Suppression of epithelial–mesenchymal transition and apoptotic pathways by miR-294/302 family synergistically blocks let-7-induced silencing of self-renewal in embryonic stem cells

The embryonic stem cell (ESC)-enriched miR-294/302 family and the somatic cell-enriched let-7 family stabilizes the self-renewing and differentiated cell fates, respectively. The mechanisms underlying these processes remain unknown. Here we show that among many pathways regulated by miR-294/302, the combinatorial suppression of epithelial–mesenchymal transition (EMT) and apoptotic pathways is sufficient in maintaining the self-renewal of ESCs. The silencing of ESC self-renewal by let-7 was accompanied by the upregulation of several EMT regulators and the induction of apoptosis. The ectopic activation of either EMT or apoptotic program is sufficient in silencing ESC self-renewal. However, only combined but not separate suppression of the two programs inhibited the silencing of ESC self-renewal by let-7 and several other differentiation-inducing miRNAs. These findings demonstrate that combined repression of the EMT and apoptotic pathways by miR-294/302 imposes a synergistic barrier to the silencing of ESC self-renewal, supporting a model whereby miRNAs regulate complicated cellular processes through synergistic repression of multiple targets or pathways.Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into any cell type.¹ Therefore, they hold great potential for clinical applications in regenerative medicine. However, the molecular mechanisms regulating the self-renewal and differentiation of ESCs are still not fully understood. miRNAs are an important class of short non-coding RNAs that regulate ESC self-renewal and differentiation.² miRNA-deficient ESCs proliferate at a slower rate with a slight accumulation of cells in the G1 phase, and they cannot silence the self-renewal program when induced to differentiate.^{3, 4, 5} Introducing individual members from an miRNA family highly expressed in ESCs partially rescues the proliferation defect and reverses the G1 accumulation.⁶ The family shares a seed sequence (5′-AAGUGCU-3′) and has eight members, including miR-294 and miR-302a-d. Because of their role in influencing the ESC Cell Cycle, they have been called the ESCC family of miRNAs. In addition, ESC cell cycle regulating miRNAs (ESCC miRNAs) suppress the G1 restriction point by inhibiting retinoblastoma (Rb) family proteins, preventing ESCs from exiting the cell cycle during serum starvation or contact inhibition.⁷ In contrast to ESCC miRNAs, the introduction of let-7 family miRNAs that are enriched in somatic cells as well as several other lineage-specific miRNAs such as miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218 silences self-renewal in Dgcr8−/− (DiGeorge syndrome critical region gene 8−/−) ESCs but not wild-type ESCs.^{7, 8} Interestingly, the ESCC miRNAs prevent these miRNAs from silencing ESC self-renewal. Consistent with their roles in promoting self-renewal, ESCC miRNAs dramatically enhance the de-differentiation of human and mouse fibroblasts to induced pluripotent stem cells (iPSCs).^{9, 10, 11, 12, 13}How ESCC miRNAs maintain self-renewal in the presence of differentiation-inducing miRNAs is not clearly understood. Genomic studies have shown that these miRNAs target hundreds of mRNAs enriched in many biological processes.^{8, 14, 15, 16} Functional analysis of a small number of targets chosen based on their known roles has begun to give some insights into their functions in reprogramming somatic cells to iPSCs.^{10, 11, 17} However, due to the inherent differences between the maintenance and establishment of pluripotency,¹⁸ what targets or pathways underlie the antagonism between the two opposing families of miRNAs in regulating ESC self-renewal remains unknown. Recent work showed that while the miR-294/302 family suppresses and let-7 induces the G1/S restriction point, this cell cycle function cannot explain their antagonistic roles in maintaining pluripotency.⁷ Therefore, we set out to search for additional functions of the two miRNA families that directly underlie their opposing roles in regulating pluripotency. In this study, we found that combined repression of epithelial–mesenchymal transition (EMT) and apoptotic pathways by miR-294/302 forms a synergistic barrier to block the silencing of ESC self-renewal by let-7 and other differentiation-inducing miRNAs.     

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.Induced pluripotent stem cells (iPSCs)¹ are genetically manufactured pluripotent cells obtained by the transfection of reprogramming factors. Such iPSCs were first reported in 2006 for the mouse (1) and in 2007 for humans (2, 3). Although iPSCs have already been used in the fields of drug development and disease models (4–7), basic aspects of iPSCs largely remain to be elucidated to provide us with a fuller understanding of their properties and for therapeutic applications to be developed in the field of regenerative medicine. These aspects include the need for a definitive system to be established to evaluate their properties; e.g. pluripotency, differentiation propensity, risk of possible contamination of xenoantigens, and even the potential for tumorigenesis. Cell surface glycans are often referred to as the “cell signature,” which changes dramatically depending on the cell properties and conditions (8) as a result of changes in gene expression, including epigenetic modifications of glycan-related molecules. Glycans, because of their outermost cell-surface locations and structural complexity, are considered to be most advantageous communication molecules, playing roles in various biological phenomena. Indeed, SSEA3/4 and Tra-1–60/81, which have been used to discriminate pluripotency, are cell surface glycan epitopes that respond to some specific antibodies (9–12).Glycan-mediated cell-to-cell interactions have been shown to play important roles in various biological phenomena including embryogenesis and carcinogenesis (13–16). This might also be the case for the acquisition and maintenance of iPSC and ESC pluripotency, although there remains much to clarify concerning the roles of cell surface glycans in these events. Thus, the development of novel cell surface markers to evaluate the properties of iPSCs and ESCs is keenly required. Toward this goal, a glycomic approach has been made by several groups (17–20). In our previous study using an advanced lectin microarray technique (21), thirty-eight lectins capable of discriminating between iPSCs and SCs were statistically selected, and the characteristic features of the pluripotent state were obtained. The glycan profiles of the parent SCs, derived from four different tissues, were totally different from one another and from those of the iPSCs. Despite this observation, the technique used lacks the ability to determine detailed glycan structures or allow their quantification. For this purpose, a conventional approach based on high performance liquid chromatography (HPLC) combined with matrix-assisted laser desorption-ionization (MALDI) - time of flight (TOF) mass spectrometry (MS) was undertaken for both the definitive identification of glycan structures and their quantitative comparison, which remained unclear in the previous analysis (21).We report here structural data on N-linked and O-linked glycans derived from the human iPSC 201B7 cell line (2) and human dermal fibroblasts (SC) representing undifferentiated and differentiated cells, respectively. For quantitative comparison, the glycans were liberated by gas-phase hydrazinolysis from similar numbers of cells (22–25) fluorescently tagged with 2-aminopyridine (2-AP) at their reducing terminus (26, 27), following which the derived pyridylaminated (PA-) glycans were purified by multiple-mode (i.e. anion-exchange, size-fractionation and reverse-phase) HPLC. Their structures were determined and quantified by HPLC mapping assisted with MALDI-TOF-MS and exoglycosidase digestion analyses. This report thus provides the first structural evidence showing the occurrence of a dynamic “glycome shift” upon induction of pluripotency.