290 EFFECT OF EXOGENOUS Oct4 PROTEIN ON DIRECT CONVERSION OF HUMAN FIBROBLASTS INTO NEURAL STEM CELLS

Abstract

The generation of neural stem cells (NSC) from somatic cells may provide unlimited source of neuronal cells for autologous transplantation to patients of neurological disorders. Recently, direct conversion of fibroblasts into NSC by epigenetic reprogramming has been reported (Han et al. 2012 Cell Stem Cell 10, 465–472; Thier et al. 2012 Cell Stem Cell 10, 473–479; Ring et al. 2012 Cell Stem Cell 11, 100–109). These reprogrammed cells are referred to as induced neural stem cells (iNSC) and they share the characteristics of NSC in their morphology, molecular marker expressions, and capacity to differentiate into neurons, astrocytes, and oligodendrocytes. One of the procedures to convert fibroblasts into iNSC is restriction of Oct4 activity to the initial phase of reprogramming, while Sox2, Klf4, and c-Myc are constitutively expressed. In the present study, we examined the effect of Oct4 in reprogramming of human fibroblasts into iNSC. Oct4 protein was modified by the addition of poly-arginine protein transduction domain to easily penetrate into the cell membrane. We transduced Oct4 protein, in contrast to the previous reports where the Oct4 gene was virally introduced. First, human fibroblasts were transfected by retroviral vectors carrying the genes encoding Sox2, Klf4, and c-Myc. Then, transfected cells were cultured in ReNcell NSC maintenance medium containing Oct4 protein. After 4 days, Oct4 protein was removed from the medium. With Oct4 protein transduction, 21 flat colonies were formed from 4 × 10⁵ fibroblasts. These colonies were picked and passaged for subculture and later became iNSC. However, in the absence of Oct4 protein, no colonies were obtained from the same number of fibroblasts that were initially plated. Approximately 40 days after transduction of reprogramming factors, cluster of iNSC were obtained. These cells expressed molecular markers of human NSC, including Nestin, Sox2, Pax6, and Blbp. Moreover, these iNSC could differentiate into neurons, astrocytes, and oligodendrocytes in vitro. Results of the present study demonstrate that transduction of exogenous Oct4 protein may be essential to the direct conversion of human fibroblasts into iNSC using a combination of reprogramming factors Sox2, Klf4, and c-Myc.