In the current study we compared two different genetic backg

In the current study, we compared two different genetic backgrounds on the induction and maintenance of multipotency by generating clonal iNSC lines from two distinct mouse strains (C57BL/6 vs. C3H). Clonal iNSC lines from distinct genetic backgrounds exhibited the distinct levels of conversion efficiency, endogenous NSC markers, transgene expression, and in vitro differentiation although iNSC lines from both strains shared typical features of NSCs such as morphology, gene expression pattern, and epigenetic status, indicating that the genetic backgrounds influence the cell fate transition into an iNSC state. In addition, the comparative analysis of clonal iNSC lines could serve as a platform for screening the most suitable and functional iNSC line for clinical translation of direct conversion technology.
Materials and methods
Results
Discussion To understand the mechanism underlying the induction of pluripotency, a number of inhibiting and boosting factors have been identified throughout screening multiple epigenetic and genetic factors potentially influencing iPSC generation. However, the factors that positively or negatively affect the induction of multipotency or unipotency via the direct conversion process, remain elusive. In this study, we tried to elucidate the effect of the genetic background on the induction and maintenance of iNSCs. Although we were able to generate stably expandable iNSC lines from both mouse strains tested (C57BL/6 and C3H), these iNSC lines exhibited substantial differences in both the induction and maintenance phases of multipotency. First, the conversion efficiency as assessed by the number of SSEA1-positive population was significantly different between the two mouse strains. Furthermore, iNSCs from the C57BL/6 strain showed relatively poor induction of an iNSC state, as evidenced by impaired activation of endogenous Sox2 and incomplete silencing of transgenic Sox2. Thus, these data suggest that the genetic background influences the induction of multipotency in a neuronal lineage. Second, the survival rates of sorted SSEA1-positive single Kinase Inhibitor Library was also significantly different between iNSC clones from the two strains, indicating that the maintenance of iNSC identity after successful conversion into the SSEA1-positive iNSC state is also influenced by the genetic backgrounds. Moreover, all clonal iNSC lines derived from C57BL/6 strain displayed the limited in vitro differentiation potential into neurons, astrocytes, and oligodendrocytes, indicating that the functionality of the iNSCs was also impaired in C57BL/6 iNSCs. We observed the abnormally increased proliferation of C57BL/6 iNSC clones compared to C3H iNSCs and cNSCs (data not shown). In the previous study (Graham et al., 2003), it was demonstrated that the ectopic expression of Sox2 could induce increased proliferation and also block the proper differentiation of NSCs into their daughter cell types. Thus, the increased proliferation rate and impaired differentiation ability of C57BL/6 iNSCs can be explained by the residual expression of exogenous Sox2 in C57BL/6 iNSCs (Fig. 2A, Fig. 3C and Fig. S3). It would be interesting to further decipher the mechanism governing the strain-dependent regulation of both the endogenous NSC program and transgenes, which might be tightly linked to the functionality of iNSCs. Finally, it would be also interesting to examine whether the effect of the genetic background on the induction and maintenance of other cell types (e.g. induced hepatocytes and induced neurons) directly converted from somatic cells by defined transcription factors. Recent studies have described that the distinct combinations of transcription factors can induce direct conversion of various human cells into an iNSC-like state (Lu et al., 2013; Ring et al., 2012; Wang et al., 2013; Yu et al., 2015; Zhu et al., 2014). Although the data provided in these studies support the cellular identity of human iNSCs, their molecular and functional features were not precisely characterized at the clonal level. Therefore, comparing the induction and maintenance of the NSC fate on human somatic cells at the clonal level might allow us to elucidate the underlying mechanism of direct conversion into NSCs.