Wenwen Jia

MiR‐138 Promotes Induced Pluripotent Stem Cell Generation Through the Regulation of the p53 Signaling

Induced pluripotent stem (iPS) cells, especially those reprogrammed from patient somatic cells, have a great potential usage in regenerative medicine. The expression of p53 has been proven as a key barrier limiting iPS cell generation, but how p53 is regulated during cell reprogramming remains unclear. In this study, we found that the ectopic expression of miR‐138 significantly improved the efficiency of iPS cell generation via Oct4, Sox2, and Klf4, with or without c‐Myc (named as OSKM or OSK, respectively), without sacrificing the pluripotent characteristics of the generated iPS cells. Exploration of the mechanism showed that miR‐138 directly targeted the 3′ untranslated region (UTR) of p53, significantly decreasing the expression of p53 and its downstream genes. Furthermore, the ectopic expression of p53 having a mutant 3′‐UTR, which cannot be bound by miR‐138, seriously impaired the effect of miR‐138 on p53 signaling and OSKM‐initiated somatic cell reprogramming. Combined with the fact that miR‐138 is endogenously expressed in fibroblasts, iPS cells, and embryonic stem cells, our study demonstrated that regulation of the p53 signaling pathway and promotion of iPS cell generation represent an unrevealed important function of miR‐138. STEM Cells2012;30:1645–1654

Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway.

Murine embryonic stem cells (ESCs) are pluripotent cells that differentiate into multiple cell lineages. It was recently observed that all-trans retinoic acid (RA) provides instructive signals for the commitment of the germ cell lineage from ESCs. However, little is known about the molecular mechanisms by which RA signals lead to germ cell commitment. In this study, we determined if RA induced ESC differentiation to the germ lineage through modulation of the (bone morphogenetic protein) BMP/Smad pathway activity. In a monolayer culture, RA significantly induced both the expression of the early germ-specific genes, Stra8, Dazl and Mvh, and prolonged activation of Smad1/5 (for at least 24h). Meanwhile, dorsomorphin (a BMP-Smad1/5 specific inhibitor) significantly reduced the RA-induced germ-specific gene expression and completely blocked the RA-induced activation of Smad1/5. Moreover, RA-induced germ-specific gene expression was significantly increased by treatment with the potential activator of Smad1/5, SB431542. Furthermore, the biochemical manipulation of Smad1/5 expression through shRNA knockdown significantly reduced RA-mediated up-regulation of germ-specific gene expression. Our results clearly demonstrate that the Smad1/5 pathway is specifically required at an early stage of germ cell differentiation, corresponding to the RA-dependent commitment of ESCs.

The functions of microRNAs and long non-coding RNAs in embryonic and induced pluripotent stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold immense promise for regenerative medicine due to their abilities to self-renew and to differentiate into all cell types. This unique property is controlled by a complex interplay between transcriptional factors and epigenetic regulators. Recent research indicates that the epigenetic role of non-coding RNAs (ncRNAs) is an integral component of this regulatory network. This report will summarize findings that focus on two classes of regulatory ncRNAs, microRNAs (miRNAs) and long ncRNAs (lncRNAs), in the induction, maintenance and directed differentiation of ESCs and iPSCs. Manipulating these two important types of ncRNAs would be crucial to unlock the therapeutic and research potential of pluripotent stem cells.

MicroRNA-200a Regulates Grb2 and Suppresses Differentiation of Mouse Embryonic Stem Cells into Endoderm and Mesoderm

The mechanisms by which microRNAs (miRNAs) affect cell fate decisions remain poorly understood. Herein, we report that miR-200a can suppress the differentiation of mouse embryonic stem (ES) cells into endoderm and mesoderm. Interestingly, miR-200a directly targets growth factor receptor-bound protein 2 (Grb2), which is a key adaptor in the Erk signaling pathway. Furthermore, high levels of miR-200a dramatically decrease Grb2 levels and suppress the appearance of mesoderm and endoderm lineages in embryoid body formation, as well as suppressing the activation of Erk. Finally, Grb2 supplementation significantly rescues the miR-200a-induced layer-formation bias and the Erk suppression. Collectively, our results demonstrate that miR-200a plays critical roles in ES cell lineage commitment by directly regulating Grb2 expression and Erk signaling.

Human Amniotic Fluid Stem Cells Suppress PBMC Proliferation through IDO and IL-10-Dependent Pathways

Human amniotic fluid stem cells (hAFSCs) can be readily isolated from human amniotic fluid and display multi-differentiation potential and immunomodulatory properties. The mechanism of hAFSCs immunoregulation has not been defined. Here, we explore the immunomodulatory effects of hAFSCs derived from human amniotic fluid and evaluate the role of IL-10 and the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in mediating the immunosuppressive actions of hAFSCs. Flow cytometry showed that hAFSCs were positive for the mesenchymal stem cell markers CD29, CD44, CD105, HLA-ABC, and more than 84% of the hAFSCs were positive for SSEA-4, which is a typical marker of embryonic stem cell (ESCs), and negative for HLA-DR. The RT-PCR and immunostaining results revealed that the multipotent stem cells expressed OCT-4, Nanog, CD44, SOX2 and SSEA-1. In vitro differentiation assays demonstrated that hAFSCs underwent osteogenic differentiation. We examined the immunomodulatory function of hAFSCs using a co-culture system with phorbol 12-myristate 13-acetate (PMA) stimulated peripheral blood mononuclear cells (PBMCs). PBMC proliferation was suppressed by the hAFSCs in a dose-dependent manner. The inhibitory effect was caused by increased IL-10 and IDO induction after co-culture. Neutralizing the IL-10 activity or blocking the function of IDO partially abolished the immunosuppressive action of the hAFSCs. In conclusion, these results suggest that the hAFSCs possess immunomodulatory properties, and IL-10 and IDO are involved in immunosuppression by hAFSCs.

Possible role of the 'IDO-AhR axis' in maternal-foetal tolerance

The induction and maintenance of immunologic tolerance at the feto‐maternal interface is necessary for a successful pregnancy. The most accepted hypothesis for the mechanism underlying this tolerance is that pregnancy‐induced foetal antigen‐specific maternal T regulatory (Treg) cells mediate maternal tolerance to the foetus. The aryl hydrocarbon receptor (AhR), which is highly expressed in the placenta, is widely studied in female reproductive biology and immunology. Activation of AhR can promote immune tolerance by controlling the differentiation of Treg cells in some autoimmune disorders. However, the specific mechanisms underlying tolerance are poorly understood. Indoleamine 2,3‐dioxygenase (IDO) is the initial and rate‐limiting enzyme of tryptophan catabolism in human placental trophoblasts. IDO produces kynurenine, an endogenous AhR ligand that directly activates AhR and is proposed to be central to the establishment and maintenance of immunologic tolerance at the maternal‐foetal interface. We propose that kynurenine activates AhR, leading to the AhR‐dependent Treg cells generation, which in turn critically regulates immunological tolerance at the feto‐maternal interface. This hypothesis must be tested and the proof of this hypothesis may provide a potential therapeutic target for the treatment of infertility and other adverse pregnancy outcomes resulted from inadequate immunological tolerance at the feto‐maternal interface.

Dysregulated expression of IDO may cause unexplained recurrent spontaneous abortion through suppression of trophoblast cell proliferation and migration.

In pregnancy, trophoblast proliferation, migration and invasion are important for the establishment and maintenance of a successful pregnancy. Impaired trophoblast function has been implicated in recurrent spontaneous abortion (RSA), a major complication of pregnancy, but the underlying mechanisms remain unclear. Indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan along the kynurenine pathway, is highly expressed in the placenta and serum during pregnancy. Here, we identified a novel function of IDO in regulating trophoblast cell proliferation and migration. We showed that IDO expression and activity were decreased in unexplained recurrent spontaneous abortion (URSA) compared to normal pregnancy. Furthermore, blocking IDO in human trophoblast cells led to reduced proliferation and migration, along with decreased STAT3 phosphorylation and MMP9 expression. Increased STAT3 phosphorylation reversed the IDO knockdown-suppressed trophoblast cell proliferation and migration. In addition, the overexpression of IDO promoted cell proliferation and migration, which could be abolished by the STAT3 signaling inhibitor (AG490). Finally, we observed similar reductions of STAT3 phosphorylation and MMP9 expression in URSA patients. These results indicate that the level of IDO expression may be associated with pregnancy-related complications, such as URSA, by affecting trophoblast cell proliferation and migration via the STAT3 signaling pathway.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation.

Distinct Roles for CBP and p300 on the RA-Mediated Expression of the Meiosis Commitment Gene Stra8 in Mouse Embryonic Stem Cells

In mammalian germ cells, meiotic commitment requires the expression of Stimulated by retinoic acid gene 8 (Stra8), which is transcriptionally activated by retinoic acid (RA). However, little is known about the epigenetic mechanism by which RA induces Stra8 expression. Utilizing a chromatin immunoprecipitation assay (ChIP), we showed that RA increases histone acetylation at the Stra8 promoter in murine embryonic stem cells (ESCs), a model for germ cell differentiation. Furthermore, we explored whether two coregulators with histone acetyltransferase (HAT) activity, Creb-binding protein (CBP) and p300, are involved in the activation of Stra8. The lentiviral shRNA knockdown of endogenous CBP led to Stra8 repression, while the overexpression of CBP enhanced Stra8 expression at both the mRNA and protein levels. ChIP analysis confirmed that CBP is the crucial coactivator for RA-mediated Stra8 transcription and that it enhances the level of histone acetylation and recruits RNA polymerase II to establish transcriptionally active chromatin. Furthermore, shRNA of p300 enhanced Stra8 expression, and the overexpression of p300 reduced Stra8 expression, independently of its HAT activity. ChIP showed that the knockdown of p300 significantly increased the level of CBP at the Stra8 promoter. These findings demonstrate that CBP and p300 play distinct roles in RA-mediated Stra8 gene transcription.

Pwp1 Is Required for the Differentiation Potential of Mouse Embryonic Stem Cells Through Regulating Stat3 Signaling

Leukemia inhibitory factor/Stat3 signaling is critical for maintaining the self‐renewal and differentiation potential of mouse embryonic stem cells (mESCs). However, the upstream effectors of this pathway have not been clearly defined. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD‐40 repeat‐containing protein associated with histone H4 modification, is required for the exit of mESCs from the pluripotent state into all lineages. Knockdown (KD) of Pwp1 does not affect mESC proliferation, self‐renewal, or apoptosis. However, KD of Pwp1 impairs the differentiation potential of mESCs both in vitro and in vivo. PWP1 chromatin immunoprecipitation‐seq results revealed that the PWP1‐occupied regions were marked with significant levels of H4K20me3. Moreover, Pwp1 binds to sites in the upstream region of Stat3. KD of Pwp1 decreases the level of H4K20me3 in the upstream region of Stat3 gene and upregulates the expression of Stat3. Furthermore, Pwp1 KD mESCs recover their differentiation potential through suppressing the expression of Stat3 or inhibiting the tyrosine phosphorylation of STAT3. Together, our results suggest that Pwp1 plays important roles in the differentiation potential of mESCs. Stem Cells 2015;33:661–673