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

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 (eiNSCs) 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. Neural stem cells (NSCs),3 somatic stem cells of the brain and spinal cord, could differentiate into three major cell types (neurons, astrocytes, and oligodendrocytes) in the central nervous system (CNS) in response to relevant signaling pathways (1). A number of in vitro transplantation studies with disease animal models have demonstrated that the engrafted NSCs can functionally rescue disease-related phenotypes, proving their therapeutic potential (2-5). Thus, NSCs have long been considered as a potential source for replacing damaged cells and tissues often found in various neurodegenerative diseases (2-5). However, major concerns associated with these cells may preclude expediting advances into their therapeutic application. First, the accessibility of NSCs is largely restricted in vitro, due to their limited origins. Second, the autologous cell transplantation is not feasible using NSCs from their in vivo origins, and furthermore, allogeneic transplantation of NSCs may raise the potential for immune rejection. Finally, with current culture conditions, it is technically challenging to homogeneously maintain human NSCs in vitro (6, 7). Thus, NSC-like cells generated from easily accessible patient somatic cell types such as fibroblasts and blood cells could serve as an autologous source for therapeutic applications, thereby overcoming current obstacles to NSC-mediated translation research.We and others have demonstrated the direct conversion of somatic fibroblasts into self-renewing and multipotent induced neural stem cells (iNSCs) or induced neural progenitor cells (iNPCs) by the forced expression of different sets of transcription factors (8 -12). Recently, Thier et al. (12) have shown that the restricted expression of Oct4 by a tetracycline-dependent lentiviral vector, together with retrovirus-mediated overexpression of Sox2, Klf4, and c-Myc, could elicit the conversion of fibroblasts into iNSCs. Simultaneously, we have generated multipotent and self-renewing iNSCs from mouse fibroblasts by retrovirus-mediated ...

Take the shortcut – direct conversion of somatic cells into induced neural stem cells and their biomedical applications

“…Using this excisable reprogramming vector system, the authors could derive transgene-free iNSCs by direct conversion of human adult peripheral blood monocytes, as well as dermal and fetal pancreas fibroblasts. Similar oriP/EBNA of EBV-based episomal vectors were used to express OKSM and LIN28 in combination with a small hairpin against p53 in adult human fibroblasts [42] or Brn4, Klf4, Sox2, and c-Myc in mouse embryonic fibroblasts [58]. Mauksch et al reported the generation of iNSCs by nonviral means employing plasmid transfection and recombinant protein transduction using SOX2 and PAX6 [54].…”

“…As a matter of fact, the various protocols for the generation of iNSCs give rise to distinct iNSC populations that slightly differ in self renewal capacity, marker expression, and regional identity, as well as in vitro and in vivo differentiation potential. While Kim et al [11] reported passage of their mouse iNSCs for 3-5 times only, more recent studies demonstrated maintenance of NSC characteristics for more than 30 passages [13,14,28,34,36,38,58,73]. In order to demonstrate selfrenewal potential and clonal growth ability, iNSCs were either cultivated as primary and secondary neurospheres [33,34], analyzed in colony formation assays [28,33,36,39,42,48,72], and/or passaged several times [11,13,14,26,30,32,33,38,57,59,60,73].…”

“…While Kim et al [11] reported passage of their mouse iNSCs for 3-5 times only, more recent studies demonstrated maintenance of NSC characteristics for more than 30 passages [13,14,28,34,36,38,58,73]. The neural expansion media were supplemented with either LIF and small molecules like CHIR99021, SB431542, purmorphamine, A83-01, and/or ascorbic acid [25,26,48], or basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF) [14,30,32,34,36,38,[57][58][59][60]62], or even a combination of them [33,35,41]. Among the various publications reporting iNSC generation, there is extensive accordance in terms of expression of bona fide neural stem cell markers, such as SOX1, SOX2, PAX6, NESTIN, CD133, and BLBP.…”

“…However, the differentiation potential can vary depending on the iNSC population, as well as the in vitro differentiation protocol used (e.g., spontaneous, undirected, and directed in vitro differentiation approaches) ( Table 2). Most iNSC populations were shown to be tripotential giving rise to neurons, astrocytes, and oligodendrocytes [13,25,28,32,34,57,58,77]. Yet, there seems to be a bias toward neurons and astroglia as differentiation into oligodendrocytes was less efficient [13,14,58].…”

“…Wang et al [57] generated integration-free and expandable iNSCs from human urine-derived epithelial cells using oriP/EBNA episomal vectors encoding OCT4, SOX2, KLF4, and SV40LT, in combination with the microRNA cluster MIR302-367. Similar oriP/EBNA of EBV-based episomal vectors were used to express OKSM and LIN28 in combination with a small hairpin against p53 in adult human fibroblasts [42] or Brn4, Klf4, Sox2, and c-Myc in mouse embryonic fibroblasts [58]. In 2013, Sendai virus (SeV) was employed as a nonintegrating viral vector for the generation of iNSCs [25].…”