miR-135b enhances reprogramming of MEFs to iPSCs. (A) miR-135b enhances Oct4-GFP+ colony formation. The indicated microRNA mimics were transfected at a final concentration of 50 nM into MEFs on Day 0 and again on Day 5 after 4F transduction. GFP+ colonies were counted at Days 11–12. Data represent two independent experiments with triplicate wells. Let-7a was used as a control. (*) P < 0.05. (B) miR-135b increases the percentage of Oct4-GFP+ cells. Cells from the indicated treatments were harvested at Day 14 post-infection with 4F and paraformaldehyde-fixed prior to FACS analysis to determine the percentage of GFP+ cells. Data represent two independent experiments with triplicate wells. (*) P < 0.05. (C) Blocking of miR-135b compromises reprogramming. MicroRNA inhibitors were transfected into MEFs on Days 0 and 5 post-infection with 4F. GFP+ colonies were counted at Days 11–12 post-infection. Data represent two independent experiments with triplicate wells. (*) P < 0.05. (D) miR-135b iPSCs reach a fully reprogrammed state. miR-135b-transfected iPSCs were fixed with paraformaldehyde and stained for alkaline phosphatase, Nanog, and SSEA1 expression. Endogenous Oct4 expression was monitored by GFP expression. (E) Teratoma formation confirms the pluripotency of miR-135b iPSCs. 1 × 106 iPSCs were injected into athymic nude mice and tumors were harvested for H&E staining 3–4 wk later. (F) miR-135b iPSCs show expression profiles similar to mES cells. Total RNA from miR-135b iPSCs was used for mRNA expression profile analysis and compared with original MEFs and with mES cells. The three tested miR-135b iPSC clones (clones 1, 3, and N1) showed similar expression patterns to mES cells, which were quite different from the expression profile of the original starting MEFs. (G) Chimeric mouse from miR-135b iPSC clone 4. (H) miR-135b iPSC could contribute to the germline of recipient embryos (miR-135b iPSC clone 4).
