Profiling of naïve and primed human pluripotent stem cells reveals state-associated miRNAs

Abstract

Naïve human pluripotent stem cells (hPSC) resemble the embryonic epiblast at an earlier time-point in development than conventional, 'primed' hPSC. We present a comprehensive miRNA profiling of naïve-to-primed transition in hPSC, a process recapitulating aspects of early in vivo embryogenesis. We identify miR-143-3p and miR-22-3p as markers of the naïve state and miR-363-5p, several members of the miR-17 family, miR-302 family as primed markers. We uncover that miR-371-373 are highly expressed in naïve hPSC. MiR-371-373 are the human homologs of the mouse miR-290 family, which are the most highly expressed miRNAs in naïve mouse PSC. This aligns with the consensus that naïve hPSC resemble mouse naive PSC, showing that the absence of miR-371-373 in conventional hPSC is due to cell state rather than a species difference.Naïve human pluripotent stem cells (hPSC) are cells in vitro resembling the inner cell mass of human embryonic day (E) 6-7 preimplantation blastocysts 1,2 . Conventional hPSC are not considered naïve but instead are referred to as primed, since they resemble a later stage found in the post-implantation epiblast [2][3][4][5][6][7][8] . A plethora of methods currently exist to generate naïve hPSC 9 , which can be evaluated using various molecular markers, particularly gene and transposon expression profiles, cell surface protein expression and genome-wide DNA demethylation (reviewed in 10 ).MicroRNAs (miRNAs) are a class of small non-coding RNAs (∼ 22 nucleotides) which inhibit complementary mRNAs by binding to the 3′ untranslated region (UTR) and either flagging it for degradation or blocking translation. Early miRNAseq experiments in mouse embryonic stem cells (mESC) have shown that pluripotent stem cells have a miRNA expression patterns dominated by a few miRNAs which have been defined as the ESCspecific cell cycle-regulating (ESCC) miRNAs. ESCCs have the seed sequence AAG UGC and make up 20-50% of all miRNAs in mESC [11][12][13][14][15] . ESCC miRNAs are specifically expressed in naïve and primed pluripotent states and are downregulated upon differentiation. Upregulation of these miRNAs in somatic cells has also been associated with proliferation and cancer in mouse and human (reviewed in 16 ).In murine cells both in vivo and in vitro, there is a switch in dominant ESCC miRNA expression in pluripotent cells from the preimplantation/naïve miR-290 family to the postimplantation/primed miR-302 family both containing the seed sequence AAG UGC [17][18][19][20][21] . However, the miR-290 family does not exist in the same genomic context in the human. The human homolog, the miR-371-373 cluster, is reported to be variably, if at all, expressed in conventional human PSC and this discrepancy has previously been viewed as a key difference between the two species 13,14,22 . Conversely, high expression of the miR-302 family has been reported as a key marker of primed human ES and iPS cells 13,14,23 and mouse EpiSC 11 .Here we present a comprehensive miRNAseq dataset of naïve, intermediate and primed hPSC. This ...

Hsa-miRNA-143-3p Regulates Differentiation of Human Stem Cells From The Apical Papilla Through Targeting Nuclear Factor I-C

“…So this study considers that the regulation of miR-143-3p is involved in specific signaling events that are unique to root formation, and reduced miR-143-3p may activate the NFIC signalling pathway during initiation of SCAP differentiation. MiR-143-3p was identified miRNA markers of the human naive, that are specifcally expressed in naive and primed pluripotent states and are downregulated upon diferentiation [31] , which is in line with our observation of downregulation during root development, that is, miR-143-3p can maintain the stemness of SCAPs and degrade its expression in the process of differentiation into odontoblasts or pulp cells.…”

Building Pluripotency Identity in the Early Embryo and Derived Stem Cells

“…On the contrary, miRNA markers of primed state are miR-363-5p and several members of the miR-17 and miR-302 families [274]. The conversion of mESCs from naïve to the primed condition is shepherded by an immediate upregulation of miRNA-363-3p and miRNA-205-5p, as well as miR-17 family members miR-18b-3p, -20b-5p, -20b-3p and -106a-5p [274]. During this transition, quantitative variations of both miR-290-295 and the miR-302/367 clusters were also detected.…”

“…The second cluster, named miR-290-295, is less conserved and includes miR-290, miR-291a, miR-291b, miR-292, miR-294 and miR-295 and miR-293 (without the AAGUGC seed sequence). The human orthologue comprises miR-371 and the AAGUGC seed-containing miR-372 and miR-373 (miR-371-373 cluster) [274]. In both clusters, other miRNAs with the AAGUGC seed are present, as well as other miRNAs with a different seed [275].…”

“…miRNA markers of human naïve cells are miR-143-3p, -22-3p and the miR-371-373 cluster, the latter very highly expressed. On the contrary, miRNA markers of primed state are miR-363-5p and several members of the miR-17 and miR-302 families [274]. The conversion of mESCs from naïve to the primed condition is shepherded by an immediate upregulation of miRNA-363-3p and miRNA-205-5p, as well as miR-17 family members miR-18b-3p, -20b-5p, -20b-3p and -106a-5p [274].…”

“…PSCs express a characteristic set of lncRNAs [290,291], which is actively involved in the maintenance of the pluripotency status [274,275,292]. More than one thousand pluripotency-associated lncRNAs act as "regulators of regulators" by interacting with the PGRN, or operate as "pivots of pluripotency" acting as a barrier against differentiation [292,293].…”