Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts.

Authors:
Kim SM, Kim JW, Kwak TH, Park SW, Kim KP, Park H, Lim KT, Kang K, Kim J, Yang JH, Han H, Lee I, Hyun JK, Bae YM, Schöler HR, Lee HT, Han DW.
In:
Source: J Biol Chem
Publication Date: (2016)
Issue: 291: 27
Research Area:
Neurobiology
Stem Cells
Regenerative medicine
Cells used in publication:
Embryonic fibroblast, mouse (MEF)primary
Species: mouse
Tissue Origin: embryo
Platform:
4D-Nucleofector™ X-Unit
Experiment

To generate e-iNSCs, 1 x 106 of MEFs were transfected using Amaxa P4 primary cell 4DNucleofector kit (Lonza) according to the manufacturer’s instructions. Briefly, 1.5 µg of each episomal vector was mixed with 82 µl of P4 primary cell solution and 18 µl of supplement 1. The mixture of MEFs and episomal vectors was then transferred into NucleocuvetteTM Vessel and electroporated with CZ-167 program. The transfected cells were plated onto the gelatincoated dish in MEF medium. Starting on the next day, the cells were cultured in NSC medium, which was replaced every other day with fresh medium until initial clusters were observed.

Abstract

The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (e-iNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability.