br Experimental Procedures br Author Contributions

Experimental Procedures
Author Contributions
Acknowledgments We thank Y. Sasai (RIKEN, Japan) for the RAX-GFP mESC line, M. Wegner (University of Erlangen, Germany) for SOX9, E. Tanaka (CRTD, Germany) for RAX and CRX, and D. Forrest (NIDDK, USA) for TRβ2 antibodies. We thank J. Brzezinski (University of Colorado Denver), S. Oakeley (Basel, Switzerland), G. Kempermann (DZNE), M. Ader, F. Calegari, N. Ninov, and E. Tanaka (all CRTD) for helpful comments on the manuscript. We thank M. Obst, the light microscopy facility, and the animal facility for excellent support. This work was kindly supported by the Funding Programs for DZNE Helmholtz (M.K.), TU Dresden CRTD (M.K.), DFG (KA2794/3-1; SPP1738) (M.K.), MedDrive TU Dresden UKD-Medical Faculty (M.K.), research award Novartis Pharma GmbH (M.K.), Volkswagen Foundation Freigeist fellowship (V.B.), and the European Union\'s sixth Framework Program ESTOOLS (K.A.).
Introduction Differentiated annexin v can be reprogrammed to become induced pluripotent stem cells (iPSCs) by exogenous supplementation of defined factors (Takahashi and Yamanaka, 2006). The iPSCs provide an interminable source of a broad range of differentiated cells for applications such as in vitro disease modeling, drug development, toxicity testing, and cell-replacement therapies. Mature neurons and neural stem cells (NSCs) are among the most clinically useful cells that can be produced from pluripotent stem cells (PSCs) (Nemati et al., 2011). However, their clinical utility has been hampered by the tumorigenic potential elicited by the residual PSCs in the differentiated cell population, the lengthy and inefficient differentiation process (Hu et al., 2010), and genomic instability (Weissbein et al., 2014). In the process of trans-differentiation, one mature somatic cell type can be converted into another functional mature or progenitor cell type without undergoing an intermediate pluripotent state by using a variety of inducers, such as transcription factors, epigenetic modifiers, and microRNAs (Moradi et al., 2014; Pournasr et al., 2011). Mature neurons have been successfully trans-differentiated from several cell sources (Ambasudhan et al., 2011; Ladewig et al., 2012; Marro et al., 2011; Vierbuchen et al., 2010). However, their inability to proliferate and survive for long periods of time in culture conditions limits their use. An alternative approach is to convert somatic cells into NSCs, which are expandable in vitro and have the potential to differentiate into major neural cell types, such as neurons, oligodendrocytes, and astrocytes. Ectopic expression of several combinations of genes via lentiviruses with or without small molecules have been used to produce induced NSCs (iNSCs) from somatic cells (Cassady et al., 2014; Han et al., 2012a; Lujan et al., 2012; Ring et al., 2012; Thier et al., 2012; Wang et al., 2013). Most of these NSC-induction cocktails depend on the use of potentially tumorigenic pluripotency-associated factors in reprogramming or a multi-factor strategy that increases the intricacy of the approach. Hence, the use of a single reprogramming factor for generation of iNSCs may represent a more controllable, easier, and safer approach. Here, we demonstrate that iNSCs could be generated from human fibroblasts by ectopic expression of a single neurogenic factor, zinc-finger protein 521 (Zfp521). Our data indicate that Zfp521 alone is sufficient for conversion of fibroblasts into iNSCs, which may serve as an alternative and more accessible source of cells for neural cell-replacement therapies as well as in vitro disease modeling, toxicity testing, and drug development.
Results
Discussion In this study, we demonstrate that iNSCs can be successfully generated from human fetal, neonatal, and adult fibroblasts by ectopic expression of a single Dox-inducible, lentivirally encoded neurogenic transcription factor Zfp521. These iNSCs display morphological characteristics of endogenous NSCs, are clonogenic, exhibit rostral regional specificity, and maintain their self-renewal ability and tripotency over prolonged passaging with retention of a stable karyotype following serial passaging. Therefore, Zfp521 seems to be sufficient to trigger a self-sustaining gene regulatory network that confers a rostral NSC identity to fetal, neonatal, and adult human fibroblasts (in the presence of a specific cocktail of small molecules), which is concordant with its reported permissive and rostralizing role in differentiation of neuroectodermal cells from undifferentiated ESCs and epiblast-stage embryos (Kamiya et al., 2011). Analysis of a global iNSC transcriptome confirmed the acquisition of neural gene expression profiles and activation of biological processes associated with neuronal differentiation and development in Zfp521-iNSCs. Although many of these genes were also expressed at similar levels in endogenous human NSCs isolated from fetal brain, these two cell types differed in the expression of a subset of genes that were differentially expressed between each NSC type and HNFs. However, GO analysis of these DE genes revealed that they still belonged to functional categories related to nervous system development and neurogenesis, supporting the conclusion that differences in expression profiles observed between iNSCs and WT-NSCs are more of a quantitative than of a qualitative nature and may reflect technical variance rather than biological differences.