Overexpression of miRNAs during in vitro differentiation. (A): Kinetics of the induction of the endogenous miRNAs miR-9, -22, -124a, and -125b. Northern blot assays showing an induction of these miRNAs during expansion of NPs (stage 4) and at early differentiation (stage 5 days 1–6). (B): Overexpression of two duplexes (miR-9/9* and miR-124a) and of four duplexes (miR-9/9*, -22, -124a, and -125b) (Mix of 4) in NPs (stage 4 day 2). Northern blots demonstrating overexpression of the specific miRNAs 48 hours post-transfection. Abbreviations: EB, embryoid body; NP, neural precursor.

Effects of overexpression of miRNAs on neuronal and astroglial cell differentiation and cell death. (A): Percentages of Tuj1⁺ (black) and GFAP⁺ (white) cells among the differentiating neural progenitor cells in the different miRNA transfection conditions: untransfected, mock-transfected, mix-transfected (miR-9/9*, -22, -124a, and -125b), miR-124a+miR-9/9*, and miR-125b+miR-22. The average of three culture replicates for each combination is shown, with error bars indicating the standard deviation (using analysis of variance, p < .03 for the comparisons of GFAP⁺ cells in mock- and mix-transfected cells, and for mock- and miR-124a+miR-9/9*-transfected cells). In independent repetitions of these experiments, similar decreases in the relative number of GFAP⁺ cells were observed in cultures overexpressing miR-9/9* and miR-124a when compared to the mock-transfected cultures. However, between individual experiments, the absolute percentages of Tuj1⁺ and GFAP⁺ cells differed, which was attributed the heterogeneity of the ES cells. (B): To compensate for variations between individual experiments, the ratio of Tuj1⁺ to GFAP⁺ cells in cultures overexpressing the different combinations of specific miRNAs was determined. In all experiments, there was a significant increase of the Tuj1⁺/GFAP⁺ ratios after transfections of four miRNA duplexes or miR-124a+miR-9/9* combined transfections. (C): Percentages of PI-positive (black) and PI-negative (white) cells at stage 5 day 6 of culture in the different miRNA transfection conditions: untransfected, mock-transfected, mix-transfected (miR-9/9*, -22, -124a, and -125b), miR-124a+miR-9/9*, and miR-125b+miR-22. The average of three culture replicates for each combination is shown, with error bars indicating the standard deviation. The treatment of the cultures with the miRNAs did not affect the percentages of PI-positive or PI-negative cells. Abbreviation: PI, propidium iodide.

Effect of miRNA overexpression and inhibition on STAT3 Tyr705 phosphorylation. Western blot analyses with anti-STAT3 and anti-phospho-STAT3 antibodies showed a reduction of phosphorylated STAT3 after transfection with miR-124a and miR-9/9*. Conversely, inhibition of miR-9 by specific 2′-O-methyloligonucleotide (2′-OMe) led to a significant increase of phosphorylated STAT3 levels when compared to mock-transfected controls (p < .05). The lower panel demonstrates relative levels of phopho-STAT3 normalized for total STAT3.

miRNA expression during ES cell–derived neurogenesis in vitro. (A): Schematic view of the five-stage ES cell differentiation protocol used in this study [19-22]. The different stages are indicated as follows: ES; EB; Precursors, selection of Nestin⁺ cells; Expansion, expansion of Nestin⁺ cells; Differentiation, neural differentiation into mature phenotypes. Times of transfection with miRNAs and analyses of their effects on cell development are indicated. (B): Immunocytochemistry demonstrating the development of the main components in the ES cell differentiation protocol: neurons (Tuj1, green) and astrocytes (GFAP, red). Inset shows composite image. Cells were stained at stage 5 day 12. (C): Representative view of an oligonucleotide array corresponding to stage 5 of ES cell differentiation. (D): Clusters of miRNAs induced during the course of differentiation. Three independent RNA samples corresponding to each stage of differentiation and to PNs were analyzed. After normalization, the three data sets were averaged to produce a set of expression levels at a given stage of differentiation. Colors from light to dark blue indicate relative intensities of miRNAs upregulated from stage 1 to stage 5 of differentiation (light blue corresponds to low intensity and dark blue to high intensity). (E): Validation of miRNAs induced from stage 3 (NP) to stage 5 (ND) by Northern blottings. Some of the miRNAs (miRNA-9*, -22, and -125b) maintained their expression levels during the entire stage 5 (ND) of the differentiation protocol, whereas others (miR-9 and -124a) were only transiently elevated. MiR-19 and miR-292–3′ demonstrate alternative expression patterns. 5S rRNA was detected by ethidium bromide staining of the gels prior to transfer to verify equal loading of total RNA. Abbreviations: EB, embryoid body; ES, embryonic stem cell; GFAP, glial fibrillary acid protein; ND, neural differentiation; NP, neural precursor; PN, primary neuron.

Representative example of a typical FCM analysis of cells harvested at stage 5 day 6 of culture. The upper row shows distribution of cells by FSC/SSC (left panel) and the determination of the fraction of PI-positive cells staining in the PI/SSC analysis (middle and right panels). The lower row demonstrates the fraction of Tuj1⁺ (green line) and GFAP⁺ (blue line) cells in mock-transfected cultures (left panel); in miR-124a and miR-9/9* cotransfected cultures (right panel); and in miR-124a, miR-9/9*, miR-22, and miR-125b cotransfected cultures (middle panel). Only the PI-negative cells (upper right panel) were included in this analysis. Abbreviations: PI, propidium iodide; FCM, flow cytometry; FSC, foward scatter; SSC, side scatter.

Effects of miRNA inhibition on neural lineage differentiation. (A): Northern blot experiments showing selective inhibition of the endogenously induced miRNAs miR-9, miR-124a, or miR-125b in NPs with sequence-specific 2′ -O-methyloligonucleotides 48 hours after transfection (stage 4 day 2). (B): The effect of miRNA inhibition on neural differentiation was analyzed by FCM as described in Figure 3, and the ratio of Tuj1⁺ to GFAP⁺ cells was determined. There was a significant reduction of the Tuj1⁺/GFAP⁺ cell ratio when miR-9 was inhibited alone (2′-OMe-9) or together with miR-124a (using analysis of variance, p < .05). Shown is the average of three culture replicates for each condition, with error bars indicating standard deviations. Abbreviations: miRNA, microRNA; FCM, flow cytometry.