STEM-10L-myc EXPRESSION PROMOTES HUMAN NEURAL STEM CELL MULTIPOTENCY

Abstract

Several preclinical studies indicate that neural stem cells can limit or reverse CNS damage by cell replacement, regeneration or delivery of various factors and therapeutic agents to sites of degeneration or tumor. Allogeneic NSC lines have several advantages over autologous ones, including off the shelf usage, greater availability to patients, and cost effectiveness. Genetic modification of NSCs for immortalization and retention of stem cell properties with c-myc or v-myc gene has been shown to be clinically useful in trials for stroke (ReNeuron), and brain tumors (Aboody et al). Here, we propose to express L-myc variants in human fetal brain derived NSCs, to extend NSC lifespan and stem cell characteristics. We have isolated and grown NSCs from freshly dispersed 10 week old human fetal brain tissue and propagated in culture. We then transduced these cells with retroviral vectors containing the L-myc gene. NSCs stably expressing L-myc gene (hNSC.Lmyc) were isolated for further characterization. hNSC.Lmyc cells propagated through passage 15, demonstrating a normal karyotype, expression of stem cell markers (Sox-2, A2B5 and nestin) and low expression of differentiation markers (Olig 2 and vimentin) in vitro. We have also demonstrated that hNSC.Lmyc cells retain their tumor-specific migratory abilities in vitro and in vivo for longer periods of time as compared to untransduced primary NSC pools. We further characterized these hNSC.Lmyc cells for gene expression profiling and genome wide DNA sequencing analysis. Somatic mutation analysis comparing early and late passages of hNSC.Lmyc cells showed no change in the genomic sequences. hNSC.Lmyc cells were able to self renew and differentiate into neurons, oligodendrocytes and astrocytes in vitro. In this study we generated and characterized hNSC.Lmyc line that has potential therapeutic use in numerous central nervous system (CNS) diseases.