Taken together, these results suggest that DSG2 plays an essential role in the acquisition of pluripotency during somatic cell reprogramming. DSG2 Regulates the EMT/Mesenchymal-to-Epithelial Transition Process in PSCs through Hoechst 33258 analog 5 the Regulation of Slug Expression EMT is an important process for embryonic development and is involved in maintaining self-renewal or inducing differentiation in PSCs (Thiery et?al., 2009). a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal. and safety concerns related to teratoma development expression was regulated at the transcriptional level (Figure?2C). In addition, we analyzed the expression level of DSG2 in RA-treated hPSCs and in comparison with that of the three germ-layer markers, namely glial fibrillary acidic protein (ectoderm), -fetoprotein (endoderm), and -smooth muscle actin (mesoderm). DSG2 expression was also markedly downregulated upon RA treatment, whereas that of the three germ-layer markers Hoechst 33258 analog 5 was increased after RA treatment (Figure?2D). To determine whether DSG2 expression is also downregulated upon mESC differentiation, we next examined the expression of mDSG2 in mESC-derived EBs and evaluated the differentiation status, followed by an analysis of SSEA-1 cell surface expression and differentiation-related gene expression (Figures S2A and S2B). Consistent with the hPSC results, mDSG2 expression was also downregulated upon EB formation (Figures S2C and S2D). To further clarify the specificity of DSG2 expression in the undifferentiated hPSCs, we compared the expression of DSG2 between fibroblasts and iPSCs during reprogramming. As shown in Figures 2E and S2E, unlike the hPSC surface markers E-cadherin, EpCAM, and TRA-1-60, DSG2 expression was rapidly increased at the early stage of reprogramming in human foreskin fibroblasts (HFFs). These results suggest that DSG2 takes precedence over conventional surface markers in determining whether PSCs are differentiated or undifferentiated. DSG2 is an adhesion molecule of desmosome complexes. Therefore, we next compared the expression of different members of desmosome between differentiated and undifferentiated cells. As shown in Figures 2F and S3A, DSG2 was highly expressed in undifferentiated PSCs and rapidly downregulated upon EB formation, whereas the expression?of different desmosome components was reversely increased in differentiating cells. To further evaluate DSG2 as a highly specific surface marker of undifferentiated PSCs among the desmosome components, we examined its expression in all human cell types by querying the Amazonia expression atlas (Assou et?al., 2007). is indeed highly expressed in various hESC and human iPSC (hiPSC) lines, as well as in human embryonic carcinoma cell lines, but is absent in more than 250 samples of somatic tissues (Figure?S3B). Together, these results clearly demonstrate that DSG2 is a unique surface marker for undifferentiated hPSCs and is only pluripotent specific among desmosome components. DSG2 Is Essential for Self-Renewal and Suppressing Differentiation Self-renewal involves proliferation with a concomitant suppression of differentiation (Thomson et?al., 1998). To elucidate the role of DSG2 in the self-renewal of undifferentiated hPSCs, we generated stable DSG2-depleted hESC lines via transduction with lentiviral particles harboring short hairpin RNA (shRNA) plasmids targeting DSG2. hESC lines stably exhibiting 85% and 96% downregulation at the mRNA and protein levels, respectively, were selected (Figure?3A), and the effect of DSG2 on the proliferation of hESCs was evaluated by bromodeoxyuridine (BrdU) incorporation and cell-cycle analysis. As shown in Figure?3B, BrdU-positive cells accounted for approximately 86% of the total control shRNA-transfected hESC population. Interestingly, Ntrk1 stable depletion of DSG2 decreased the BrdU-positive cell population compared with that in the control cells (Figure?3B). In addition, cell-cycle analysis revealed that DSG2 downregulation resulted in a smaller S-phase population (Figure?3C). Consistently, cyclin A1, B1, and D1 expression was markedly downregulated in DSG2-depleted hESCs, whereas the cell-cycle inhibitor p27 was markedly upregulated (Figure?3D). Together, these results indicate that DSG2 has an essential role in the proliferation of undifferentiated hPSCs. Open in a separate window Figure?3 DSG2 Is Essential for Self-Renewal and Suppressing Differentiation (A) Expression level of DSG2 in shCtrl and shDSG2 cells was determined by qPCR (top panel) and immunoblotting (bottom panel). Relative expression Hoechst 33258 analog 5 levels of analyzed by qPCR are shown on the graph. ?p? 0.05 versus shCtrl. (B) Cell proliferation was assessed using the BrdU incorporation assay. The graph represents the findings of five independent experiments in which 10 colonies of BrdU-positive cells were counted under each condition. ?p? 0.01 versus shCtrl. Scale bar, 50?m. (C) Cell-cycle distribution was analyzed by FACS using BrdU and propidium iodide incorporation. (D) The expression level of cell-cycle regulators was analyzed by immunoblotting. (E) DSG2-KD hESCs spontaneously differentiated. Phase-contrast images showing Hoechst 33258 analog 5 shCtrl and shDSG2 ESCs. Scale bar, 200?m. (F and G) The gene expression level of core pluripotency transcription factors (F) and three germ-layer markers (G) was assessed by qPCR. (H) Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies. The K6-1 antibody was used for DSG2 detection. Undifferentiated PSC colonies typically have clear borders from the feeders and contain small round cells, with spaces between them, and.