We further show that Zfp iNSCs lines established from
We further show that Zfp521-iNSCs lines established from fetal, neonatal, and adult fibroblasts have the capacity to differentiate in vitro into the three main cell types: neurons, astrocytes and oligodendrocytes. This trilineage differentiation potential was also retained in three clonally derived iNSC lines that were tested, which further confirms that these cells are bona fide self-renewing multipotent stem cells. Functional analyses revealed that neurons derived from Zfp521-iNSCs exhibit resting membrane potentials, express sodium and potassium currents, and fire APs with characteristics indistinguishable from human iPSC-NSC-derived neurons that were used as controls and human PSC-derived neurons described by others (Pre et al., 2014; Song et al., 2013; Telias et al., 2014). iNSCs transplanted into the neonatal mouse hdac inhibitor integrated into brain tissue and retained the capability to differentiate into neurons and astrocytes after 4 weeks. When transplanted into the adult rat brain, iNSCs survived and started migrating into the neighboring brain tissue. Nonetheless, in contrast to cells transplanted into neonatal brain, they retained the characteristics of neural precursors 3 weeks after transplantation. This result is in agreement with published data, which showed that a much longer time interval of several months is required for formation of mature neurons from transplanted human iNSCs and PSC-derived NSCs (Hemmer et al., 2014; Koch et al., 2009). Several gene combinations have been successfully used for reprogramming human somatic cells to an NSC-like state (Cassady et al., 2014; Corti et al., 2012; Han et al., 2012a; Lu et al., 2013; Ring et al., 2012; Thier et al., 2012). These strategies, however, mostly rely on the use of multiple pluripotency-associated factors for reprogramming and the resultant cells have been characterized inadequately or may have limited self-renewal capacity. Our approach bypasses these limitations, as it is based on a single reprogramming factor, which provides a safer tool to generate iNSCs applicable for potential cell-based therapies and a unique platform for unraveling the mechanisms of iNSC reprogramming. Moreover, Zfp521-iNSCs could be stably propagated for more than 60 passages and were confirmed to be clonogenic. Furthermore, in the present study, we showed that Zfp521 enables the direct conversion of fetal and neonatal fibroblasts into stable iNSC lines, however NSCs from adult human fibroblasts can be derived by combinatorial treatment of Zfp521 and a cocktail of small molecules to overcome restrictions on neural fate conversion. Recently, it was also reported that inhibition of the let-7 microRNA, or expression of its target HMGA2, facilitates SOX2-mediated direct reprogramming of human adult fibroblasts and blood cells into iNSCs (Yu et al., 2015). Moreover, ectopic expression of OCT4 in the presence of SB431542, LDN-193189, Noggin, and CHIR99021 cocktail resulted in the generation of iNSCs from human neonatal and adult peripheral blood progenitors (Lee et al., 2015). Zfp521 is a transcriptional co-repressor protein, the expression of which seems to be restricted to immature/multipotent cells of the body, including mesenchymal stem cells and hematopoietic stem cells (Bond et al., 2008; Han et al., 2012b). The highest expression levels of Zfp521 in the nervous system have been observed in the cerebellum, NSCs, and striatonigral neurons (Lobo et al., 2006). The importance of Zfp521 for neural development is demonstrated by the finding that NSCs/neuroectoderm cells cannot be generated from ESCs/epiblast when Zfp521 is abrogated (Kamiya et al., 2011). In addition, although homozygous Zfp521-deficient embryos apparently develop normally and are phenotypically indistinguishable from normal littermates at the time of birth, they exhibit behavioral abnormalities, lose weight over time, and are unable to survive for more than 10 weeks post-partum (Ohkubo et al., 2014) indicating the indispensable roles for Zfp521 in post-natal neurogenesis and development. Zfp521 exhibits a conserved amino acid and protein domain composition across diverse animal species (>90% identity), which suggests indispensable and universal roles for Zfp521 in organismal development (Shen et al., 2011). Notably, in this study, mouse Zfp521 has been used for conversion of human cells. Thus, it will be worthwhile to determine the mechanism underlying the regulatory role of Zfp521 during reprogramming in future studies.