Figure 4

Knockdown of miR-128a during in vitro differentiation of murine Isl1-Cre/ROSA26^mTmG induced pluripotent stem cells (iPSCs) (iITG-iPSCs). (A) iITG-iPSCs (scale bar: 500 µm) were differentiated using the hanging drop method. Transfections of LNA probes for knockdown of miR-128a (LNA-128) and corresponding controls (LNA-Ctr) were performed after 0.75 weeks (wks) and after 2 wks of differentiation. Analysis was performed on respective timepoints between 1 and 3 wks. Abbreviations: EB: Embryoid bodies; FACS: Flow cytometry; (B) MiR-128a was significantly downregulated by LNA-128 from 1 wk until 3 wks compared to respective LNA-Ctr (n = 8,7, Mann–Whitney tests, 3 assays in triplicates). (C) Isl1-GFP-positive CPCs after 2 wks (upper panel, scale bars: 500 µm) and after 3 wks (lower panel, scale bars: 500 µm) of in vitro differentiation after transfection with either LNA-Ctr or LNA-128. Images are an overlay between phase contrast and fluorescent microscopic pictures. (D) The frequency of Isl1-GFP-positive CPCs after miR-128a knockdown was significantly increased to the double amount compared to LNA-Ctr (n = 9, p = 0.024, t-test; 3 assays in triplicates) after 2 wks. At later timepoints, the Isl1-CPC frequency was not significantly affected by miR-128 knockdown (3 assays in triplicates at 2.25 wks, 2 assays in triplicates at 3 wks). (E) Isl1 expression was tendentially upregulated during iITG-iPSC differentiation after miR-128 knockdown (n = 6, 2 assays in triplicates). (F) Beating frequency (beats per minute, bpm) of differentiated iITG-iPSC EBs was reduced after miR-128 knockdown (2 and 2.25 wks: 3 assays in triplicates, 2.75 (t-test) and 3 wks (Mann–Whitney test): Two assays in triplicates, 4–9 videos per condition, 3 independent observers). Data are represented as means ± SEM. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.