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

Abstract

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. 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. The innate immune system is the first line of an organism’s defense against a broad range of pathogen invasions. Although innate immunity consists of different mechanisms, the antiviral response is one of the most critical components and is presumably developed in most, if not all, mammalian cells (1Sen G.C. Virues and infections.Annu. Rev. Microbiol. 2001; 55: 255-281Crossref PubMed Scopus (756) Google Scholar, 2Kumar H. Kawai T. Akira S. Toll-like receptors and innate immunity.Biochem. Biophys. Res. Commun. 2009; 388: 621-625Crossref PubMed Scopus (785) Google Scholar). However, a series of our recent studies have demonstrated that mouse embryonic stem cells (ESCs) have an attenuated innate immune response. In particular, they do not express type I interferons (IFN) and lack response to inflammatory cytokines. Similar observations have been made in human ESCs and induced PSCs (iPSCs) (3Guo Y.L. Carmichael G.G. Wang R. Hong X. Acharya D. Huang F. Bai F. Concise reviews: Attenuated innate immunity in embryonic stem cells and its implications in developmental biology and regenerative medicine.Stem Cells. 2015; 33: 3165-3173Crossref PubMed Scopus (17) Google Scholar, 4Guo Y.L. Utilization of different antiviral mechanisms by mammalian embryonic stem cells and differentiated cells.Immunol. Cell Biol. 2017; 95: 17-23Crossref PubMed Scopus (11) Google Scholar). Therefore, this is a common property shared by all types of PSCs. It appears that ESCs in the early embryo are immunologically divergent from the traditional view of “innate immunity” established in somatic cells of developed organisms. The biological implications of the attenuated innate immune responses in ESCs have been speculated from different perspectives. Immune response is a double-edged sword: it serves as a critical part of the defense mechanism, but it can also cause immunologic toxicity to tissues since IFN and inflammatory cytokines negatively impact cell proliferation and viability (5Hertzog P.J. Hwang S.Y. Kola I. Role of interferons in the regulation of cell proliferation, differentiation, and development.Mol. Reprod. Dev. 1994; 39: 226-232Crossref PubMed Scopus (75) Google Scholar, 6Garcia M.A. Meurs E.F. Esteban M. The dsRNA protein kinase PKR: Virus and cell control.Biochimie. 2007; 89: 799-811Crossref PubMed Scopus (405) Google Scholar, 7Samuel C.E. Antiviral actions of interferons.Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (1998) Google Scholar). this could be by tissues of developed it could cause to ESCs in an early this an attenuated response could as a mechanism in ESCs by cytotoxicity early of Y.L. The innate immunity in embryonic stem The molecular and biological from early Reprod. PubMed Scopus Google Scholar). the of IFN response not to be with the pluripotency of ESCs, as demonstrated by a recent that of the IFN can cause of and in ESCs D. M. I response of stem S. PubMed Scopus Google Scholar). are by and are not However, they biological if the in IFN not the defense of ESCs. antiviral mechanisms have been ESCs a of that are of IFN X. Huang D. Wang S. T. M. immunity of stem PubMed Scopus Google they the RNA interference antiviral that not be in differentiated mammalian cells antiviral responses in differentiated PubMed Scopus Google Scholar). Although RNAi is as a major antiviral mechanism in a has not been demonstrated in RNAi activity detected in mouse ESCs, to the that RNAi could be an antiviral mechanism in ESCs, in which the IFN system is deficient F. Antiviral RNA interference in mammalian PubMed Scopus Google Scholar). However, the of RNAi as an antiviral mechanism in ESCs S. RNA interference a innate antiviral immune response in 14: PubMed Scopus Google Scholar). Dicer is the key enzyme for and D−/−ESCs could be used as a to the of RNAi since they have the of ESCs and the to express pluripotency they and S. of embryonic stem S. PubMed Scopus Google Scholar, S. R. T. mouse embryonic stem cells are in and Dev. PubMed Scopus Google Scholar). it that D−/−ESCs to express and show increased antiviral activity S. mouse embryonic stem cells a PubMed Scopus (11) Google Scholar). our studies ESCs as a to express from synthetic have to the role of Dicer in the regulation of antiviral responses in these of a protein from its synthetic is an to M. immune with PubMed Scopus Google Scholar). major biological with this is that synthetic to the cells is detected as RNA and antiviral to cell viability and of the cells M. immune with PubMed Scopus Google Scholar). However, this is not a in ESCs to attenuated antiviral responses as we demonstrated in a recent R. Wang Huang F. embryonic stem cells have antiviral mechanisms that can be for the of Dev. PubMed Scopus Google Scholar). of this in ESCs, we to protein from its synthetic as a in assay to RNAi activity in this RNAi activity detected in wild-type ESCs, but not in D−/−ESCs, as expected. However, D−/−ESCs increased antiviral responses to RNA transfection. RNA IFN response by with receptors and I receptors to IFN of and S. H. H. Akira S. M. S. RNA is the for PubMed Scopus Google Scholar, T. Akira S. Toll-like receptors and with innate receptors in and PubMed Scopus Google Scholar, H. T. for Rev. PubMed Scopus Google Scholar). In RNA can also antiviral mechanisms, as protein kinase of PKR of cellular and protein this it also cell proliferation antiviral PubMed Scopus Google Scholar). PKR is constitutively in cells and is by dsRNA by cellular but it can be by IFN as a part of the IFN response. the IFN system can multiple and a antiviral response C.E. Antiviral actions of interferons.Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (1998) Google Scholar). it is that the of ESCs in expressing IFN is to the underlying molecular of this is poorly understood. In this study, we that D−/−ESCs have gained the ability to express not the type I IFN but also constitutively active which to the cell proliferation and cell viability of D−/−ESCs. a critical role of Dicer as a repressor of antiviral responses in ESCs, which a mechanism essential for ESCs to proliferation and to cell resulting from RNA assay to the RNAi activity in ESCs. first cells it to The cells with a synthetic dsRNA corresponding to the of as Based on the of be to that detected as early as transfection. with dsRNA of with a to as by by that in ESCs by by in with However, in D−/−ESCs, and the of to a for these be that the different and in ESCs is to of by RNAi which is abolished in D−/−ESCs. The effects in the of by and in ESCs and D−/−ESCs are to the of demonstrated the of RNAi activity in ESCs, which is in with the from as an S. of the type I double-stranded RNA interference in mammalian PubMed Scopus Google Scholar). However, the most is that of D−/−ESCs with and to a with cell as by the increased of cells the cytotoxicity by dsRNA in ESCs is and to that D−/−ESCs have increased to the cytotoxicity of The which has a that can M. M. S. H. Kawai T. H. of by short double-stranded RNA as in of 2009; PubMed Scopus Google Scholar). Therefore, the RNA used in the can with most, if not all, RNA and that can most of the common antiviral Therefore, cellular effects of RNA on ESCs and D−/−ESCs the of antiviral responses. The increased antiviral response in D−/−ESCs is a finding that we with of and in this the of Dicer on the IFN we the cells with and the of and as of the of the type I IFN in the of the not increased in ESCs, but they in D−/−ESCs. Similar cells with a synthetic dsRNA used as a RNA in our studies R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar, R. Wang Acharya D. Bai F. Huang F. Y.L. Antiviral responses in mouse embryonic stem of cellular mechanisms in type I and Biol. PubMed Scopus Google not It is that cellular RNA with dsRNA from and of can antiviral responses and cause cellular in the of M. M. nuclear Dicer the of double-stranded Biol. PubMed Scopus Google Scholar, a kinase for cellular PubMed Scopus Google Scholar, Carmichael G.G. of and on the cellular response to double-stranded Biol. Rev. PubMed Scopus Google Scholar). The B2 short interspersed nuclear is a major type of in mouse The cellular impact and of and B2 2009; PubMed Scopus Google Scholar). which has features of dsRNA of the and mechanism of B2 an repressor of RNA 2007; Scopus Google is in mouse ESCs. in synthetic B2RNA to and in and in ESCs and D−/−ESCs. It is that the B2RNA in D−/−ESCs in ESCs, its in D−/−ESCs Dicer is for it could the B2 RNA as this we ESCs and D−/−ESCs with B2RNA and the of by that detected in D−/−ESCs in ESCs with the of B2RNA in D−/−ESCs also the of the RNA receptors that the effects of the of which is in D−/−ESCs in ESCs, the dsRNA receptors are ESCs are to to and but they are to express the cytokines R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar, R. Wang Acharya D. Bai F. Huang F. Y.L. Antiviral responses in mouse embryonic stem of cellular mechanisms in type I and Biol. PubMed Scopus Google Scholar). if Dicer the of ESCs to IFN, we the cells with and of and in ESCs and D−/−ESCs. induced of all with induced in D−/−ESCs in ESCs It is also that the of all are in D−/−ESCs in ESCs. the protein detected in but it induced by in ESCs and D−/−ESCs. PKR also in ESCs and D−/−ESCs a of the IFN we from ESCs and D−/−ESCs The is that if the IFN, it as the effects of In this we used since they are to IFN ESCs Acharya D. Wang R. Wang Bai F. Y.L. of antiviral innate immunity in of mouse embryonic stem Dev. PubMed Scopus Google Scholar). in from D−/−ESCs, but not that from ESCs induced the of in that from D−/−ESCs IFN, as by of in the these that D−/−ESCs have a type I IFN the of the major that the effects of in that most of these in ESCs and D−/−ESCs for and the different The PKR is in ESCs as we demonstrated with and R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar). if this is also in D−/−ESCs, we the cells with Although dsRNA is the of RNA with a can also PKR M. M. nuclear Dicer the of double-stranded Biol. PubMed Scopus Google Scholar, a kinase for cellular PubMed Scopus Google Scholar, Carmichael G.G. of and on the cellular response to double-stranded Biol. Rev. PubMed Scopus Google Scholar). Therefore, we speculated that B2RNA be to PKR since it has a secondary structure with and of the and mechanism of B2 an repressor of RNA 2007; Scopus Google Scholar). PKR is by the of which is a PKR M.A. Meurs E.F. Esteban M. The dsRNA protein kinase PKR: Virus and cell control.Biochimie. 2007; 89: 799-811Crossref PubMed Scopus (405) Google and by the of PKR which is the active of in detected in ESCs and D−/−ESCs with and but of in D−/−ESCs in ESCs. However, the most is that and detected in D−/−ESCs but detected in ESCs that PKR is constitutively active in D−/−ESCs. ESCs are by cell proliferation rate to constitutively by of and R. S. S. D. S. cell are by and PubMed Scopus Google Scholar). D−/−ESCs have a rate ESCs that the of and to be in D−/−ESCs in ESCs not However, major cell and which are in ESCs, are in D−/−ESCs the cell proliferation in D−/−ESCs. PKR is to cell proliferation, in ESCs as we demonstrated with PKR R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar). if constitutively PKR in D−/−ESCs to cell proliferation, we ESCs and D−/−ESCs with an PKR inhibitor which a to that PKR R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar). in increased the cell of D−/−ESCs in a with the the of but this not in ESCs. the cells by ESCs have the and cell the and they are However, in D−/−ESCs have increased cell and cell which are of cell Treatment of D−/−ESCs with the cell and D−/−ESCs have a cell with a of and cells to ESCs. Although the cell in ESCs by the cell not with the cell proliferation rate The of on the of PKR by PKR with its to increased cell proliferation to the of In line with the from in that and in D−/−ESCs in ESCs, but not by The of and and not in D−/−ESCs ESCs by not However, a protein that activates and to cell T. Huang S. The role of in the regulation of cell proliferation and PubMed Scopus Google is in D−/−ESCs could to the of cells by in D−/−ESCs. In cell it is that are cells cell in D−/−ESCs in ESCs. In particular, D−/−ESCs are ESCs to the cytotoxicity of RNA as in the of cells with different of our demonstrated that D−/−ESCs are to the cytotoxicity of and all the of PKR to the cytotoxicity by RNA we cells with to PKR to RNA transfection. in the cytotoxicity by and B2RNA can be by have that not have effects on proliferation and viability of ESCs R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar, R. Wang Acharya D. Bai F. Huang F. Y.L. Antiviral responses in mouse embryonic stem of cellular mechanisms in type I and Biol. PubMed Scopus Google D−/−ESCs not the of to the cytotoxicity in the cells with we first ESCs and D−/−ESCs with IFNβ-neutralizing by cell with The of this is that the activity of by the cells be by the antibodies its in IFNβ-neutralizing antibodies antibodies the of dsRNA on viability. However, IFNβ-neutralizing but not the cytotoxicity of and on D−/−ESCs Similar the cells with B2RNA not that contributes to the cytotoxicity in D−/−ESCs in which it can be but not in ESCs that are deficient in expressing this these that PKR and to the cytotoxicity with antiviral responses in D−/−ESCs. from its synthetic as an we detected RNAi activity in ESCs, a to the in a from a S. of the type I double-stranded RNA interference in mammalian PubMed Scopus Google Scholar). However, the features of synthetic RNA as RNA to different antiviral responses ESCs and D−/−ESCs. Dicer to the of the ability to express type I IFN and PKR in D−/−ESCs. features D−/−ESCs to the cytotoxicity with antiviral from RNA from cellular not the molecular underlying the of D−/−ESCs, but also the biological of Dicer in the regulation of and the in expressing type I IFN is a common of all types of it appears that this is an to the (3Guo Y.L. Carmichael G.G. Wang R. Hong X. Acharya D. Huang F. Bai F. Concise reviews: Attenuated innate immunity in embryonic stem cells and its implications in developmental biology and regenerative medicine.Stem Cells. 2015; 33: 3165-3173Crossref PubMed Scopus (17) Google Scholar). However, from this that lack of IFN in ESCs is not by but it is also by Dicer since D−/−ESCs have and pluripotency and in to the S. of embryonic stem S. PubMed Scopus Google Scholar, S. R. T. mouse embryonic stem cells are in and Dev. PubMed Scopus Google Scholar). features presumably made D−/−ESCs a to RNAi in mammalian cells in the with an IFN antiviral However, it is to that D−/−ESCs have gained the ability to express type I IFN as we demonstrated in this and in a recent in which increased activity against also in D−/−ESCs by S. mouse embryonic stem cells a PubMed Scopus (11) Google Scholar). finding D−/−ESCs as an system to RNAi antiviral activity in ESCs as we and the of PKR in these cells a role of Dicer in antiviral responses by cellular The constitutively PKR in D−/−ESCs have biological The PKR is in ESCs and is by cellular dsRNA in a H. PKR is by cellular and acts as a Dev. PubMed Scopus Google Scholar). that PKR proliferation, which can be partly by PKR with by PKR inhibitor R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar). The of replicated with synthetic dsRNA and B2RNA in this In to PKR can be by cellular RNA with features of as RNA and of M. M. nuclear Dicer the of double-stranded Biol. PubMed Scopus Google Scholar, a kinase for cellular PubMed Scopus Google Scholar, Carmichael G.G. of and on the cellular response to double-stranded Biol. Rev. PubMed Scopus Google Scholar, R. of innate immunity RNA structure and the protein kinase Biol. PubMed Scopus Google Scholar). Dicer is a key in the for and It also plays a critical role in the inflammatory by in that are and active in early D. the of the embryonic stem and 2015; Scopus Google Scholar). In human Dicer to of increased antiviral and M. M. nuclear Dicer the of double-stranded Biol. PubMed Scopus Google Scholar, H. S. M. RNA toxicity in PubMed Scopus Google Scholar, M. D. a of pluripotency and in mouse embryonic stem 2017; PubMed Scopus Google Scholar). Furthermore, PKR is by H. PKR is by cellular and acts as a Dev. PubMed Scopus Google Scholar). B2RNA is the mouse of in The cellular impact and of and B2 2009; PubMed Scopus Google Scholar). in D−/−ESCs, could be one of cellular from Dicer that to the of to cell and cell viability in D−/−ESCs. the molecular mechanisms underlying D−/−ESCs have gained the ability to type I IFN to be we can that in be a In particular, ESCs express a of as that are critical for the maintenance of the stem cell in embryonic stem cell and Dev. PubMed Scopus Google Scholar). Although we have the that the innate immunity of ESCs, the most are that of the of and the of the of pluripotency by Cells. PubMed Scopus Google Scholar). could to the of in ESCs since and are the key that IFN H. of Rev. Biophys. PubMed Scopus Google Scholar). and can be in D−/−ESCs by but not in ESCs S. mouse embryonic stem cells a PubMed Scopus (11) Google Scholar). Furthermore, protein a that IFN as a of in ESCs as in a recent S. mouse embryonic stem cells a PubMed Scopus (11) Google Scholar). The cell proliferation rate of ESCs is by of and and the of cell R. S. S. D. S. cell are by and PubMed Scopus Google Scholar). and the of that activity M. Cell regulation of stem cells by Cell Rev. 14: PubMed Scopus Google and this the of and in D−/−ESCs and rate of It appears that PKR by in D−/−ESCs not the of However, this increased the of a protein that activates and to cell T. Huang S. The role of in the regulation of cell proliferation and PubMed Scopus Google which the increased cell proliferation of D−/−ESCs in the of It be that a of PKR activity on the of cell is to and the cell However, it is that by PKR of M.A. Meurs E.F. Esteban M. The dsRNA protein kinase PKR: Virus and cell control.Biochimie. 2007; 89: 799-811Crossref PubMed Scopus (405) Google could be a major mechanism of of dsRNA and the of IFN responses to the of D−/−ESCs is also to the that IFN can effects in the from the with IFNβ-neutralizing antibodies that the of type I can the of which the of of PKR The that D−/−ESCs can express type I IFN is since ESCs do not these cytokines. are not of studies that the of Dicer knockout on IFN response in differentiated somatic However, it has been that Dicer with in cells in an increased IFN response P.J. an response in Res. PubMed Scopus (11) Google Scholar). It is also that Dicer knockout mouse embryonic and cells show cell proliferation rate S. R. F. are for mouse embryonic induced stem PubMed Scopus Google Scholar, and of and human PubMed Scopus Google Scholar). that Dicer in ESCs and differentiated However, Dicer play and roles in ESCs in which antiviral responses and a rate of cell proliferation are for the and of an early In the in this the that antiviral responses could negatively impact have Dicer as a repressor of the IFN system and the PKR in ESCs the However, it be that Dicer cellular The mechanisms that to the of the ability to express type I IFN and PKR in D−/−ESCs to be The of mouse ESCs have been with cell and in our studies R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar, R. Wang Acharya D. Bai F. Huang F. Y.L. Antiviral responses in mouse embryonic stem of cellular mechanisms in type I and Biol. PubMed Scopus Google Scholar). of wild-type mouse ESCs (ESCs) and Dicer knockout ESCs (D−/−ESCs) used for most to the as cell and by S. of embryonic stem S. PubMed Scopus Google and and by RNA role in mouse embryonic stem S. 2007; PubMed Scopus Google Scholar). ESCs and D−/−ESCs in mouse that as Y.L. Huang F. mouse embryonic stem cells can to and 2007; PubMed Scopus Google Scholar). differentiated from in in the of as R. Wang Acharya D. Bai F. Huang F. Y.L. Antiviral responses in mouse embryonic stem of cellular mechanisms in type I and Biol. PubMed Scopus Google Scholar, Acharya D. Wang R. Wang Bai F. Y.L. of antiviral innate immunity in of mouse embryonic stem Dev. PubMed Scopus Google Scholar). cells in a with RNA by in to the that have been R. Wang Huang F. embryonic stem cells have antiviral mechanisms that can be for the of Dev. PubMed Scopus Google Scholar). the for from a by The resulting the for in Wang Huang F. of RNA from the Res. PubMed Google Scholar, F. of and RNA by in Res. PubMed Google the with a M. the in mammalian Biol. PubMed Scopus Google and the of in from the in the of the in our to The RNA by resulting in as double-stranded RNA dsRNA that the of and dsRNA of as and by the and RNA from of the but with a in a dsRNA B2RNA is a RNA corresponding to the B2 that is in the mouse The cellular impact and of and B2 2009; PubMed Scopus Google Scholar). It can a secondary structure with and of the and mechanism of B2 an repressor of RNA 2007; Scopus Google Scholar). B2RNA by from mouse and with the and The a of a by our developed in F. Huang In of to in is as as 2017; Google Scholar). by the of the B2 in a from which B2RNA by with RNA B2RNA but in the of synthetic that acts as a to RNA Huang F. for RNA by and of in RNA Res. 33: PubMed Scopus Google ESCs and D−/−ESCs to and for the RNA the cells with for and and for and the in The PKR inhibitor used to PKR activity as R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar). the effects of on ESCs and D−/−ESCs with the cells with IFNβ-neutralizing antibodies antibodies for to RNA transfection. The cellular responses to type I IFN with mouse The cells and used for in with Y.L. of embryonic stem as stem cells with and attenuated innate Cell Res. PubMed Scopus Google Scholar). ESCs and D−/−ESCs with the and cells with The cells in that for an from cells with used as The and for and the and used for the of to the ESCs and D−/−ESCs with in with the with a and for protein a mouse to the Cell and viability with an the of of in cells a The with a on the The cell expressing by with an R. Wang Huang F. embryonic stem cells have antiviral mechanisms that can be for the of Dev. PubMed Scopus Google Scholar). Cell by the cell from to that from of with as R. Wang Huang F. embryonic stem cells have antiviral mechanisms that can be for the of Dev. PubMed Scopus Google Scholar). The viability of ESCs and D−/−ESCs by cell and by cell The of cells with a The which with the of used as an of cell proliferation viability. Cell with an the cells with The cell PKR and to the cells with a of The cells for and the on cell proliferation the by to our R. Wang Acharya D. Bai F. Huang F. Y.L. embryonic stem cells are deficient in type I in response to and double-stranded Biol. PubMed Scopus Google Scholar). The antibodies against and from and antibodies from Cell RNA by on an system as Y.L. Huang F. mouse embryonic stem cells can to and 2007; PubMed Scopus Google Scholar). The from the for in Res. 2001; PubMed Scopus Google Scholar). used to of for The of the are in used for in a with a and are as the are by to are as in in this are in the in the The that they have of with the of this and for ESCs and D−/−ESCs. also of for the of the by the of of F. M. and M. F. and F. M. in part by the of

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