Articles in PresS. Am J Physiol Heart Circ Physiol (August 10, 2012). doi:10.1152/ajpheart.00338.2012
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Title:
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microRNAs in ES cell differentiation
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Emanuele Berardi1,*, Matthias Pues1,*, Lieven Thorrez1 and Maurilio Sampaolesi1,2
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Keywords: miRNA; embryonic stem cells; early differentiation
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Running title: miRNAs and ES cells
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1Laboratory
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Regeneration, Katholieke Universiteit Leuven, Herestraat 49 O&N4 bus 814, 3000
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Leuven,
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Neuroscience, Experimental and Forensic Medicine, University of Pavia, Via Forlanini 8,
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27100 Pavia, Italy; *EB and MP contributed equally to this review.
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Address correspondence to:
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Lieven Thorrez, PhD and Maurilio Sampaolesi, Ph.D
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Translational Cardiomyology Laboratory
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Department of Development and Regeneration
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KU Leuven, 49 Herestraat. 3000 Leuven, Belgium
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Tel: +32-(0)163-30295
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Fax: +32-(0)163-30294
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e-mails: [email protected]
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of Translational Cardiomyology, SCIL, Dept. of Development and
Belgium;
2Human
Anatomy
Institute,
Dept.
of
Public
Health,
[email protected]
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Berardi E et al. microRNAs in ES cell differentiation
Copyright © 2012 by the American Physiological Society.
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Abstract
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MicroRNAs (miRNAs) are small sequences of non-coding RNAs that regulate
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gene expression by two basic processes: direct degradation of mRNA and translation
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inhibition. miRNAs are key molecules in gene regulation for embryonic stem cells
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(ESCs), since they are able to repress target pluripotent mRNA genes, including Oct4,
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Sox2 and Nanog. miRNAs are unlike other small non-coding RNAs in their biogenesis,
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since they derive from precursors that fold back to form a distinctive hairpin structure
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whereas other classes of small RNAs are formed from longer hairpins or bimolecular
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RNA duplexes (siRNAs) or precursors without double stranded character (piRNAs). An
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increasing amount of evidence suggests that miRNAs may have a critical role in the
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maintenance of the pluripotent cell state and in the regulation of early mammalian
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development.
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This review gives an overview of the current state of the art of miRNA expression
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and regulation in ESC differentiation. Current insights on controlling stem cell fate
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towards mesodermal, endodermal and ectodermal differentiation and cell reprogramming
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are also highlighted.
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Introduction
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Embryonic stem cells can be isolated from the inner cell mass of the blastocyst
52
(54, 61) and they can be expanded in vitro for several passages without losing their
53
pluripotency. In fact, they can be differentiated into all germ layer cells at late passages
54
upon appropriate culture conditions. ES-like cells, termed induced pluripotent stem cells
55
(iPSCs) can be obtained by forcing the expression of pluripotency genes including Oct4
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(official gene symbol Pou5f1), Sox2, Klf4, Nanog, Lin28 and c-Myc. ESCs and iPSCs,
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referred to as pluripotent stem cells (PSCs) are a promising source for drug screening and
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cell therapy in degenerative diseases (56). However, developmental biologists and stem
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cell researchers concur with the concerns regarding our poor knowledge of molecular
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mechanisms regulating the ESC switch between pluripotency and differentiation. The
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molecular programs responsible for different PSC fates are mediated by internal
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regulatory elements, such as transcription factors, but these programs are influenced by
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external elements such as physical interactions with the stem cell niche.
64
Most of these transcription factors are regulated at the posttranscriptional level by
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the presence of miRNAs in the ESCs, often grouped as family or cluster. microRNA
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clusters are located on polycistronic transcripts and transcribed as a single primary
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precursor, while miRNA families consist in a collection of miRNAs sharing high
68
sequence homology.
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miRNAs are around 22 nucleotides in length and are present in the genome as
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independent transcription units or as clusters. Occasionally, miRNAs are located in the
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exonic/intronic sequences and regulated by the host gene transcription. miRtrons are
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referred to as miRNAs hosted in the intronic sequences of protein-coding genes. They are
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co-transcribed with the host gene or regulated by their own specific promoters, similar to
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those of protein-coding genes. However, the majority of characterized miRNAs is
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intergenic and often oriented antisense to neighboring genes. Therefore, they are
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suspected to act as independent units. In fact, the miRNA promoter consists of a core
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promoter containing binding sites for RNA polymerase and transcription factors, a
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proximal promoter containing regulatory binding sites for specific transcription factors
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and CpG islands for methylation, and a distal domain for secondary regulatory elements.
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Typically, miRNAs are able to bind and repress mRNAs through complementary
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base pairing, and miRNA-mRNA duplexes are generated with at least six consecutive
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nucleotides, that undergo base pairing to establish a miRNA-mRNA duplex. The miRNA
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seed sequence, localized between nucleotides 2–8, is responsible to specifically bind the
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complementary sites of target mRNAs mostly located in the 3’ untranslated regions (3’-
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UTRs). miRNAs are transcribed by RNA polymerase II into primary miRNAs (pri-
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miRNAs) that are several kilobases long and characterized by hairpin structures. RNA
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polymerase III is also able to generate pri-miRNAs although limited to the miRNA
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cluster of the human chromosome 19 among repetitive Alu sequences. The double-
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stranded specific endonuclease RNaseIII DROSHA processes the pri-miRNAs into a 60-
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70nt stem loop precursor (pre-miRNA) to be exported into the cytoplasm by nuclear
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transport receptor Exportin-5 (Fig. 1).
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The final 22nt miRNA/miRNA* duplex is finally generated in the cytoplasm by
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the action of ds-RNaseIII DICER. The guide strand at the 5’ end pairing is recruited by
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Argonaute proteins (AGOs) and incorporated into the RNA-induced silencing complex
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(RISC). The less stable strand (miRNA* or passenger) is then degraded. After integration
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in the RISC complex, miRNAs are able to target specific mRNAs, although the entire
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process is still largely unknown (2, 4) (Fig.1).
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The core RISC components interact with several proteins responsible for RNA
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remodeling and the generation of foci known as processing bodies (P-bodies), or glycine-
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tryptophan bodies (GW-bodies) accountable for mRNA decapping, deadenylation and
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degradation (66). Recently some authors reported that circulating HDL particles (59),
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exosomes (58) and liposomes (22) transport endogenous miRNAs to recipient cells for
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functional mRNA targeting in a non cell-autonomous manner. It has recently been
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reported that miRNAs can be regulated by long noncoding RNAs (19).
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In the last decade several reports identified miRNAs as key players in ESC
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differentiation towards ectodermal, mesodermal and endodermal cell types (15). In this
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review, we highlight the emerging role of miRNAs in ESC differentiation and describe
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the molecular mechanisms controlling miRNA expression and function during those
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particularly complex processes.
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miRNAs regulating early differentiation
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When stem cells give rise to their progenies during differentiation, the transition
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at the cellular level resembles closely the developmental progression at the organismal
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level that is the progression from the inner cell mass to the three germ layers and
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eventually to fully functional tissues and organs. Embryonic development requires a
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precise spatial-temporal control of cell fate decision factors. Two main events are
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necessary to promote the switch from a pluripotent to a differentiating state: reduction of
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proliferation rate and cell lineage commitment. Cell cycle control plays a key role during
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these events and the contribution of miRNAs has been reported. Self–renewal capacity
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and pluripotency in ESCs are maintained by a shortening of their cell cycle that leads to a
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rapid cell division (23, 39, 50). At this stage, cell cycle is characterized by a truncated G1
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phase, elevated expression of G1-associated cyclins, active cyclin-dependent kinases
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(CDKs), and low levels of inhibitory cell cycle proteins such as p21, p27, and p57 (5).
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As pluripotent cells adopt particular fates, lineage specific genes are
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transcriptionally activated. In addition, it is equally critical to suppress the expression of
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genes that would otherwise drive differentiation toward alternative fates or inhibit cell
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type specific processes, so called disallowed genes (55). While this occurs at the
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transcriptional level, miRNAs also contribute to this process by clearing mRNAs
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expressed in a previous developmental phase.
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Let-7
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Studies on lin-4 and let-7 miRNAs provided the first evidence that miRNAs have
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essential roles in development. Let-7, the “anti-stemness” miRNA, promotes the exit
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from cell-cycle by repressing CDK6, CDC25A and CCND2 that control G1/S and G2/M
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cycle progression (33). Inhibition of let-7 production by Lin28 also has an important role
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in the maintenance of ESCs. In turn, upon activation, let-7 targets the 3’UTR of some
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stemness factors, including c-Myc, Sall4 and Lin28, destabilizing the self-renewing
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capacity and promoting the differentiation of ESCs (35) (Fig. 3). Besides let-7, several
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other miRNAs control the expression of pluripotency markers during cell differentiation.
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141
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Others
hESC-enriched miRNAs, including the miR-17 and -520 families, and the miR371 cluster are down-regulated rapidly in response to differentiation (49).
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Differentiation of ESCs to embryoid bodies in mouse is correlated with G1 phase
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elongation, up to 6-8 hours longer than as observed in adult cells. (63). During
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differentiation of human ESCs, P53 drives active expression of P21, which supports
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elongation of G1 phase and counteracts cell division. When activated, p53 targets miR-
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34a and miR-145, which inhibit the expression of several stem cell factors and antagonize
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pluripotency (Fig. 3). miR-145, in particular, is expressed at low levels in self-renewing
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human ESCs, but is substantially upregulated during differentiation, directly targeting
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and suppressing Oct4, Sox2 and Klf4 and thus acting as a key antagonist of ESC
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maintenance (64). Also miR-134, miR-296 and miR-470 directly inhibit Nanog, Oct4 and
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Sox2 by targeting in various combinations their coding sequences (53) (Fig. 3).
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Recently, the neural precursor cell enriched miR-762, was shown to downregulate
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Amd1, a key enzyme in the polyamine synthesis pathway, which is highly expressed in
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ESCs (67).
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In the last few years emerging evidence pointed to a key role for miRNAs during
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embryonic germ layer specification. Human hESCs derive from the inner cell mass
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(ICM) of the embryonic blastocyst stage and are characterized by pluripotency and self-
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renewal (43, 54). Differentiation of hESCs into each one of the germinal layers (primitive
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endoderm, mesoderm and ectoderm) is a tightly regulated process and correlates with
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temporally and spatially regulated expression of specific markers, such as Brachyury and
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FoxF1 in the mesoderm, Mixl1 and Sox17 in the endoderm and Pax6 in the ectoderm.
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miRNAs play a pivotal role in embryonic development by posttranscriptional silencing of
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specific genes involved in the differentiation programs (3, 27, 44, 46). Although several
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studies investigated the role of miRNAs in the early development of ESCs, their
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contribution to specification into the three germ layers is still probably underestimated.
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Here we summarize the knowledge to date available about the role of miRNAs in this
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process in vertebrates (Table 1, Fig. 2).
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miRNAs in ectodermal differentiation
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Until the eight-cell stage of mouse development, blastomeres are totipotent and
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retain the ability to contribute to all embryonic and extra-embryonic cell lineages (24,
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65). At the late eight-cell stage, blastomeres increase cell-cell interactions to form a
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compacted morula. Subsequent asymmetric cell divisions produce two distinct cell
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populations: an outside population in contact with the environment and an inside
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population completely surrounded by the outside cells (25). As development proceeds to
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the blastocyst stage, the outside cell population becomes more epithelial-like and gives
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rise to the trophectoderm. The inside cell population forms the ICM, which subsequently
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segregates into two populations: the epiblast (which gives rise to all the tissues of the
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embryo) and the primitive endoderm (65).
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During trophectoderm development (between the morula and blastocyst stages of
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embryonic development), specification of trophectoderm in mouse ESCs is sustained by
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miR-297, miR-96, miR-21, miR-29c, let-7, miR-214, miR-125a, miR-424 and miR-376a.
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Those miRNAs are responsible of ectodermal progression by targeting key proteins of
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Ras and MAPK/p38 pathways (Fig. 2) (60).
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In human ESCs the early ectodermal differentiation is controlled by miR-30b and
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miR-30c, which regulate the embryonic ectoderm development protein (EED), involved
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in (patho-)physiological neural tube formation (48).
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Functional analysis of several miRNAs involved in neural commitment showed
an important contribution by two isoforms of miR-125 by regulation of SMAD4 (7).
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miRNAs in endodermal differentiation
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During early embryo development, cells migrate inward along the archenteron to
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form the inner layer of the gastrula, which then develops into the endoderm. A potential
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role for miR-338-5p and miR-340-3p has been reported. These miRNAs promote
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definitive endodermal differentiation in murine ESCs by direct involvement of histone
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deacetylase (Hdac) activity (14).
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In 2008, Tzur and colleagues compared miRNA expression in two different ESC
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lines (HES1 and HES2), during endodermal differentiation triggered by sodium butyrate,
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an hdac inhibitor. Comparative analysis showed a key role for miR-10a and miR-24.
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miRNA expression profiling also revealed the involvement of several liver-enriched
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miRNAs, including miR-122 and miR-192 (57).
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Further studies identified miR-375, implicated in pancreatic development, as an
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important player involved in commitment towards definitive endoderm by direct
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targeting of TIMM8A mRNA in differentiating human ESCs (21).
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miRNAs in mesodermal differentiation
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During gastrulation, inward migrating cells contribute to the mesoderm, an
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additional layer between the endoderm and the ectoderm.
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miR-302 cluster
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In vertebrates, the miR-302 family is exclusively expressed during early
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embryogenesis, suggesting a unique miRNA signature. In fact, miR-302 overexpression
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sustains pluripotency and promotes mesendoderm specification, while preventing
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neuroectoderm differentiation (44).
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miR-200 family
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Five members of the miR-200 family (miR-200c, miR-141, miR-200b, miR-
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200a, and miR-429) have been proposed to have a role in Epithelial-to-Mesenchymal
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Transition (EMT), occurring at several stages of development, including gastrulation in
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mouse (Fig.2 and Table 1). Members of the miR-200 family are counteracted by Snail to
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promote EMT and early mesoderm commitment at day 2 of ESC differentiation (16).
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Also a general cytoprotective effect of miR-210, mediated by reducing mitochondrial
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ROS production in cardiomyocytes has been described (38).
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miR-17 family
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miR-17 family members (miR-17-5p, miR-20a, miR-93, and miR-106a) are
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expressed during embryonic development, and regulate cell differentiation by targeting
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3′-UTR of specific mRNAs, such as Signal Transducer and Activator of Transcription 3
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(STAT3) (13) (Fig.2 and Table 1). Further evidences in human confirm the involvement
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of miRNAs in early mesoderm formation.
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Others
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Lee and colleagues found an active role of miR-124 during gastrulation by in
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vitro analysis of human embryoid body differentiation. miR-124, which targets two key
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regulators of dynamic rearrangement of cytoskeleton and cell migrations (SLUG and
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IQGAP1), is highly expressed in hESC and gradually degraded during cell migration
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occurring in mesoderm development (28). In addition, it has been reported that miR-145
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promotes mesodermal and ectodermal differentiation by inhibition of self-renewal and
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pluripotency factors, such as OCT4, SOX2 and KLF4 (64) (Table 1). miR-302 promotes
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the mesendoderm lineage at the expense of neuroectoderm formation (44).
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miRNAs regulating pluripotency
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Pluripotent stem cells preserve their identity by promoting self-renewal and
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preventing differentiation. miRNAs have been highlighted as integral part of the gene
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network that regulates self-renewal, pluripotency and cell-type specification in ESCs.
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Indeed, Dicer1-null ESCs do not fully down regulate pluripotency markers and show
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alterations in the differentiation program (37). Also when a second major miRNA-
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processing gene, DGCR8, is ablated in ESCs, similar defects in differentiation are
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observed (62).
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Several miRNAs are expressed preferentially in ESCs and help maintain the ESC
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phenotype by rapid cell proliferation and cell-cycle progression. ESCs are defined by a
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specific miRNA signature and the majority of these miRNAs are transcribed from two
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clusters in the human genome: the miR-371 and the miR-302 clusters.
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miR-290/371 cluster
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The miR-371 cluster is found within a 1050 bp region on chromosome 19 and
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encodes miR-371, miR-372, miR-373, and miR-373* (52). In mouse, the ortholog of this
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cluster is the miR-290-295 cluster, encoding miR-290, miR-291a, miR-291b, miR-292,
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miR-293, miR-294 and miR-295. In ESCs the overexpression of the miR-290 cluster
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withholds them from early differentiation and ensures their high proliferation rate (30).
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This effect is mediated by preventing an accumulation in G1 phase. The miR-290 cluster
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assists G1-S progression and also regulates the G2-M transition. The cell cycle regulators
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Wee1 and Fbxl5 have been identified in vitro as targets of miR-290-295 miRNAs (30).
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Embryonic stem cells lack the G1 checkpoint in their cell cycle (8) and show very short
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G1 phase. A short G1 and a long S phase assist stem cells in maintaining their
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pluripotency. The G1/S transition in cells is regulated by two cyclin/Cdk complexes:
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Cyclin D/CDK4,6 and Cyclin E/CDK2. In ESCs, the Cyclin D/CDK4,6 complex is not
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present, while the Cyclin E/CDK2 complex is constitutively active. Mouse ES cell-
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specific members of the miR-290 family promote G1/S transition by suppression of
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several key inhibitors of the cyclinE-Cdk2 pathway including Cdkn1a, Rbl2 and Lats2
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(Fig. 3) (61). Members of the miR-290 cluster also inhibit ESC differentiation by
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targeting Dkk1, a Wnt pathway inhibitor, and increase pluripotency, thus preventing
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mesodermal formation (68). Many of the defects in Dicer-deficient cells can be reversed
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by transfection with miR-290 family miRNAs (47). Furthermore, some miRNAs are also
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regulators of epigenetic effects. De novo DNA methylation in differentiating mouse ES
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cells is controlled by the miR-290 cluster, by targeting repressors of DNA methyl
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transferases (Fig. 3) (6, 47).
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miR-302 cluster
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The miR-302 gene, located within a 700 bp region on chromosome 4, encodes a
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cluster of eight miRNAs (miR-302b*-b-c*-c-a*-a-d-367) that are specifically expressed
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in hESCs and embryonal carcinoma cells (52). In human ESCs and iPSCs, miR-302 is the
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most predominant miRNA species (52). In ESCs, key transcriptional regulators such as
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Oct4, Sox2, Nanog and Tcf3 specifically occupy the promoters of both active and silent
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miRNA genes to activate and repress their expression, respectively (34). Oct4 and Sox2
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are required for the transcriptional regulation of miR-302 (Fig. 3) (9). The expression of
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miR-302a also promotes G1/S transition, mediated by repression of cyclin D1 (9). MiR-
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302b and miR-372 repress multiple target genes, with downregulation of individual
290
targets only partially recapitulating the total miRNA effects. These targets regulate
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various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT),
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epigenetic regulation and vesicular transport (Fig. 3) (51). It was suggested that the
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relative expression of miR-302b might serve as diagnostic indicator in defining the
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developmental state of embryonic cells and other stem cell lines, such as iPSCs (49). In
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mouse, miR-302b is expressed at higher levels in murine epiblast stem cells (mEpiSC) as
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compared with an earlier developmental state, mouse ESCs. Similarly, in human ESCs
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and iPSCs, the miR-302 family is mildly down-regulated when the cells regress to an
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earlier developmental state.
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miR-520 cluster
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Some species-specific miRNAs involved in human ESC propagation, such as the
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miR-520 cluster, have been reported to be critical for cell proliferation and chromatin
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remodeling in human ESCs (42, 49). The miR-520 cluster is a large microRNA cluster,
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located on chromosome 19 and contains 21 miRNAs (miR-518b, miR-518c, miR-519b,
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miR- 519c, miR-520a, miR-520c, miR-520e, miR-520g, miR-524*, miR-515-5p, miR-
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517a, miR-517b, miR-517c, miR-519e, miR-520b, miR- 520d, miR-520f, miR-520h,
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miR-521, miR-525-3p, and miR-526b*). The miR-520 cluster members share a
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consensus 7-mer seed sequence with the miR-302 members and have many common
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targets (42).
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miR-17 cluster
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The miR-17 family consists of 14 mature miRNAs located on chromosomes 13, X
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and 7 in humans (13). miR-17 family members, miR-17-5p, miR- 20a, miR-93, and miR-
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106a have been identified as microRNAs that are specifically expressed in
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undifferentiated or differentiating ESCs (13). The miR-17 cluster is amplified in
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lymphoma and solid tumors (40).
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miRNAs for reprogramming differentiated cells to pluripotent stem
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cells
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Several methods to reprogram somatic cells to iPSCs already exist, but they are
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hampered by various drawbacks. These include low efficiencies, high costs, genetic
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insertions, and the requirement of forced expression of one or more pluripotent stem cell
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transcription factors (17). Recent insights in miRNAs involved in pluripotency however
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have led to new and improved reprogramming methods.
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miRNAs improving retroviral reprogramming
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miR-290 cluster
328
The Blelloch group was the first to demonstrate that introduction of ESC-specific
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miRNAs could enhance the production of iPSCs (26). Briefly, mouse embryonic
330
fibroblasts
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reprogramming factors Oct4, Sox2 and Klf4 (OSK), together with mimics of the miRNAs
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miR-291-3p, miR-294 and miR-295. These miRNAs belong to the ES cell-specific cell
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cycle-regulating miRNA subset from the miR-290 cluster. The reprogramming efficiency
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enhanced significantly with all three miRNA mimics, with miR-294 displaying the
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largest effect. It increased the efficiency from 0.01-0.05% to 0.1-0.3%. MiR-294 was
336
shown to substitute, but not enhance, cMyc contribution to reprogramming efficiency,
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possibly by exercising its function downstream of cMyc.
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miR-302/367 cluster
(MEFs)
were
reprogrammed
by
retroviral
transfection
with
the
339
Later, they also showed that addition of synthetic mimics of the miRNAs from the
340
miR-302/367 cluster enhances retroviral reprogramming of human fibroblasts by OSK
341
and OSK plus c-Myc (OSKM) (51). This cluster (consisting of miR-302a/b/c/d and miR-
342
367) silences AOF2 (a lysine-specific histone demethylase), HDAC2
343
deacetylase 2) and MECP1/2 (methyl CpG-binding proteins) activities. Together with a
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(histone
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subsequent degradation of DNMT1 (DNA (cytosine-5)-methyltransferase 1), this
345
ultimately results in total genomic DNA demethylation and H3K4 modification. These
346
epigenetic reprogramming events induce expression of OCT3/4, SOX2 and NANOG,
347
necessary for somatic cell reprogramming (29). miR-302/367 also functions through
348
mesenchymal-epithelial transition (MET) by targeting TGF-β receptor and RHOC, a part
349
of the RAC subfamily of the RHO family of GTPases (51).
350
Others
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MEFs obtained from miR-34a knockout mice are more susceptible to
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reprogramming, resulting in a 4.5 fold increase in reprogramming efficiency, and iPSC-
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like colonies appeared after 5 instead of 7 days (10). miR-34 is a major downstream
354
transcriptional target of the tumor suppressor p53, which is known for repressing
355
reprogramming (18). miR-34 does not repress cell proliferation, but acts (at least in part)
356
by direct suppression of the pluripotency genes Nanog, Sox2 and N-Myc. Another group
357
of miRNAs, namely miR-93 and miR-106b, was shown to play a role in reprogramming
358
(29). Both of them have a very similar seed sequence, and miRNA mimics improved the
359
reprogramming efficiency of MEFs by retroviral transfection with OSK or OSKM 4-6
360
times. miR-93 and miR-106b directly target TGF-β receptor II. This receptor is partially
361
responsible for the mesenchymal-to-epithelial transition (MET), a hallmark during the
362
initial stage of reprogramming.
363
A library screen of murine miRNAs revealed a new family (miR-130/301/721) of
364
miRNAs that is able to improve early-phase reprogramming of MEFs (41). These
365
miRNAs, all with the same seed sequence, target Meox2, a transcription factor interfering
366
with Tgf-β, and thereby promote the MET. MET is also induced by bone morphogenic
367
protein (Bmp) which functions by inducing miR-205 and the miR-200 family of
368
miRNAs. Indeed, addition of Bmp7 or mimics of miR-200b and miR-200c (two of its
369
downstream miRNAs) was shown to synergize with the OSKM transcription factors to
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accelerate the progression through the initiation phase of reprogramming (45).
371
Previous discussed papers provide ample evidence of the great potential of
372
miRNAs in somatic cell reprogramming. Nonetheless, above methods merely ameliorate
373
the existing method of retroviral transduction of OSK and/or OSKM transcription factors.
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This leads to higher efficiencies, but doesn’t circumvent the other disadvantages.
375
Reprogramming using only miRNAs
376
miR-302/367 cluster
377
In 2008, Lin and colleagues discovered the possibility of reprogramming human skin
378
cancer cells (and later human hair follicle cells) to iPSCs, by mere transfection of
379
miRNas from the miR-302/367 cluster, without the need of transcription factors (31, 32).
380
Anokye-Danso and colleagues demonstrated a similar result (1). Both mouse and
381
human fibroblasts were reprogrammed solely by forced expression of the miR-302/367
382
cluster. The efficiency amounted up to nearly 10%, almost two orders of magnitude
383
greater than with lentiviral transfection of OSKM, and at much higher speed.
384
Others
385
The approach utilized by Miyoshi et al. completely circumvents vector-based
386
gene-transfer, by direct transfection of mature double-stranded miRNAs in mouse and
387
human cells (36). They used a combination of miR-200c, the miR-302 and miR-369
388
families. Despite the lower reprogramming efficiency, this methodology entails great
389
benefits for clinical and translational applications, especially for large scale
390
reprogramming. In fact, the production of miRNA mimics is fairly easy and at relative
391
low cost.
392
The amount of miRNAs known to be involved in iPSC reprogramming is already
393
significant, however it seems that only a tip of the iceberg has been unveiled, with new
394
discoveries being published in fast succession. Protocols involving miRNAs, completely
395
steering clear of viral reprogramming and transcription factors, will make it possible to
396
generate iPSCs on a much larger scale. The lack of genomic insertions, makes it possible
397
to consider these cells for use in a clinical setting.
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Berardi E et al. microRNAs in ES cell differentiation
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Open questions and future directions
402
403
Although miRNAs are definitely relevant for the transcriptomic changes
404
occurring in ESC differentiation, the exact contribution of each of the individual miRNAs
405
is still questionable. The understanding of such regulation could be informative also for
406
ESC handling and directing to a specific cell fate.
407
Since the miR-290 cluster in mouse ESCs is relatively well known it could be
408
interesting to see if the homologous human clusters, miR-371/2/3 and mir-302 could have
409
similar target mRNAs and effects.
410
In addition, this information could be useful to verify potential roles for those
411
miRNAs in regulating the cell cycle of adult multipotent stem cells.
412
The regulation of miRNA expression during ESC differentiation and in the terminally
413
differentiated cell types are also open questions (20).
414
Furthermore, manipulating the expression of pluripotent related miRNAs, as mir-290 and
415
miR-302 clusters known to be active on TCF3, NANOG, SOX2 and OCT4 promoters
416
pave potential clinical applications. In fact, overexpression of those miRNAs could be
417
useful for the expansion of adult stem cells while their inhibition could be relevant for
418
controlling tumor progression. Finally, since our group recently identified a miRNA
419
family involved in muscle lineage switch of adult stem cells (11, 12), it could be
420
interesting to verify if this phenomenon can be reproduced in pluripotent cell types.
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Berardi E et al. microRNAs in ES cell differentiation
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Conclusion
424
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Pluripotent stem cells (PSCs) hold tremendous promise in regenerative medicine
426
applications since they univocally are capable to differentiate in any cell type and can be
427
easily expanded in large amounts. ESC self-renewal and differentiation depend on
428
soluble signals, which activate pathways responsible for changes in gene transcription
429
signature and cell differentiation state. In addition, cell-cell contact is also a critical
430
parameter, since at low cell densities cell growth rates diminish, while at high cell
431
densities spontaneous differentiation occurs. In this staggering scenario miRNAs seem to
432
have a prominent role in complex molecular mechanisms responsible for ESC self-
433
renewal and differentiation.
434
Discovering novel pluripotency genes and stem cell pathways regulated by
435
miRNAs will be the future directions in this scientific area. It is likely that some of those
436
genes and pathways will be relevant to overcome hurdles facing the utilization of ESCs,
437
such as the lack of reliable methods to differentiate ESCs to desired cell types in large
438
amount.
439
Molecular and cellular mechanisms promoting miRNA processing, localization
440
and turnover are not well understood and more basic research and preclinical
441
investigation are required to move a step forward for future clinical application of
442
miRNAs in pluripotent stem cell technology.
443
444
445
446
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448
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Berardi E et al. microRNAs in ES cell differentiation
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Acknowledgment
452
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This work was supported in part by: FWO- Odysseus Program n. G.0907.08; Research
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Council of the University of Leuven n. OT/09/053 and GOA11/012; CARE-MI n.242038
455
FP7-EC grant; FWO n. G.0606.12; Project Financiering 10/019; the Italian Ministry of
456
University and Scientific Research grant n. PRIN 2008RFNT8T_003 and CARIPLO
457
2008-2005. We are grateful to Catherine Verfaillie for continuous support, Danny
458
Huylebroeck, and An Zwijsen for helpful discussion; EB, LT and MP are supported by
459
F+11/039, KULeuven Biomedical and FWO founds respectively. We thank Christina
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Vochten for the professional secretarial service and Paolo Luban for a kind donation.
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FIGURE LEGEND
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Figure 1. miRNA biogenesis. miRNAs are transcribed as long transcripts (pri-miRNAs)
680
by RNA polymerase II or III, processed into hairpin RNAs (pre-miRNAs) by Drosha and
681
then exported to the cytoplasm by exportin. The distal domain contains secondary
682
regulatory elements; the proximal promoter is responsible for specific transcription factor
683
bindings as well as the core promoter, in which the RNA polymerase binding sites are
684
also located. Pre-miRNAs are further cleaved into a 22nt miRNA/miRNA* duplex by
685
Dicer. The weakest strand is loaded into the RISC complex and functions as a guide for
686
mRNA target recognition and final degradation.
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688
Figure 2. Expression pattern of miRNAs involved in mammalian germ layer
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specification. In the early stages of embryonic development, trophectoderm specification
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is promoted by several miRNAs whose expression and activity is increased in ESCs
691
during gastrulation. In late gastrulation phases, specific miRNAs contribute to the
692
development of both mesoderm and endoderm.
693
694
Figure 3. Diagram showing the basic regulatory network of miRNA interactions with
695
pluripotency factors. On the left, miR-17, miR-302, miR-371-373 cluster (or the ortholog
696
miR-290-295 cluster in mouse), and miR-520 are shown to regulate a large number of
697
genes involved in cell signaling and epigenetic regulation. In turn, these genes, directly or
698
indirectly activate the core pluripotency factors depicted in the middle. The anti-stemness
699
Let-7 miRNA promotes the exit from cell-cycle by repressing MYC, LIN28 and cyclin-
700
dependent kinases. Conversely, the inhibition of let-7 by LIN28 has a critical role in the
701
maintenance of ESCs. To the right, microRNAs that antagonize pluripotency and thus
702
induce early differentiation are depicted. Pointed arrows represent activation; blunted
703
arrows indicate repression.
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Table 1. Summary of miRNA expression in germ layers derived from activated ESC
Germ layers
Species
miRNAs
Target genes
Ref.
Ectoderm
Human
miR-125
SMAD4
(7)
miR-30b, miR-30c
Embryonic ectoderm development protein (EED)
(48)
miR-297, miR-96, miR-21, miR29c, let-7, miR-214, miR-125a,
miR-424 and miR-376a
All Ras and MAPK (p38 pathways)
(60)
miR-200a,b,c, miR-141, miR-429
Snail
(16)
miR-145
OCT4, SOX2, KLF4
(64)
miR-10a
Hoxa1
(57)
miR-24
Noch1-MAPK14
(57)
miR-375^
TIMM8A, Mtpn, Jak2, C1qbp, Usp1, Adispor2
(21, 57)
miR-122*, miR-192*
CAT1, SIPI1
(57, 21)
miR-93
STAT3
(13)
miR-338-5p, miR-340-3p
Members of Hdac and related pathways
(14)
miR-302, miR-427
Lefty1, Lefty2 (TGFβ/Nodal Smad2/3)
(44)
miR-145
OCT4, SOX2, KLF4
(64)
miR-290
Dkk1 (affecting Wnt pathway)
(68)
miR-200a,b,c, miR-141, miR-429
Snail
(16)
miR-93, miR-17-5p
STAT3
(13)
Mouse
Endoderm
Human
Mouse
Mesoderm
Human
Mouse
*liver or ^pancreatic specific miRNAs.