The RNAi machinery functions in mouse iPSC induction. (A) Knock-down of mouse RNAi machinery gene Ago2 by shRNAs. MEFs were transduced with lentiviral shRNAs plus 4 μg/μl polybrene, and total RNAs or proteins were harvested at day 3 post-transduction. mRNA and protein levels of targeted genes were analysed by RT–qPCR and western blotting, respectively. pLKO is the empty vector control for the shRNA lentiviral vectors. pGIPZ is a lentiviral vector expressing a non-targeting shRNA. (B) Knock-down of Ago2 dramatically decreases iPS induction by 4F. Primary MEFs were transduced with the four reprogramming factors (OSKM (4F)) plus shRNA Ago2. Colonies were stained at day 14 post-transduction for alkaline phosphatase (AP), which is a marker for mES/iPS cells. pLKO and pGIPZ vectors served as negative controls. (C) Knock-down of Ago2 decreases iPS induction by OSK. Colonies were stained and quantified for AP at day 21 post-transduction. Error bar represents s.d. of duplicate wells. (D) GFP+ colony quantification of iPSC with shAgo2. GFP+ colonies were quantified at day 21 post-transduction. Error bar represents s.d. of duplicate wells.

miR-93 and miR-106b greatly enhance iPS induction. (A) Reprogramming assay timeline. miRNA mimics or inhibitors were transfected at a final concentration of 50 nM on days 0 and 5 of reprogramming. GFP+ colonies were quantified at day 11 for 4F induction and days 15–20 for OSK induction. (B) Representative images of GFP+ colonies from reprogrammed Oct4-GFP MEFs transfected with miRNA mimics. Arrows indicate GFP+ colonies. (C) miR-93 and miR-106b mimics enhance iPS induction with 4F induction. Oct4-GFP MEFs were transfected with 50 nM of the indicated miRNAs at days 0 and 5 of reprogramming. GFP+ colonies were quantified at day 11 post-transduction. Fold induction and error bars were calculated from three independent experiments in triplicate wells. ^***P<0.0001. (D) The enhancing effect of miR-93 and miR-106b is observed using the OSK system. miRNA mimics were transfected as in 4F experiments. GFP+ colonies were quantified on days 15–20. Error bars represent s.d. of three independent experiments in triplicate wells. ^***P<0.0001. (E) miR-93 and miR-106b inhibitors dramatically decrease reprogramming efficiency. miRNA inhibitors were transfected at a final concentration of 50 nM. The experimental timeline was the same as in miR mimic transfections. Error bars represent s.d. of three independent experiments in triplicate wells. ^***P<0.0001. (F) miR-106b promotes the MET transition during 4F-mediated reprogramming. miR-106b mimic was transfected into MEFs and cells were harvested at different time points to analyze E-Cadherin expression. Fold induction of ECad was normalized to day 4 samples after 4F infection. Error bars represent s.d. of three independent experiments. ^*P<0.001. (G) miR-106b promotes the MET transition in OSK-infected cells. The experimental procedures were the same as in (F). Fold induction of ECad was normalized to day 4 samples after OSK infection. Error bars represent s.d. of three independent experiments. ^*P<0.001. (H) Inhibition of miR-106b decreases induction of MET process. The experimental procedures were the same as in (F), except anti-miR oligos were transfected instead of miR mimics. Fold induction of ECad was normalized to day 4 samples after 4F infection. Error bars represent s.d. of three independent experiments. ^*P<0.001.

miR-93 and miR-106b directly target mouse p21 and Tgfbr2. (A) miR-93 and miR-106b transfection decreases p21 protein levels. Oct4-GFP MEFs were transfected with 50 nM miR mimics and harvested 48 h after transfection for western analysis. Actin was used as the loading control. (B) p21 is knocked down efficiently by siRNA. p21 siRNA- and control-transfected MEFs were harvested at 48 h and RT–qPCR, and western blotting was undertaken to verify p21 expression. p21 mRNAs were normalized to GAPDH. (C) Knock-down of p21 by siRNA enhances iPSC induction. MEFs were infected with 4F virus, and siRNAs were transfected following the same timeline as miRNAs mimic transfection. GFP+ colonies were quantified at day 11. Error bars represent three independent experiments in triplicate wells. (D) miR-93 and miR-106b transfection decreases TGFBR2 expression. Transfected cells were harvested at 48 h for western blotting. (E) Tgfbr2 is knocked down by siRNAs. Relative Tgfbr2 mRNA levels were normalized to those of GAPDH. (F) Knock-down of Tgfbr2 by siRNAs enhances iPSC induction. Error bars represent four independent experiments in triplicate wells.

miR-17, miR-25, miR-106a and miR-302b clusters are induced during early stages of reprogramming. (A) Induction of 10 miRNA clusters in the early stages after transduction with the four reprogramming factors. miR RT–qPCR was used to quantify expression levels of representative miRNAs from clusters highly expressed in ES cells. Total RNAs from day 0 MEFs and from MEFs transduced with reprogramming factors at day 4 post-infection were analysed. Blue bars: day 4 MEFs; white bars: day 0 MEFs. Asterisks indicate induced miRNAs. (B) Seed region comparison of different miR clusters induced at day 4 post-reprogramming factor transduction. Red indicates similar seed regions. (C) Representative miRNAs can be induced with different combinations of reprogramming factors. miRNA expression was quantified at 4 days post-transduction.

Characterization of iPS clones derived from miR mimic experiments. (A) Derived clones activate endogenous Oct4-GFP expression. Colonies were picked starting at day 12 post-OSKM transduction with miRNA mimics and maintained on irradiated MEF feeder plates. Green fluorescence is GFP signal from the endogenous pou5f1 locus. (B) Clones shown in (A) are positive for alkaline phosphatase staining and immunostaining of ES-specific markers based on Nanog and SSEA1 staining. Hoechst 33342 was used for nuclear staining. (C) RT–PCR of endogenous ES markers. Total RNAs were isolated from iPS cell lines at day 3 post-passage. ES cell-specific markers such as ERas, ECatI, Nanog and endogenous Oct4 expression were analysed by RT–PCR. (D) Cells from all three germ layers can be obtained in embryoid body (EB) assays using derived iPS clones. iPS cells were cultured for EB formation at ∼4000 cells/20 μl drop for 3 days, and EBs were then reseeded onto gelatin-coated plates for further culture until day 12–14, when beating cardiomyocytes were observed (Supplementary Video 1). Cells were immunostained with different lineage markers: β-tubulin III, ectoderm marker; AFP, endoderm marker; α-actinin, mesoderm marker. (E) Teratomas form from injected iPS cells. In total, 1.5 million cells were injected into each mouse, and tumours were harvested 3–4 weeks after injection for paraffin embedding and H&E staining. Structures representing different lineages are labeled. Representative pictures are from miR-106b clone 1#. (F) Derived clones can be used to generate chimeric mice. iPS cells were injected into blastocysts from albino or black C57B6 mice (NCI) and the contribution of iPSCs can be seen with agouti or black coat colour.

Reprogramming is enhanced by other family miRNAs. (A) miR-17 and miR-106a can also enhance reprogramming efficiency. miR-17 and miR-106a mimics were transfected into MEFs at a final concentration of 50 nM. GFP+ colonies were quantified at day 11 post-transduction. Error bars represent three independent experiments in triplicate wells. (B) miR-17 and miR-106a also target p21. p21 western blotting was performed 2 days after transfection of miRNA mimics into MEFs. (C) miR-17 and miR-106a target TGFBR2 expression. miRNA mimics were transfected into MEFs at 50 nM final concentration. Western blotting was performed 2 days post-transfection. (D) Model for the role for miRNAs during iPS induction. Several miRNAs, including miR-17, miR-25 and miR-106a clusters, are induced during early stages of reprogramming. These miRNAs facilitate full reprogramming by targeting factors that antagonize the process, such as p21 and other unidentified proteins. Up and down represent the potential different stages and barriers during reprogramming process and dashed line indicates that barriers for reprogramming which are lowered upon miRNAs induction in reprogrammed cells.