A miR-372/let-7 Axis Regulates Human Germ Versus Somatic Cell Fates

Abstract

The embryonic stem cell cycle (ESCC) and let-7 families of miRNAs function antagonistically in the switch between mouse embryonic stem cell self-renewal and somatic differentiation. Here, we report that the human ESCC miRNA miR-372 and let-7 act antagonistically in germline differentiation from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). hESC and iPSC-derived primordial germ cell-like cells (PGCLCs) expressed high levels of miR-372 and conversely, somatic cells expressed high levels of let-7. Manipulation of miRNA levels by introduction of miRNA mimics or knockdown with miRNA sponges demonstrated that miR-372 promotes whereas let-7 antagonizes PGCLC differentiation. Knockdown of the individual miR-372 targets SMARCC1, MECP2, CDKN1, RBL2, RHOC, and TGFBR2 increased PGCLC production, whereas knockdown of the let-7 targets CMYC and NMYC suppressed PGCLC differentiation. These findings uncover a miR-372/let-7 axis regulating human primordial germ cell (PGC) specification. Stem Cells 2016;34:1985-1991.

Keywords: Embryonic stem cells; Primordial germ cells; let-7; microRNA; mir-372.

Figure 1

Modeling human PGCLC formation in vitro. (A) Differentiation of representative H9 hESCs into PGCLCs as shown with representative flow cytometry analysis at days 0 and 7 following differentiation. Percentage of double positive (DP) cells ± SD cells expressing both SSEA-1 and C-Kit shown in top right corner (n=5). (B) H9 hESCs were either cultured in media alone, media with 20% FBS, or media with increasing concentration of RA for 4–10 days. Highest rate of SSEA-1+/c-Kit+ cells (PGCLCs), DP cells, was detected in the 10⁻⁸M RA group by flow cytometry analysis after 7 days of differentiation. * p<0.05 relative to all groups. (C) Comparison of percent DP cells resulting from differentiation of various pluripotent human stem cell lines in retinoic acid for 7 days: H1 (XY) hESC, H9 (XX) hESCs, and two independent iPSC lines (BJ3 and BJ4), derived from BJ fibroblasts (XY), shown as mean ± SD (n=5).

Figure 2

PGCLCs express known markers of germ cells. (A) Flow cytometric analyses of intracellular VASA expression of representative H9 derived PGCLCs (n=3) presented as histogram. (B) VASA expression of in representative H9 derived DP cells assessed by confocal microscopy. DAPI (blue) and VASA (red). (C) qRT-PCR analysis of germ cell expressed genes in DP cells differentiated from hESC (H9, H1) and iPSC (BJ3, BJ4) lines (n=3). Expression levels are shown as mean ± SD relative to DN (somatic-like) cells. All are statistically significant against DN cells, p<0.05. (D) Western blot for DAZL comparing H9 and H1 derived DP to DN cells. Gonads = human testicular tissue. (E) qRT-PCR analysis for SYCP1 expression in DP cells derived from H9, H1, BJ3, and BJ4 cells shown as mean ± SD (n=3). (F) SYCP3 expression in H9 derived DP cells assessed by confocal microscopy. DAPI (blue) and SYCP3 (green).

Figure 3

miR-372/let-7 axis in human PGCLC formation. (A) qRT-PCR of mature miRNAs, miR-372 and let-7 family members in H9 derived DP and DN populations shown as mean ± SD, normalized over H9 hESCs (n=3). Let-7 was not detected in DP cells. (B) Percent DP cells obtained from differentiation of H9 and H1 hESCs (day 7) following introduction of miR-372 and let-7 mimics at day 0 of differentiation shown as mean ± SD (n=3). Mutant miR-294 was used as negative control. * p<0.05 relative to control (CT). (C) Percent DP cells obtained from differentiation of H9 and H1 hESCs transduced with control, let-7 sponge, miR-372 sponge, or let-7 sponge followed by transfection with miR-372 mimics at day 0 shown as mean ± SD (n=3). * p<0.05 relative to control. (D) qRT-PCR for germ cell markers in DP cells derived from H9 hESCs transfected with control, miR-372, or let-7 at day 0. Levels shown relative to DN cells as mean ± SD (n=3). * p<0.05 relative to control. (E) qRT-PCR for HOXA1 and HOXB1 genes in DN and DP cells derived from H9 hESCs transfected with mimics as in (D). Levels shown relative to wt-H9 hESCs as mean ± SD (n=3). * p<0.05 (F) Fraction of 100 H9 derived DP cells that stained positively for SYCP3 following differentiation in presence of control miRNA or miR-372 mimic shown as mean ± SD (n=3). (G) Bisulfite sequencing of differentially methylated regions (DMRs) at the H19, PEG1, and SNRPN loci in H9 derived DP cells following introduction of miR-372. Compare to no miRNA control in (Fig S1B).

Figure 4

Effects of miR-372 and let-7 in human PGCLC formation. (A) qRT-PCR of germ cell markers as in Fig 3D, but following introduction of miRNAs at different time points of differentiation H9 hESCs: day 0 (D0) or day 3 (D3) shown as mean ± SD (n=3). * p<0.05 relative to control (CT) DP cells, ** p<0.05 relative to both control and D3 miR-372 DP cells. # p>0.4 between the two groups. (B) Percent of H9 derived DP cells expressing phospho-Histone H3 (PHH3) as assessed by immunofluorescence, shown as mean + SD (n=6). *p<0.05 relative to both CT and let-7 derived DP cells. (C) Percent of H9 derived DP cells following transfection of siRNAs to indicated miR-372 target mRNAs at D0 of differentiation shown as mean ± SD (n=3). * p<0.05. (D) Percent of H9 derived DP cells following transfection of siRNAs to indicated let-7 target mRNAs, CMYC and NMYC shown as mean ± SD (n=3). * p<0.05. (E) Model showing how ESCC and let-7 miRNAs modulate the differentiation of pluripotent cells to hPGCLCs.