Figure 4. Knockdown of Mbnl proteins enhances reprogramming efficiency and kinetics.

a) Experimental scheme. b) qRT-PCR quantification of mRNA expression levels of endogenous Oct4 and Nanog (data for additional genes in Supplementary Fig. 11a). Secondary MEFs were transfected with control siRNAs (siControl), siRNAs targeting Mbnl1 and Mbnl2 (siMbnl1+2) or Oct4 (siOct4) and treated with doxcycline (Dox) for 3 days (blue bars) or 5 days (red bars) before analysis. Empty bars, secondary MEFs without Dox induction. Values represent Means ± Range (n=3). c) Top, quantification of SSEA1-stained area change relative to siControl at day 5 post Dox-induction; values represent Means ± Range (n=3); bottom, representative images of SSEA1 staining. Scale bar = 100 µm. d) Top, quantification of Dox-independent iPSC colony formation. Secondary MEFs were treated with Dox for 8 days followed by 5 days of Dox withdrawal and counting of alkaline phosphatase (AP)-positive colonies; bottom, representative images of AP staining. e) Teratoma assay assessing the pluripotency potential of iPSCs derived from secondary MEFs following knockdown of Mbnl proteins. Hematoxylin and eosin staining, with additional staining/immunolabeling using periodic acid-Schiff (PAS; for detection of glycogen or glycoprotein producing cells), Safranin O (SafO; for detection of cartilage), or antibody to neuronal nuclear antigen (NeuN); additional teratoma analysis and chimera testing of the pluripotency potential of siMbnl iPSCs in Supplementary Figs. 12 and 13. Scale bar = 100 µm. f) Top, experimental scheme for clonal analysis. Upon Dox removal at day 21, clones derived from single cells either survive and form iPSCs (“transgene-independent”) or do not survive (“transgene-dependent”). Bottom, analysis of total Mbnl1/Mbnl2 mRNA expression (left) and percentage of total PSI change (right) for Foxp1 exon 16b in transgene-independent (red) and transgene-dependent (blue) clones, at day 21, where total PSI change is the PSI difference between MEFs and iPSCs during reprogramming. g) Quantification (by morphological examination) of human iPSC colonies formed by reprogramming BJ fibroblasts expressing shRNA targeting GFP (shGFP) or MBNL1 (shMBNL1). h) Immunostaining of human iPSCs derived from shMBNL-expressing BJ fibroblasts for TRA1-60, NANOG, SSEA4 and OCT4 pluripotency markers. Scale bar = 50 µm. Additional characterization of human iPSCs in Supplementary Fig. 15. p-values of one-sided t-tests shown for all comparisons in Fig 4. i) Model for the role of MBNL proteins in the regulation of ESC-differential AS, pluripotency and iPSC reprogramming.