Embryonic stem cell-specific microRNAs promote induced pluripotency - PubMed (original) (raw)

Embryonic stem cell-specific microRNAs promote induced pluripotency

Robert L Judson et al. Nat Biotechnol. 2009 May.

Abstract

This report demonstrates that introduction of microRNAs (miRNAs) specific to embryonic stem cells enhances the production of mouse induced pluripotent stem (iPS) cells. The miRNAs miR-291-3p, miR-294 and miR-295 increase the efficiency of reprogramming by Oct4, Sox2 and Klf4, but not by these factors plus cMyc. cMyc binds the promoter of the miRNAs, suggesting that they are downstream effectors of cMyc during reprogramming. However, unlike cMyc, the miRNAs induce a homogeneous population of iPS cell colonies.

PubMed Disclaimer

Figures

Figure 1

Figure 1

The ESCC miRNAs promote three-factor, but not four-factor induced pluripotency. (a) Fold increase of day 10 Oct-GFP+ colonies with retroviruses expressing Oct4, Sox2, and Klf4 (OSK) together with 16nM miRNA mimic relative to transfection reagent only (Mock). N=3. Raw data in Supplementary Table 1. (b) Sequence of miR-290 cluster, miR-302d, and miR-294 seed sequence mutant. Bold indicates seed sequence. Capitals indicate point mutations. Grey box highlights ESCC seed-sequence. (c) Fold increase in day 10 Oct4-GFP+ colonies with addition of mimic to OSK in the presence (light grey) or absence (dark grey) of cMyc retrovirus. Bars represent the number of GFP+ colonies after mimic transfection divided by the number of GFP+ colonies after mock transfection. N= 6, 26, 2, 5, & 3 left to right. Asterisk indicates p-value ≤ 0.0001. Raw data for bars 1&2 in Supplementary Table 2. (d) Percent day 10 Oct4-GFP colonies for OSK plus 1.6, 16 and 160nM transfected miR-294 mimic or 160nM miR-1 relative to OSKM. (e) Quantitative RT-PCR for endogenous pluripotency markers in control (V6.5) ES cells, MEFs, and miR-294-iPS lines. N=3, 3, & 5. RPL7 was used as input control. Data was normalized to ES expression. (f) Quantitative RT-PCR for exogenous Oct4, Sox2, and Klf4 in MEFs 6 days after viral infection, control (V6.5) ES cells, and MEFs (each N=3) and 5 individual miR-294-iPS lines. Horizontal black bars indicate Ct > 40. RPL7 was used as input control. Data was normalized to MEF expression 6 days after viral infection. (g) X-gal staining demonstrates miR-294-iPS chimeric contribution to ectoderm (neural tissue, N), endoderm (lung, L), and mesoderm (cartilage, C). (h) GFP expression in genital ridges of E12.5 chimera demonstrates Oct4-GFP miR-294-iPS contribution to germline. All error bars indicate standard deviation.

Figure 2

Figure 2

Characterization of the relationship between Myc and miR-294. (a) cMyc (blue) and nMyc (yellow) bind the miR-290 cluster promoter. ChIP-seq data reads were aligned to the mm9 assembly of the genome and peaks were generated with Findpeaks. Vertical hash marks denote the positions of the miR-290 cluster miRNAs. (b) Quantitative RT-PCR for total mature miR-294 expression in control (V6.5) ES cells, MEFs, and MEFs infected with viruses expressing Sox2 (S), Oct4 (O), Klf4 (K) or cMyc (M). RNA was collected on days 2 and 6. N=3. Horizontal black bars indicate Ct > 40. Sno202 was used as input control. Data was normalized to ES cells. (c) H3K4me3 (green) and H3K27me3 (red) surrounding the miR-290 cluster in MEFs. Chip-seq data were analyzed as described in a. (d) Quantitative RT-PCR for total mature miR-294 expression in control (V6.5) ES cells (E), MEFs (M), MEFs infected with either Oct4, Sox2, Klf4 (OSK); Oct4, Sox2, Klf4, and cMyc (OSKM); or OSK+miR-294, and established iPS lines resulting from these conditions (iPS). RNA was collected on days 2, 6, and 10 of reprogramming. Three independent experiments are shown. Horizontal black bars indicate Ct > 40. Sno202 was used as input control. Data was normalized to ES cells. (e) Total cell number during reprogramming. Cells were counted on day 7 after infection with OSKM or OSK +/− miRNA mimic. Concentrations of miR-294 mimic: 1.6, 16 and 160nM. Concentration of miR-1 mimic: 160nM. (f) GFP negative colonies in presence of cMyc. Oct4-GFP+, ES-like colonies (black arrow) and GFP-negative, nonES-like colonies (white arrow). (g) Quantification of number of day 10 GFP-negative colonies after infection with OSKM or OSK +/− miR-294 mimic. All error bars indicate standard deviation of N=3.

Similar articles

Cited by

References

    1. Marson A, et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell. 2008;134:521–533. - PMC - PubMed
    1. Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specific MicroRNAs. Dev Cell. 2003;5:351–358. - PubMed
    1. Wang Y, et al. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat Genet. 2008;40:1478–1483. - PMC - PubMed
    1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. - PubMed
    1. Blelloch R, Venere M, Yen J, Ramalho-Santos M. Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell. 2007;1:245–247. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources