br Experimental Procedures br Author Contributions M C
Author Contributions M.C. designed, performed, and analyzed all experiments. S.Y.L. performed MO injections and WISH. W.K. conducted embryo treatments and qPCR. K.A. provided cyp24a1, cyp2r1, and cyp27 plasmids. W.G. provided guidance and edited the manuscript. T.E.N. guided the project and composed the manuscript with M.C.
Acknowledgments We thank S. Alhgren (Tg(Gli-d:mCherry)), N. Lawson (Tg(EPV.Tp1CMmu.Hbb:EGFP)), and P. and K. Crosier (Tg(Runx1P2:eGFP)) for transgenic lines; S. Wente and W. Chen for CRISPR-Cas9 plasmids; and the BIDMC Flow Cytometry Core for technical assistance. This study was supported by a V-Foundation Scholar Award (T.E.N.) and the Harvard Stem Cell Institute (T.E.N.).
Introduction Somatic AKT inhibitor VIII manufacturer can be reprogrammed to a pluripotent state (induced pluripotent stem [iPS] cells) by defined transcription factors (Oct3/4, Sox2, Klf4, and c-Myc, hereafter referred to as OSKM) (Takahashi and Yamanaka, 2006). iPS cells are almost equivalent to embryonic stem cells (ESCs) in their properties (Okita et al., 2007; Mikkelsen et al., 2008). Many studies have been done to elucidate the mechanism of reprogramming (Stadtfeld et al., 2008; Samavarchi-Tehrani et al., 2010; Buganim et al., 2012), and several factors that affect reprogramming efficiency have also been revealed (Huangfu et al., 2008; Onder et al., 2012; Costa et al., 2013). But the importance of cell-cell interactions and secreted, extracellular molecules in reprogramming has not been extensively studied. To approach this problem, we focused on EPHRIN receptor (EPH)/EPHRIN (EFN) signaling pathways. EPH receptors and EPHRINs are both anchored to the plasma membrane and form an important cell communication system with roles in normal physiology and disease pathogenesis (Pasquale, 2010). The EPH/EPHRIN signals propagate bidirectionally into both the EPH-expressing cells (forward signaling) and the EPHRIN-expressing cells (reverse signaling) to elicit different effects on each cell (Pasquale, 2008). Several EPH receptors and EPHRINs have truncated forms, which are secreted and function to propagate or repress signals (Aasheim et al., 2000; Dawson et al., 2007; Wykosky et al., 2008). Recently, some of EPH receptors and EPHRINs have been identified as regulators of stem and progenitor cell proliferation (Holmberg et al., 2005, 2006; Jiao et al., 2008; Nomura et al., 2010). However, whether EPH/EPHRIN signaling pathways regulate cell reprogramming has been unknown.
Discussion Previous studies have demonstrated that the inhibition of ERK1/2 activity plays a role in acquiring and maintaining the naive pluripotent state of ESCs and iPS cells (Silva et al., 2008; Ying et al., 2008). However, neither ERK1/2 activity changes during reprogramming nor their role in reprogramming has been addressed. Our data show that the reduction of ERK1/2 activity by truncated EPHA7 occurs in the middle phase (around day 6) of reprogramming and is essential for driving reprogramming. The expression of truncated EPHA7 during reprogramming is transient, unlike most of the reprogramming-related genes that have high expression levels in mature ESCs or iPS cells. Microarray data in earlier reports (Mikkelsen et al., 2008; Sridharan et al., 2009; Samavarchi-Tehrani et al., 2010) also indicated that EPHA7 was mainly expressed in reprograming cells but not in ESCs or iPS cells. Moreover, our results show that the regulation of ERK1/2 activity by truncated EPHA7 is specific to cell reprogramming and may not be involved in pluripotency maintenance in ESCs and that the expression of eph receptors and ephrins is quite low in ESCs and iPS cells, indicating that EPH-EPHRIN signaling might not be involved in the regulation of the ERK1/2 pathway in ESCs.
Acknowledgments This work was supported by JST, CREST (to E.N.); the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to E.N.); and the Japanese Government (MEXT) Scholarship Program (to J.L.).