Previously it was demonstrated that induced NP iNPs can be

Previously, it was demonstrated that induced NP (iNPs) can be directly converted from mouse somatic vitamin d receptor by overexpressing various TF combinations (Han et al., 2012; Kim et al., 2011; Lujan et al., 2012). Kim et al. (2011) first demonstrated that expandable iNPs could be generated from FBs via a modified pluripotency reprogramming procedure, and the resulting iNPs were able to differentiate into neurons and glial cells. Subsequently, several studies reported the generation of iNPs through the introduction of neural-enriched factors with/without iPSC factors (Han et al., 2012; Lu et al., 2013; Lujan et al., 2012), and the resulting iNPs were able to differentiate into all three major neural cell types of the CNS. Meanwhile, reports show that human iNPs can also be converted from somatic cells via the introduction of TFs (Lu et al., 2013; Ring et al., 2012; Wang et al., 2013). In these studies, several TF combinations, including at least one of the iPS factors, were used for hiNP generation (Ring et al., 2012), and the differentiation propensity of the iNPs described in the aforementioned studies was mainly restricted to CNS neurons. hESCs can be used as an in vitro differentiation model to generate neural phenotypes of various developmental stages, including embryonic NPs (ENPs) populations, and the critical neural genetic factors that contribute to the neural fate acquisition have begun to be uncovered (Hou et al., 2013; Rosa and Brivanlou, 2011; Zhang et al., 2010). Given that hESC-ENP populations possess broad differentiation potential to give rise to both CNS and PNS neural cell types, it may be possible to directly convert FBs into iNPs resembling hESC-ENPs through the use of TFs highly expressed in the hESC-ENP population.
Results
Discussion Previously, various TF combinations have been used to directly convert FBs into iNPs (Lu et al., 2013; Ring et al., 2012; Wang et al., 2013). These iNPs possessed the general properties of neural progenitors, such as neural marker/gene expression, proliferation, and differentiation propensity. Unlike the hESC-ENPs, which were demonstrated to differentiate into both CNS and PNS lineages (Elkabetz et al., 2008; Lee et al., 2007), previous reports have shown that iNPs exhibit developmental potentials primarily toward CNS subtypes (Lu et al., 2013; Wang et al., 2013). However, studies have rarely addressed whether these iNPs possess the ability to give rise to PNS neuron subtypes. In this study, we showed that iENP-6F and iENP-7F are able to differentiate into not only CNS lineages but also PNS lineages. Furthermore, they responded to the same extracellular stimuli as hESC-ENPs and gave rise to specific neuronal subtypes. In line with these observations, genome-wide transcriptome profiling also confirmed a high similarity between FB-induced ENPs and their hESC-derived counterparts. Thus, our results suggest that the iENP population reprogramed by hESC-ENP-TFs may be more similar to embryonic NPCs than adult brain-derived NPCs. Although the two iENP populations generated in this study shared similar NP characteristics, further investigation revealed that they exhibit different functional features. First, our analysis demonstrated that iENP-6F exhibited higher proliferation and reduced apoptosis compared with iENP-7F. Second, iENP-7F showed stronger differentiation potency toward neuronal lineages than glial lineages. Third, dissection of the neuronal differentiation potential of the iENP populations revealed that iENPs-7F have a regional preference toward caudal identity, whereas iENPs-6F have a regional preference toward rostral identity. The above differences between iENP-6F and 7F may be explained by the neural reporters used for TF and iENP selection. We used two neural reporters, PAX6 and SOX1, to monitor and evaluate the efficiency of neural fate conversion by hESC-ENP-nTFs, through which we identified a 6- and 7-TF combinations for iENP-6F and -7F induction, respectively. On the other hand, PAX6 and SOX1 were also used to select the iENP-6F and 7F populations, respectively. Thus, it is tempting to suggest that neural reporter selection may decide the functional characteristics of the resulting iENP populations. It is well known that hESC-derived neural rosettes and neural epithelia consist of various ENSCs/ENPs, which are responsible for the subsequent neural development of the CNS and PNS (Pankratz et al., 2007). Therefore, the originally selected 25 nTFs highly expressed in hESC-ENPs are likely essential for the formation of heterogeneous NP populations. Accordingly, induction of FBs with specific nTF combinations selected from the 25-TF pool should result in the formation of an iENP population with specific neural characteristics. Together, these results suggest that the scheme described in this study may provide an excellent way for generating desirable iENP populations through the selection of specific TF combinations from the original 25-TF pool and iENP populations using different neural reporters. Future studies will be required to determine whether specific combinations of hESC-ENP-nTFs can define the functional aspects of the resulting iENPs, and elucidate the mechanisms by which the TF combinations reprogram FBs into iENPs.