br Introduction Somatic cells can

Introduction Somatic neuronal nitric oxide synthase can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transduction of four embryonic transcriptional factors (Takahashi et al., 2007b; Takahashi and Yamanaka, 2006; Yu et al., 2007). This discovery provides not only a novel opportunity for regenerative medicine but also innovative cell therapies specific for patients or diseases (Romano et al., 2014; Sinnecker et al., 2014). However, many issues regarding iPSCs remain. One of these is their low generation efficiency. After the reprogramming induction, most cells are converted into partially reprogrammed cells (Utikal et al., 2009). Recently, some studies showed that more primitive cells such as neural stem cells are more readily converted into iPSCs than terminally differentiated cells because of their original expression of genes that are related to cell reprogramming (Kim et al., 2008; Kleger et al., 2012; Tsai et al., 2011). On the other hand, it has been reported that progenitors that are more readily differentiated than stem cells can effectively be reprogrammed into iPSCs (Guo et al., 2014). The types of cells that are sensitive to reprogramming remain controversial. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) was first reported to be a tissue stem cell marker, e.g., related to the small intestine and colon (Barker et al., 2008, 2007; Haegebarth and Clevers, 2009; Jaks et al., 2008). Many studies have recently found that Lgr5 is a marker of progenitors and not only tissue stem cells (Ng et al., 2014; Ren et al., 2014; Yee et al., 2013), both of which are reported to be sensitive to successful reprogramming. However, the significance of Lgr5-expressing cells (Lgr5+ cells) for the generation of iPSCs remains unexamined. In the present study, we evaluated the reprogramming behavior of Lgr5+ cells. Here we showed that the use of mouse Lgr5+ hair follicles resulted in the induction of less alkaline phosphatase staining-positive (AP+) cells but a greater number of Nanog-positive (Nanog+) cells than the use of Lgr5-negative (Lgr5−) cells. In addition, Lgr5+ cells emerged after reprogramming induction in mouse embryonic fibroblasts (MEFs) and normal human dermal fibroblasts (NHDFs), and both showed the strong advantage of the conversion into Nanog+ cells.
Materials and methods
Results
Discussion Although Lgr5+ cells in tissue stem cells and progenitors have been relatively well studied (Barker and Clevers, 2010a, 2010b; Barker et al., 2010, 2008, 2007; Carmon et al., 2011, 2012; Chai et al., 2011; da Silva-Diz et al., 2013; de Lau et al., 2014; Fukuma et al., 2013; Gil-Sanchis et al., 2013; Haegebarth and Clevers, 2009; Huch et al., 2013; Jaks et al., 2008; Schuijers and Clevers, 2012; Shi et al., 2012; Yee et al., 2013), the reprogramming behavior of Lgr5+ cells has not been examined. Here we showed that Lgr5+ cells derived from reprograming MEFs and NHDFs as well as mouse Lgr5+ HFs promoted the generation of Nanog+ colonies. Lgr5 expression is often observed at the site of active Wnt signaling (Carmon et al., 2011; Schuijers and Clevers, 2012). Various studies focusing on the relationship between Wnt signaling and cell reprogramming have been reported. The activation of Wnt signaling results in the promotion of reprogramming (Lluis et al., 2008; Marson et al., 2008). On the other hand, Wnt target genes (Lef1, Tcf1, Tcf3, and Tcf4) have stage-specific regulation of reprogramming to iPSCs, where suppression and promotion occurs (Ho et al., 2013). In our study, despite the similar levels of Wnt3a and Axin2 between M-Lgr5+ cells and v6.5 (less than two times), the levels of Lef1 and Hnf1a (Tcf1) were lower and the level of Tcf4 was higher in M-Lgr5+ cells than in pluripotent stem cells (Fig. 3E). Surprisingly, this balance is optimized for promoting the early rather than the late phase of reprogramming (Ho et al., 2013). Recently, it has been reported that the reprogramming process consists of sequential events; the early phase is the initiation of reprogramming, and the late phase is the maturation of reprogramming (Buganim et al., 2013; David and Polo, 2014). Cells that transiently express Lgr5+ seemed to be still under the initiation of reprogramming. In addition, M-Lgr5+ cells showed the low expression levels of genes involved in cell cycles (Fig. 3F). However, as shown in Fig. 3A right panel, M-Lgr5+ cells certainly proliferate, although they do not form crowded colonies. Thus, their reprogramming speed may be very slow. Comparing the progeny of M-Lgr5+ cells with nonprogeny, we found that the progeny represented the status of late reprogramming phase (Tcf3 and Tcf4; low) to a higher degree (Ho et al., 2013) (Fig. 4I), which is consistent with the result of Nanog expression. Overall, it appears that the MEFs passing through the Lgr5+ stage are slowly, which results in less AP+ cells, but carefully reprogrammed, which results in more Nanog+ cells, compared with nonprogeny. The reason for the slight difference in the ratio of SSEA1+ cells between them is probably that SSEA1 is an intermediate marker of initiation–maturation phase (Brambrink et al., 2008). Moreover, there is a possibility that failed reprogramming in an early stage (AP+ but Nanog−) is unlikely to occur in the Lgr5+ cells progeny. To investigate whether Lgr5 itself has an effect on reprogramming, we additionally performed experiments of Lgr5 overexpression and knockdown during MEFs reprogramming. Lgr5 overexpression from the beginning of reprogramming induction had a negative effect not only on the result of AP staining but also on Nanog expression (Fig. S4A–C). In contrast, Lgr5 overexpression and knockdown during the route to reprogramming had no effect on reprogramming status (Fig. S4A, B, E and F). It seems that the negative effect of Lgr5 overexpression from the beginning of the reprogramming reflects the mere overexpression effect of the protein, rather than the function of Lgr5 itself.