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    • The mechanisms involved in reprogramming somatic cells to

    2018-10-20

    The mechanisms involved in reprogramming somatic pde inhibitors to iPSCs by the Yamanaka factors remain poorly understood. Because of the low efficiency and slow kinetics of most reprogramming systems, molecular events that direct somatic cells to pluripotency have been difficult to define. Recent work has demonstrated that miRNAs such as miR-294, miR-302, and miR-181 family members facilitate (Judson et al., 2013; Li et al., 2011; Liao et al., 2011; Lin et al., 2011; Melton et al., 2010; Subramanyam et al., 2011), but let-7 family members inhibit, reprogramming (Melton et al., 2010; Unternaehrer et al., 2014). Therefore, it remains unclear whether miRNA activity as a whole promotes reprogramming and whether miRNAs, in particular those miRNAs shown to promote reprogramming, are necessary for the derivation of iPSCs. Here, we present data demonstrating that while miRNA activity as a whole facilitates reprogramming, the derivation of iPSC may be achieved without canonic miRNAs. Because Dgcr8Δ/Δ fibroblasts do not survive extended culture times, they must be transduced with STEMCCA virus for reprogramming 7 or 10 days after Cre expression. Our qPCR analysis detected negligible levels of miRNAs in these cells (Figure 1B), consistent with a previous report that mature miRNAs are effectively eliminated in DicerΔ/Δ MEFs 6 days after transduction of Cre-expressing lentivirus (Kim et al., 2012). Nevertheless, to exclude the possibility that residual miRNAs may be present and essential for reprogramming, we reprogrammed Dgcr8Δ/Δ NSCs, which can be propagated for longer terms to ensure exhaustion of residual miRNAs before transduction of reprogramming factors (Figure 2A). The prolonged culture of Dgcr8Δ/Δ NSCs exhausts residual miRNAs by two mechanisms. First, the Dgcr8Δ/Δ NSCs are proliferative; therefore, residual miRNAs are diluted out with each cell division. We split Dgcr8Δ/Δ NSCs at a 1:5 ratio for each passage, resulting in the expansion of any single cell to 1.9 × 106–2.4 × 108 (59–512) progeny cells and making it highly unlikely that any residual miRNAs could persist at a biological meaningful concentration by the end of 9–12 passages. Second, the sorted Dgcr8Δ/Δ NSCs were reprogrammed after a continuous culture for 45–60 days, which is a sufficient duration to achieve complete degradation of residual miRNAs. Therefore, our data conclusively demonstrate that reprogramming of NSCs may be achieved solely by transcriptional factors without any miRNA activities. Kim et al. (2012) reported that iPSCs could not be isolated from MEFs 6 days after disruption of Dicer, which is inconsistent with our data on reprogramming Dgcr8Δ/Δ fibroblasts (Figure 1). DICER is required for the biogenesis of not only canonical miRNAs but also other small RNA species, such as endogenous siRNAs, shRNAs, mirtrons, and short interspersed nuclear element-derived RNAs (Figure S1) (Babiarz et al., 2008). The discrepancy between the data on reprogramming of Dicer-deficient cells and those of Dgcr8-deficient cells probably reflects the activities of some DICER-dependent but DGCR8-independent small RNAs. Alternatively, the poorer proliferation capacity of DicerΔ/Δ fibroblasts may contribute to the failure of iPSC derivation (Kim et al., 2012), which is known to be proliferation dependent (Smith et al., 2010). Recently, Zhang et al. (2013) reported that they were unable to isolate iPSCs from human foreskin fibroblasts that were null for the endogenous miR-302/367 cluster. These data suggested the miR-302/367 cluster is required for human somatic cell reprogramming. Although this result is not consistent with our findings, the discrepancy may be explained by the potential difference in somatic cell reprogramming and/or in the self-renewal of human and mouse pluripotent stem cells (Nichols and Smith, 2009). Alternatively, the discrepancy may be caused by the different miRNA deficiencies of the reprogrammed fibroblasts. In our study, the Dgcr8Δ/Δ fibroblasts lacked miRNAs both promoting reprogramming, such as the miR-290s and miR-302s, and inhibiting reprogramming, such as the let-7s; however, the fibroblasts used by Zhang et al. (2013) were only deficient in the reprogramming-promoting miR-302/367 cluster. The fine balance between pluripotency-promoting and differentiation-inducing miRNAs has been demonstrated to play critical roles in the maintenance of the ground state of pluripotency (Kumar et al., 2014), which could be similarly required in reprogramming. Nonetheless, this is an interesting observation that deserves further investigation.