Embryonic stem cell–specific microRNAs promote induced pluripotency

Abstract

This report demonstrates that introduction of physiologically relevant miRNAs can enhance somatic cell reprogramming. The mouse embryonic stem (ES) cell specific microRNAs (miRNA) miR-291-3p, miR-294, and miR-295 enhanced the efficiency of Klf4, Oct4 and Sox2 induced pluripotency. These miRNAs did not further enhance reprogramming in the presence of cMyc. cMyc binds the promoter of these miRNAs, suggesting that they are downstream effectors of cMyc promoted pluripotency. However, unlike exogenous cMyc, these miRNAs induced a homogeneous population of reprogrammed colonies suggesting overlapping and independent functions of cMyc and the miRNAs.The miR-290 cluster constitutes over 70% of the entire miRNA population in mouse ES cells1. Its expression is rapidly down-regulated upon ES cell differentiation2. A subset of the miR-290 cluster, called the embryonic stem cell cycle (ESCC) regulating miRNAs, enhances the unique stem cell cell cycle3. This subset includes miR-291-3p, miR-294, and miR-295. To test whether ESCC miRNAs could promote the induction of pluripotency, we introduced these miRNAs along with retroviruses4 expressing Oct4, Sox2, and Klf4 (OSK) into mouse embryonic fibroblasts (MEFs). The MEFs carried two reporters: an Oct4-GFP reporter that activates GFP with the induction of pluripotency and ubiquitous expression of a β-galactosidase/neo fusion from the Rosa26 locus5. MiRNAs were introduced on days 0 and 6 post-infection by transfection of synthesized double-stranded RNAs that mimic their mature endogenous counterparts. This method transiently recapitulates ES-like levels of the miR-290 cluster miRNAs ( Supplementary Fig. 1).OSK plus miR-291-3p, miR-294, or miR-295 consistently increased the number of Oct4-GFP+ colonies as compared to controls transduced with OSK plus transfection reagent (Fig. 1a). The miR-294 mimic showed the greatest effects, increasing efficiency from 0.01-0.05% to 0.1-0.3% of transduced MEFs. Introduction of a chemically synthesized miR-294 premiRNA similarly enhanced reprogramming (Supplementary Figure 2). Two other members of the miR-290 cluster that are not ESCC miRNAs, miR-292-3p and miR-293, did not increase colony number (Fig. 1a). The ESCC miRNAs share a conserved seed sequence, which largely specifies target mRNAs (Fig. 1b). MiR-302d, a member of another miRNA cluster that has the same seed sequence also enhanced reprogramming (Fig. 1b&c). Mutation of the seed sequence in miR-294 blocked the increase in colony number (Fig. 1b&c). In summary, together with Oct4, Sox2, and Klf4, the ESCC miRNAs and related

Mechanism and methods to induce pluripotency

“…In ESCs, cMyc was found to occupy promoters of active genes and ESC specific microRNAs (miRNAs) such as miR- 291-3p, miR-294, miR-295, miR-141, miR-200, and miR-429 (Kim et al, 2008;Judson et al, 2009;Lin et al, 2009). Overexpression of these miRNAs either promoted iPSC formation or decreased mouse ESC differentiation (Judson et al, 2009;Lin et al, 2009). It is likely that cMyc creates a permissive cellular state by recruiting multiple chromatin remodelers, such as histone acetyltransferase GCN5 and histone demethylase Lid.…”

miRNA Control of Tissue Repair and Regeneration

“…In this context, it should be noted that the promoter region of the miR-290-295 cluster may be bound by c-MYC. 55 Similar studies with ectopic expression of miR-93, miR-106b, and miR-302 cluster were successful in enhancing cell reprogramming.…”

Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

“…The miRNAs increase the efficiency of reprogramming by Oct4, Sox2 and Klf4, but not by these factors plus cMyc; induce a homogeneous population of iPS cell colonies comparing with cMyc Judson et al, 2009 MicroRNAs Inhibition of the let-7 family let-7 miRNAs highly expressed in somatic cells-can suppress self-renewal in Dgcr8 -/-but not wild-type ESCs , let-7 inhibits whereas ESCC miRNAs indirectly activate numerous self-renewal genes and inhibition of the let-7 family promotes de-differentiation of somatic cells to induced pluripotent stem cells P53 OSKM together with p53 siRNA+UTF1 increase the efficiency of highly AP-positive colony by more than 200 times compared with just using OSKM; The suppression of p53 also increased the efficiency of mouse and human iPS cell generation; In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 and p21, whereas, in human fibroblasts, INK4a is more important than ARF. Zhao et al, 2008;Hong et al, 2009;Kawamura et al, 2009;Li et al, 2009a;Marion et al, 2009;Utikal et al, 2009 Signaling pathway TGF-β Inhibition of TGF-β signaling promotes the completion of reprogramming through induction of the transcription factor Nanog.…”

“…To this end, microRNAs expressed within ES cells that possibly suppress differentiation programs have emerged as possible enhancers of reprogramming. Indeed, several ES-expressed microRNAs, such as miR-291-3p, miR-294, miR-295, and miR-302, are capable of enhancing reprogramming efficiency when overexpressed (Lin et al, 2008;Judson et al, 2009;Melton et al, 2010) (Table 4).…”