A miR-372/let-7 Axis Regulates Human Germ Versus Somatic Cell Fates.
ABSTRACT: The embryonic stem cell cycle (ESCC) and let-7 families of miRNAs function antagonistically in the switch between mouse embryonic stem cell self-renewal and somatic differentiation. Here, we report that the human ESCC miRNA miR-372 and let-7 act antagonistically in germline differentiation from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). hESC and iPSC-derived primordial germ cell-like cells (PGCLCs) expressed high levels of miR-372 and conversely, somatic cells expressed high levels of let-7. Manipulation of miRNA levels by introduction of miRNA mimics or knockdown with miRNA sponges demonstrated that miR-372 promotes whereas let-7 antagonizes PGCLC differentiation. Knockdown of the individual miR-372 targets SMARCC1, MECP2, CDKN1, RBL2, RHOC, and TGFBR2 increased PGCLC production, whereas knockdown of the let-7 targets CMYC and NMYC suppressed PGCLC differentiation. These findings uncover a miR-372/let-7 axis regulating human primordial germ cell (PGC) specification. Stem Cells 2016;34:1985-1991.
Project description:The embryonic stem cell-specific cell cycle-regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGF?-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.
Project description:In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGF? and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.
Project description:The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC-like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50-fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome-wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic "blank slate" of the germ line, an immediate precursory state for sexually dimorphic differentiation.
Project description:The transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated in vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells.
Project description:microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.
Project description:MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal, pluripotency, and differentiation. However, with respect to induced pluripotent stem cells (iPSC), in which embryonic-like cells are reprogrammed from adult cells using defined factors, the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge, we use miRNA microarrays to compare the "microRNA-omes" of human iPSCs, hESCs, and fetal fibroblasts. We confirm the presence of a signature group of miRNAs that is up-regulated in both iPSCs and hESCs, such as the miR-302 and 17-92 clusters. We also highlight differences between the two pluripotent cell types, as in expression of the miR-371/372/373 cluster. In addition to histone modifications, promoter methylation, transcription factors, and other regulatory control elements, we believe these miRNA signatures of pluripotent cells likely represent another layer of regulatory control over cell fate decisions, and should prove important for the cellular reprogramming field.
Project description:Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.
Project description:NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14-HSPA2-BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.
Project description:Expression of the mouse miR-290-295 cluster and its miR-371-373 homolog in human is restricted to early embryos, primordial germ cells, the germ line stem cell compartment of the adult testis and to stem cell lines derived from the early embryonic lineages. Sequencing data suggest considerable seed diversification between the seven homologous pre-miRNAs of miR-290-295 but it is not clear if all of the implied miR-290-295 seeds are also conserved in the human miR-371-373 cluster, which consists of only three homologous pre-miRNAs. By employing miRNA target reporters we show that most, if not all, seeds in miR-290-295 are represented in miR-371-373. In the mouse, pre-miR-290, pre-miR-292 and pre-miR-293 express subsets of the miRNA isoforms processed from the single human pre-miR-371. Comparison of the possible miR-290-295/miR-371-373 seed repertoires in placental mammals suggests a model for the evolution of this miRNA cluster family, which would be otherwise difficult to deduce based solely on pre-miRNA sequence comparisons. The conservation of co-expressed seeds that is characteristic of miR-290-295/miR-371-373 should be taken into account in models of the corresponding miRNA-target interaction networks.
Project description:MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.