br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments
Introduction MicroRNAs (miRNAs) are short, endogenous, non-coding RNAs that repress gene expression post-transcriptionally by destabilizing and/or repressing translation of target mRNAs. In the canonical biogenesis pathway, primary microRNA transcripts (pri-miRNAs) are processed in the nucleus by the microprocessor complex, which consists of the RNase III enzyme DROSHA and the double-stranded RNA-binding protein DGCR8, to generate ∼70-nt precursor miRNAs (pre-miRNAs). The pre-miRNAs are then exported to the cytoplasm by EXPORTIN-5 and further processed by another RNase III enzyme, DICER, to generate ∼22-nt mature miRNAs (Figure S1) (Kim et al., 2009). More than 400 miRNAs have been identified in the human (Landgraf et al., 2007), and up to 60% of all human genes may be regulated by miRNAs (Friedman et al., 2009). Given the potentially vast regulatory influence of miRNAs on gene expression and the critical roles of these molecules in embryo development (Bartel, 2009; Sun and Lai, 2013), it is not surprising that miRNAs have emerged as important regulators in reprogramming somatic GDC-0152 Supplier into induced pluripotent stem cells (iPSCs). Together with the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashi and Yamanaka, 2006), co-expression of the miRNA cluster 302/367 or 106a/363; members of the miR-302, miR-294, or miR-181 family; or miR-93 and miR-106b greatly enhance iPSC derivation efficiency (Judson et al., 2013; Li et al., 2011; Liao et al., 2011; Lin et al., 2011; Subramanyam et al., 2011). Furthermore, expression of the miR-302/367 cluster or miR-200c, miR-302, and miR-369 without the Yamanaka factors is sufficient to reprogram human and mouse fibroblasts (Anokye-Danso et al., 2011; Miyoshi et al., 2011). How these miRNAs promote reprogramming is only partially understood. Several mechanisms have been proposed, such as acceleration of mesenchymal to epithelial transition and antagonism of the activities of let-7 family miRNAs, MBD2, NR2F2, and/or other reprogramming suppressors (Hu et al., 2013; Judson et al., 2013; Lee et al., 2013; Liao et al., 2011; Melton et al., 2010). In addition to the miRNAs that promote reprogramming, several miRNAs that inhibit reprogramming, such as the let-7 family members, have been reported (Melton et al., 2010; Unternaehrer et al., 2014). Therefore, it remains unclear whether miRNA activity as a whole facilitates reprogramming and whether miRNAs are required to convert somatic cells into iPSCs. Previous attempts to reprogram Dicer null mouse embryonic fibroblasts (MEFs) were unsuccessful (Kim et al., 2012); however, this observation cannot rule out a requirement of miRNAs in reprogramming because DICER is also critical for the biogenesis of several other small RNAs, such as endogenous small hairpin RNAs (shRNAs), mirtrons, and endogenous small interfering RNAs (siRNAs) (Figure S1) (Babiarz et al., 2008). In this study, we addressed the question of whether miRNAs are required for generating iPSC by reprogramming mouse cells that lack Dgcr8, a factor required specifically for the biogenesis of canonical miRNAs (Figure S1), including all miRNAs implicated in reprogramming (Babiarz et al., 2008; Judson et al., 2013; Wang et al., 2007). We report that Dgcr8-deficient fibroblasts and NSCs can be reprogrammed by the Yamanaka factors, albeit at decreased efficiencies. These results demonstrate that while canonical miRNAs as a whole facilitate reprogramming, they may be dispensable for the derivation of iPSCs.
Results
Discussion miRNAs may confer robustness to biological systems by integrating into transcriptional regulatory circuitry to reinforce genetic programs and buffer stochastic perturbations (Ebert and Sharp, 2012; Hornstein and Shomron, 2006). Mutant mice with deletions of individual miRNA clusters often exhibit only relatively subtle phenotypic defects (Park et al., 2012). More severe phenotypes are usually observed in mutants with compound deletions of functionally redundant miRNA clusters, suggesting that the subtle defects of individual mutations are at least partially due to functional compensation (Park et al., 2012). The Dgcr8 and Dicer mutants, which have complete miRNA loss, exhibit the most extreme phenotypic defects. The mutant ESCs can self-renew and express stem cell markers but are functionally defective in spontaneous differentiation (Kanellopoulou et al., 2005; Wang et al., 2007). These results suggest that the regulatory circuitry of pluripotent cells can be sustained solely by transcription factors, while miRNAs are required to initiate and/or sustain the differentiation. Our data support this notion. Because reprogramming is generally considered to be a de-differentiation process, our data suggest that miRNA activity may not be essential for de-differentiation but is essential for normal tissue differentiation.