphere cells. Finding radiation response in established NSCs contributes to the understanding of the role of stem cells in development of tumorigenesis, and to the estimation of the risk of stem cells in regenerative medicine.
