Dicer and PKR as Novel Regulators of Embryonic Stem Cell Fate and Antiviral Innate Immunity.

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.

Dicer represses the interferon response and the double-stranded RNA-activated protein kinase pathway in mouse embryonic stem cells.

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 IFNß-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.

Pluripotent stem cells are insensitive to the cytotoxicity of TNFα and IFNγ.

Recent studies have demonstrated that embryonic stem cells (ESCs) have an underdeveloped innate immune system, but the biological implications of this finding are poorly understood. In this study, we compared the responses of mouse ESCs (mESCs) and mESC differentiated fibroblasts (mESC-FBs) to tumor necrosis factor α (TNFα) and interferons (IFNs). Our data revealed that TNFα, IFNα, IFNß, or IFNγ alone do not cause apparent effects on mESCs and mESC-FBs, but the combination of TNFα and IFNγ (TNFα/IFNγ) showed toxicity to mESC-FBs as indicated by cell cycle inhibition and reduced cell viability, correlating with the expression of inducible nitric oxide synthase (iNOS). However, none of these effects were observed in mESCs that were treated with TNFα/IFNγ. Furthermore, mESC-FBs, but not mESCs, are vulnerable to cytotoxicity resulting from lipopolysaccharide (LPS)-activated macrophages. The insensitivity of mESCs to cytotoxicity in all cases is correlated with their lack of responses to TNFα and IFNγ. Similar to mESCs, human ESCs (hESCs) and iPSCs (hiPSCs) do not respond to TNFα and are not susceptible to the cytotoxicity of TNFα, IFNß, or IFNγ alone or in combination that significantly affects human foreskin fibroblast (hFBs) and Hela cells. However, unlike mESCs, hESCs and hiPSCs can respond to IFNγ, but this does not cause significant cytotoxicity in hESCs and hiPSCs. Our findings in both mouse and human PSCs together support the hypothesis that attenuated innate immune responses could be a protective mechanism that limits immunologic cytotoxicity resulting from inflammatory and immune responses.

Discovery and Characterization of Bacteriophage LuckyBarnes.

Here, we report the genome sequence of LuckyBarnes, a newly isolated singleton siphovirus that infects Brevibacterium iodinum ATCC 15728 and has a 50,774-bp genome with 67 predicted genes.

Characterization of embryonic stem cell-differentiated fibroblasts as mesenchymal stem cells with robust expansion capacity and attenuated innate immunity.

BACKGROUND: Mesenchymal stem cells (MSCs) isolated from adult tissues (Ad-MSCs) have shown great promise for use in regenerative medicine. However, their poor in vitro expansion capacity and tissue scarcity have been major limitations. In this study, we demonstrate that mouse embryonic stem cells (mESCs) can differentiate into cells with MSC properties. METHODS: Using previously established methods that characterize Ad-MSCs, we analyzed mESC-differentiated fibroblasts (mESC-FBs), including plastic adherence, clonogenic growth, MSC marker expression, tri-lineage differentiation potential, and the capacity to express immunomodulators. RESULTS: Although previously characterized as mESC-differentiated fibroblasts (mESC-FBs), these cells exhibit major properties of Ad-MSCs. However, mESC-FBs also display unique features inherited from ESCs, including robust expansion capacity, senescence resistance, and attenuated innate immunity. In particular, mESC-FBs are insensitive to bacterial endotoxin (lipopolysaccharide, LPS) and do not express LPS-induced inflammatory molecules, in contrast to bone marrow (BM)-MSCs. We further demonstrate that mESC-FBs are resistant to the cytotoxicity associated with inflammatory cytokines, bacterial endotoxins (LPS and heat-killed bacteria), and macrophage-mediated inflammation. CONCLUSIONS: While it remains to be determined how the unique properties of mESC-FBs will affect their immunoregulatory activity under an in vivo condition, our findings demonstrate that ESCs could be used as an alternative source to generate a new class of ESC-MSCs with unique features potentially useful in regenerative medicine.

The Molecular Basis for the Lack of Inflammatory Responses in Mouse Embryonic Stem Cells and Their Differentiated Cells.

We reported previously that mouse embryonic stem cells do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mouse embryonic stem cells and mouse embryonic stem cell-differentiated cells. We demonstrate that LPS, TNF-α, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NF-κB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mouse embryonic stem cells. Similar results were obtained in human embryonic stem cells. In addition to the inactive state of NF-κB, the deficiency in the inflammatory response in mouse embryonic stem cells is also attributed to the lack of functional receptors for LPS and TNF-α. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NF-κB from its inactive to active state. However, a limited response to TNF-α and viral infection, but not to LPS, was observed in mouse embryonic stem cell-differentiated fibroblasts. We conclude that the inflammatory response mechanism is not active in mouse embryonic stem cells, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this article demonstrate that embryonic stem cells are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology, and embryonic stem cell-based regenerative medicine.

Development of Antiviral Innate Immunity During In Vitro Differentiation of Mouse Embryonic Stem Cells.

The innate immunity of embryonic stem cells (ESCs) has recently emerged as an important issue in ESC biology and in ESC-based regenerative medicine. We have recently reported that mouse ESCs (mESCs) do not have a functional type I interferon (IFN)-based antiviral innate immunity. They are deficient in expressing IFN in response to viral infection and have limited ability to respond to IFN. Using fibroblasts (FBs) as a cell model, the current study investigated the development of antiviral mechanisms during in vitro differentiation of mESCs. We demonstrate that mESC-differentiated FBs (mESC-FBs) share extensive similarities with naturally differentiated FBs in morphology, marker expression, and growth pattern, but their development of antiviral mechanisms lags behind. Nonetheless, the antiviral mechanisms are inducible during mESC differentiation as demonstrated by the transition of nuclear factor kappa B (NFκB), a key transcription factor for IFN expression, from its inactive state in mESCs to its active state in mESC-FBs and by increased responses of mESC-FBs to viral stimuli and IFN during their continued in vitro propagation. Together with our previously published study, the current data provide important insights into molecular basis for the deficiency of IFN expression in mESCs and the development of antiviral innate immunity during mESC differentiation.