Immortalization of Mammalian Central Nervous System Stem Cells From p53 Knock-out Forebrain

Abstract

Neuronal and glial cells making the central nervous system are generated from central nervous system (CNS) stem cells during the development of CNS. CNS stem cells are potentially applicable for regeneration of damaged brain, such as in Parkinson’s Disease, or Alzheimer’s Disease. Recent studies have shown that mitogen and/or oncogene keep CNS stem cells proliferating. However, the long-term expansion of CNS stem cells needs a period of crisis, when almost all cells stop dividing and presumably wait for another intrinsic genomic change. The p53 tumor suppressor gene encodes a protein that is important in the regulation of cell proliferation and apoptosis. In this study, we used p53 knock-out embryos to analyze the effect of the p53 gene on apoptosis of the CNS stem cells. In a primary culture assay, p53 knock-out CNS stem cells are continuously growing in the presence of basic Fibroblast Growth Factor (bFGF), whereas expansion of wild-type CNS stem cells stopped shortly after cultivation due to substantial apoptosis. This indicates the critical role of the p53 locus on apoptosis of CNS stem cells. In this study, we have established immortalized CNS stem cells from p53 knock-out forebrain, MSP-1. To inhibit spontaneous differentiation during cell exparsion, MSP-1 had been introduced by c-Myc-Estrogen Receptor (ER) fusion gene, in which c-Myc can be conditionally activated by adding beta-estradiol. The integration of introducing c-Myc-ER has been confirmed in MSP-1 cells by Southern blot, Northern blot, and Western blot. The MSP-1 precursor cells express nestin and proliferate exponentially under the growing media, supplemented with bFGF, beta-estradiol and 10% FCS. The vigorous growth of MSP-1 may be supported both by c-Myc and the P53 null mutation. MSP-1 cells differentiate into neuronal and glial cell types after switching to the differentiating media (N2 serum-free media without growth factor) for 6 days. MSP-1 can be expanded from just one cell. Clonally expanded MSP-1 cells differentiated into neuronal and glial cell types after withdrawal of growth factors. The availability of immortal stem cells capable of neuronal differentiation offers an unusually powerful way of identifying the molecular basis of neuronal commitment.