br Results br Discussion Different

Results
Discussion Different routes of reprogramming have been described even when starting with MEFs but these are restricted to late stages of reprogramming and have been demonstrated with different combinations of reprogramming factors (Parchem et al., 2014). Interestingly, recent reports also suggest that stalled clonal intermediates of MEF reprogramming can be obtained that express NANOG with lower levels of E-cadherin compared with fully reprogrammed iPSCs (Tonge et al., 2014). These clones also express very high levels of the reprogramming factor transgenes. An important distinction of our work is that half the reprogramming colonies, rather than a few clones that can be stabilized, transition through a cadherin-independent NANOG-expressing phase. Interestingly, the inhibition of DOT1L in NSCs boosts the total number of N+ colonies and enhances the emergence of E-cadherin, resulting in all NANOG colonies also having E-cadherin. Since our assays used fixed reprogramming cultures, we could not estimate whether these increases were due to NANOG-positive colonies gaining E-cadherin or vice versa. E-cadherin plays an important signaling role in maintaining AEG 3482 of Nanog in ESCs (Hawkins et al., 2012). However, stem cell lines can also be derived and maintained from the blastocysts that express Nanog, Oct4, and Sox2 under lower E-cadherin expression conditions (Chou et al., 2008). Taken together, these results and previously published work (Pieters and van Roy, 2014) suggest that there may be at least two independent but interlinked signaling pathways from E-cadherin to Nanog expression. During MET in reprogramming MEFs, Sox2 suppresses the expression of Snai1, which is itself a suppressor of E-cadherin (Li et al., 2010). Therefore, increased SOX2 should lead to increased E-cadherin. However, NSCs, which express enough endogenous Sox2 that they can be reprogrammed without the addition of exogenous Sox2 (Kim et al., 2008), do not readily upregulate E-cadherin. By overexpressing Sox2 in MEF using retrovirus prior to induction of OSKM with dox, we demonstrated that additional Sox2 did not alter the ratio of Nanog colonies that had E-cadherin in this setting, although it is possible that the expression levels obtained may not have been sufficient. In initial experiments, we have found that SOX2 is not pre-bound to the Nanog promoter in NSCs (data not shown), although it remains possible that N+E− cells may acquire transient binding of SOX2 to facilitate Nanog expression. In conclusion, we demonstrate that different somatic cells types have a distinct order of getting to the pluripotent state, suggesting unique barriers to the cell fate change effected by reprogramming.
Experimental Procedures
Acknowledgments We thank Prof. James Thomson and Mitch Probasco for use of the flow cytometer; Paul Ehrlich for technical assistance; and advice from Dr. Srikumar Sengupta on RNA isolation from fixed samples; Dr. Laura Moody and Prof. Albee Messing for astrocyte isolation; Dr. Weixiang Guo and Prof. Xinyu Zhao for NSC isolation. This work was supported by the UW-Madison, a Shaw Scientist award from the Greater Milwaukee Foundation, and a Basil O’Connor award from the March of Dimes Foundation to R.S. S.A.J. was partly supported by a postdoctoral fellowship from the Stem Cell and Regenerative Medicine Center of UW-Madison and K.A.T by the GRFP (NSFDGE-1256259).
Introduction End-stage heart failure is generally characterized by an insufficient number of functional cardiomyocytes (CMs) (Towbin and Bowles, 2002). At this critical stage, cell transplantation is a promising approach for increasing the number of functional CMs. Thus, transplantation with induced pluripotent stem cells (iPSCs) represents a promising treatment for this condition (Yoshida and Yamanaka, 2010, 2011); accordingly, various studies have examined the potential application of iPSCs for cell transplantation therapy in the heart (Higuchi et al., 2015; Kawamura et al., 2012; Miki et al., 2012).