GSC reprogramming takes place over multiple weeks

GSC reprogramming takes place over multiple weeks and the mechanism is largely unknown. Low efficiency and inconsistent ESL cell formation makes it difficult to study the molecular mechanism of GSC reprogramming, knowledge of which is a prerequisite for improving the efficiency. In addition, GSCs do not reprogram to ESL 7 8-dihydroxyflavone when transfected with the four Yamanaka factors typically used in somatic cell reprogramming, suggesting that GSCs exhibit unique properties constituting a barrier to reprogramming (Morimoto et al., 2012). Here, we report that high TGF-β signaling and high ZEB1 expression result in a barrier preventing the needed upregulation of CDH1 for initiation of mesenchymal to epithelial transition (MET) during GSC reprogramming. Epithelial to mesenchymal transition (EMT) and the reverse process of MET are developmental processes whereby polar epithelial cells that typically interact with a basal membrane interconvert to fibroblast-like migratory cells with mesenchymal secretory properties. SSCs are generally not thought of as being either mesenchymal or epithelial but have certain properties of both epithelial cells (CDH1 expression) (Tokuda et al., 2007) and mesenchymal cells (THY1 expression) (Kubota et al., 2003). In GSC clusters in vitro, and in undifferentiated spermatogonia in vivo, CDH1 is detected in a majority of cells (Fanslow et al., 2014; Tokuda et al., 2007). However, following dissociation of GSC clusters detection of CDH1 by flow cytometry varies depending on the exact method used. CDH1 epitopes are typically sensitive to trypsin digestion, a method commonly used for cell dissociation. We identified a population of rare CDH1+ cells that remain following trypsin digestion (hereafter referred to simply as CDH1+ GSCs). By total RNA sequencing (RNA-seq) analysis of trypsin-digested GSCs we show that CDH1- cells are mesenchymal-like and CDH1+ GSCs are epithelial-like. The presence of a rare population of GSCs with epithelial properties, typified by enhanced CDH1 expression, likely explains the occasional spontaneous emergence of ESL cells that have been observed by others in standard GSC culture conditions in the past. By focusing our study on understanding the molecular mechanism of GSC reprogramming we identify several key positive and negative regulators of the process and define manipulations that increase the CDH1+ GSC population and promote MET and likewise, formation of ESL cells. First, we show CDH1 is essential for GSC reprogramming. Increasing OCT4 promotes reprogramming by upregulating CDH1 and boosting MET. ZEB1 and TGF-β signaling reduce CDH1 and suppress MET and GSC reprogramming efficiency. During the weeks-long process of reprogramming, CDH1+ GSCs gradually increase and the enhanced reprogramming ability of isolated CDH1+ cells suggests that CDH1+ GSCs are poised in a later stage of MET. In summary, our results suggest that CDH1 upregulation constitutes a MET barrier to SSC spontaneous reprogramming that is controlled by ZEB1 and TGF-β signaling, thereby ensuring germ cells are protected from aberrant acquisition of pluripotency. Instead of relying on transfection of genetic material, we define multiple approaches that lead to improved conversion of mouse GSCs to pluripotency that may accelerate the study of human SSC reprogramming and its clinical applications.
Results
Discussion SSCs are unique among adult cell types in that they share expression of many mRNAs in common with ESCs, including all four Yamanaka factors. Although levels are lower compared with ESCs, Oct4, Klf4, and Myc mRNA and protein, and Sox2 mRNA are all expressed in SSCs, implying that SSCs may contain special protective mechanisms to prevent acquisition of pluripotency in the germline and that additional factors may be required for the initiation of SSC reprogramming (Kanatsu-Shinohara et al., 2008). Our results show that relatively high expression of ZEB1, along with relatively high TGF-β signaling in GSCs compared with ESCs, play a pivotal role in preventing MET in GSCs, thereby inhibiting their reprogramming to pluripotency (Figure 6). We show that enhanced reprogramming can be achieved with three general approaches and that each approach is correlated with an increase in the CDH1+ cell population. First, knockdown of Zeb1, but not Zeb2 or Twist2, enhances reprogramming through its effect on CDH1. Second, RepSox treatment, but not SB431542 treatment, results in a greater CDH1+ cell population while increasing reprogramming efficiency. Finally, using isolated CDH1+ cells directly for reprogramming enhances the generation of ESL cells while CDH1− cells exhibit reprogramming efficiency lower than the basal level of spontaneous reprogramming observed in controls.