Transient exposure to miR-203 enhances the differentiation capacity 
of established pluripotent stem cells (2020)
Figure 1

Figure 1. Effects of transient induction of miR-203 in iPSC and ESC pluripotency and differentiation potential.

A, miR-203 expression, as determined by qPCR, in five temporal different stages of normal early development: oocyte, 2-cell embryo, morula, compacted morula and blastocyst. RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR. RNA expression is normalized by a housekeeping miRNA (miR-16) that maintained invariable during early embryogenesis. Data represent the mean of 6 different qPCR measures (red bars). P=0.05 (Student's t-test) comparing 2C/morula versus compacted morula/blastocyst.

B, Protocol for reprogramming of miR-203 mutant MEFs into pluripotent iPSCs and subsequent differentiation into embryoid bodies. MEFs were transduced with lentiviruses expressing Oct4, Sox2, Klf4, and cMyc (OSKM) in a constitutive manner. The resulting iPSCs were then treated with doxycycline (Dox) 1 μg/ml during 5 days to induce miR-203 expression. "miiPSCs" refers to iPSCs in which miR-203 was transiently expressed during the indicated 5 days. Dox was removed for 15-30 days before starting the embryoid body generation protocol. Samples for RNA sequencing were taken 30 days after Dox withdrawal.

C, Principal Component Analysis of RNAseq data from wild-type iPSCs (n=3 clones), miiPSCs (n=4) and wild-type ESCs (n=3).

D, Enrichment plots of the 282-gene 2-cell signature Biase et al., 2014() in miiPSCs 10 and 25 days after Dox withdrawal.

E, PCA analysis of RNA-seq and microarray data from miiPSCs and known already-published PSC types. Log2 expression values were normalized to mESCs in each study. Data from miiPSCs (this study), mES cells (each study), 2C-like cells (MacFarlan et al, 2012) and epiblast stem cells (Najm et al, 2011) were analyzed and a total of 17,243 genes were selected. For miiPSCs, clones 1 and 2 were analyzed 10 days after miR-203 exposure, while clones 3 and 4 were analyzed 25 days after miR-203 exposure (see schematic shown in Figure 1B). For Tomato PSCs, Tomato fluorescence is associated to 2C-like retrotransposon expression, thus Tomato-positive cells are identified as 2C-like cells (MacFarlan et al, 2012).

F, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs or ESCs, or from miiPSC and miESCs at day 30 of differentiation. Scale bars, 500 μm.

G, Quantification of the percentage of EBs from panel (F) presenting internal large cavities and EBs beating during the indicated time course. Data are represented as mean ± s.e.m. (n=3 independent experiments). *P<0.05; ***P<0.001 (Student´s t-test).

H, Representative images of EBs derived from human iPSCs transiently transfected with either control (left) or miR-203 mimics (right), at different time points during the differentiation process. Scale bars, 500 μm.

I, Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR-203 mimics as in panel (H) at different time points during the differentiation process. The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel. Data are mean ± s.e.m. (n=3 independent experiments). ***P<0.001 (Student´s t-test).