Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D−/−ESCs) as expected. Surprisingly, D−/−ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D−/−ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D−/−ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D−/−ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D−/−ESCs. Treatment of D−/−ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.
We observed significant up-regulation of miR-196a in esophageal squamous cell carcinoma (ESCC) as compared with their adjacent normal tissue (p = .002). Receiver operating characteristics curve analysis confirmed the suitability of miR-196a as a potential tumor marker for diagnosis of ESCC. Furthermore, analysis of miR-196a levels in saliva samples determined an average of 27-fold up-regulations in ESCC patients compared with healthy group. Our results suggest that salivary miR-196a may be a suitable noninvasive biomarker for diagnosis of ESCC. In addition, molecular pathway enrichment analysis of microRNA (miR)-196a determined focal adhesion, spliceosome and p53 signaling pathways as the most relevant pathways with miR-196a targetome.
The cell–cell/cell–matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.
Embryonic stem cells (ESCs) represent a unique cell population in the blastocyst stage embryo. They have been intensively studied as a promising cell source for regenerative medicine. Recent studies have revealed that both human and mouse ESCs are deficient in expressing IFNs and have attenuated inflammatory responses. Apparently, the ability to express IFNs and respond to certain inflammatory cytokines is not “innate” to ESCs but rather is developmentally acquired by somatic cells during differentiation. Accumulating evidence supports a hypothesis that the attenuated innate immune response may serve as a protective mechanism allowing ESCs to avoid immunological cytotoxicity. This review describes our current understanding of the molecular basis that shapes the immune properties of ESCs. We highlight the recent findings on Dicer and dsRNA-activated protein kinase R as novel regulators of ESC fate and antiviral immunity and discuss how ESCs use alternative mechanisms to accommodate their stem cell properties.
The blastocyst is the preimplantation stage embryo that consists of two major components: the inner cell mass (ICM) and the trophectoderm (TE). The ICM gives rise to the fetus and some extraembryonic tissues whereas the TE contributes to development of the placenta. Previous studies have demonstrated that both human and mouse embryonic stem cells (ESCs) derived from the ICM are deficient in expressing type I IFNs in response to viral infection. In this study, we investigated the IFN response in mouse trophoblast stem cells (TSCs) and their in vitro differentiated trophoblasts (TSC-TBs). In this study, we report that, unlike ESCs, TSCs have a functional IFN system. They can express type I IFNs in response to viral stimuli and express IFN-stimulated genes in response to type I IFNs. TSC-TBs have a further developed IFN system and acquired the ability to express specialized type III IFN-λ. Furthermore, TSCs and TSC-TBs can provide ESCs with antiviral activity against Chikungunya, West Nile, and Zika virus infection, as demonstrated with a novel coculture model that simulates the temporal and spatial relationship between the ICM and the TE in a blastocyst. Taken together, our data demonstrate that mouse ESCs can respond to type I IFNs and gain IFN-based antiviral protection from TSCs and TSC-TBs via paracrine signaling mechanisms even though they themselves are unable to express type I IFNs.
Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.
An early embryo is the most vulnerable stage in the life cycle of mammals, and it faces various immunologic challenges during the course of pregnancy. In particular, the blastocyst (the pre‐implantation embryo) is exposed to inflammatory cytokines resulting from the implantation process. Embryonic cells in the blastocyst have a dedicated task of rapid cell proliferation; how they deal with inflammatory cytokines known to negatively affect cell viability and proliferation is an important yet poorly understood question. We have investigated the effects of TNFα and IFNγ, two “embryotoxic cytokines” that impair blastocyst development and the process of implantation, on embryonic cells. Our results suggest that embryonic stem cells (ESCs), the pluripotent stem cells derived from the inner cell mass of the blastocyst, are insensitive to the cytotoxicity of TNFα and IFNγ. However, when ESCs are differentiated to fibroblasts (ESC‐FBs), they become susceptible to the cytotoxicity of TNFα and IFNγ. In particular, the combination of TNFα and IFNγ (TNFα/IFNγ) significantly reduced cell proliferation and induced apoptosis of ESC‐FBs. The cytotoxic effect of TNFα/IFNγ parallels the induction of nitric oxide synthase (iNOS) in ESC‐FBs, which has been known to cause cellular damage by producing nitric oxide (NO) as a free radical. In contrast, TNFα/IFNγ does not induce the expression of iNOS in ESCs or exert any detectable negative effects on cell proliferation and pluripotency of ESCs. However, ESCs are sensitive to the cytotoxicity of sodium nitroprusside, which can chemically generate NO in cell culture. Together, our results suggest that iNOS is a key effector molecule that mediates the cytotoxicity of TNFα/IFNγ in ESC‐FBs, but not in ESCs. Therefore, the lack of iNOS induction by TNFα/IFNγ in ESCs may be a protective mechanism that allows ESCs to avoid the cytotoxicity of the two cytokines. We are currently investigating the underlying molecular mechanisms and the biological implications of this finding. Support or Funding Information This work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).
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