Human <i>Dicer</i> expression in <i>Dicer</i>-null MEFs allows generation of iPSCs.

<p>(<b>A</b>) Timeline of reprogramming <i>Dicer</i>-null MEFs rescued with human <i>Dicer</i>. Once human <i>Dicer</i> cDNA integrated into the <i>Dicer</i>^Δ/Δ MEF genome, reprogramming became possible even when factors were transduced 6 days after Cre induction. (<b>B</b>) Rescued iPSCs (ResDcr iPSC) lacked mouse <i>Dicer</i> (<i>mDcr</i>), but instead expressed human <i>Dicer</i> (<i>hDcr</i>) gene, verified by RT-PCR. (<b>C, D, E</b>) <i>Dicer</i>^Δ/Δ MEFs expressing human <i>Dicer</i> can reprogram to become iPSCs. Rescued iPSCs expressing human <i>Dicer</i> showed ESC morphology and stained for alkaline phosphatase (boxed areas represent magnified view) (<b>C</b>), and expressed stem cell markers tested by RT-PCR (<b>D</b>) and immunofluorescence (<b>E</b>). (<b>F</b>) Rescued iPSC promoters for stem cell genes <i>Oct4</i> and <i>Nanog</i> became demethylated, resembling wild-type ESCs. (<b>G</b>) Upon subcutaneous injection into SCID mice, rescued iPSCs formed teratomas that showed differentiation into all three germ layers. (<b>H</b>) Human Dicer can cleave mouse pre-miRNAs into mature miRNAs. qPCR for a panel of mature miRNAs in rescued iPSCs (ResDcr) lacking mouse <i>Dicer</i> demonstrated comparable expression levels to that of wild-type ESCs (W4). In contrast, mature miRNAs were completely depleted in <i>Dicer</i>-null ESCs (Dicer^Δ/Δ). Each value is represented relative to an assigned W4 value of 1.0 for that miRNA. Data are presented as mean +/− SD.</p

<i>Dicer</i>-null MEFs lacking miRNAs fail to reprogram.

<p>(<b>A, B</b>) Timelines of attempt at reprogramming <i>Dicer</i>-null MEFs. The main difference between the two strategies is that (<b>A</b>) transduces reprogramming transcription factors (TFs) 6 days post induction with Cre, while (<b>B</b>) transduces TFs 1 day post induction with Cre. <i>Dicer</i>-null MEFs could not be reprogrammed when reprogramming factors were transduced 6 days after induction. However, reprogramming <i>Dicer</i>-null MEFs was possible when reprogramming factors were transduced 1 day post induction with Cre. (<b>C</b>) <i>Dicer</i>^+/+, <i>Dicer</i>^Δ/+, and <i>Dicer</i>^f/f MEFs consistently reprogrammed into iPSCs with reprogramming factors. These iPSCs stained for alkaline phosphatase. <i>Dicer</i>^Δ/Δ MEFs reprogrammed to form induced stem cell-like cell colonies that stained for alkaline phosphatase when reprogramming factors were transduced 1 day post induction with Cre (Δ/Δ-1dpi). Transducing reprogramming factors 6 dpi (Δ/Δ-6dpi) or not transducing any factors (No TF) never reprogrammed <i>Dicer</i>-null MEFs. Boxed areas represent magnified view. (<b>D</b>) Genomic PCR confirmed induced stem cell-like cell colonies formed by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) having <i>Dicer</i>^Δ/Δ genotype (floxed DNA band). Control iPSC colonies (f/f iPSC) formed without Cre induction had <i>Dicer</i>^f/f genotype (flox DNA band). (<b>E</b>) Residual Dicer protein is still present 1 day after deletion of <i>Dicer</i> gene. By 6 days post induction (dpi) with Cre, residual Dicer protein is completely degraded, inhibiting cellular reprogramming. (<b>F, G</b>) Wild-type ESCs, <i>Dicer</i>^f/f iPSCs (f/f iPSC), and <i>Dicer</i>^Δ/Δ induced stem cell-like cells generated by transducing reprogramming factors 1 day post induction with Cre (Δ/Δ-1dpi) expressed all stem cell markers tested by RT-PCR (<b>F</b>), and immunofluorescence (<b>G</b>). (<b>H</b>) <i>Dicer</i>^f/f iPSCs and <i>Dicer</i>^Δ/Δ induced stem cell-like cells acquired ESC methylation patterns in <i>Oct4</i> and <i>Nanog</i> promoters.</p