Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy

Abstract

We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFN) when exposed to viral infection and double-stranded RNA. In this study, we extended our investigation and demonstrated that single-stranded RNA and protein-encoding mRNA can induce strong IFN expression and cytotoxicity in fibroblasts and epithelial cells, but none of the effects associated with these antiviral responses were observed in mESCs. Our results provided additional data to support the conclusion that mESCs are intrinsically deficient in antiviral responses. While our findings represent a novel feature of mESCs that in itself is important for understanding innate immunity development, we exploited this property to develop a novel mRNA-mediated gene expression cell model. Direct introduction of synthetic mRNA to express desired genes has been shown as an effective alternative to DNA/viral vector-based gene expression. However, a major biological challenge is that a synthetic mRNA is detected as a viral RNA analog by the host cell, resulting in a series of adverse effects associated with antiviral responses. We demonstrate that the lack of antiviral responses in mESCs effectively avoids this problem. mESCs can tolerate repeated transfection and effectively express proteins from their synthetic mRNA with expected biological functions, as demonstrated by the expression of green fluorescent protein and the transcription factor Etv2. Therefore, mRNA-based gene expression could be developed into a novel ESC differentiation strategy that avoids safety concerns associated with viral/DNA-based vectors in regenerative medicine.

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

“…This observation echoes the lack of IFN expression in virus-infected ESCs (16,19). Since NFκB is a key transcription factor that mediates a broad spectrum of immune and inflammatory responses induced by numerous pathogens and cytokines (40), we reasoned that the inactive status of NFκB in ESCs as noted in virus-infected cells could also account for the lack of immune and inflammatory response in bacteria-infected ESCs.…”

“…Indeed, it has been demonstrated that ESCs do not show immune responses typically seen in differentiated cells when infected with bacteria and viruses (14,15). Our recent studies in mouse ESCs (mESCs) (16–18) and those from other investigators in human ESCs (hESCs) and in induced pluripotent stem cells (iPSCs) (19,20) demonstrated that the IFN system, the central component of innate antiviral immunity in differentiated somatic cells (21), is not fully developed in these cells. Therefore, the lack of innate immune responses to both bacterial and viral infection appears to be an intrinsic property of all pluripotent stem cells (10).…”

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

“…Overall, our results suggested that the differentiation process could induce the IFN expression mechanism, but not to the level in naturally differentiated 10T1/2 cells. It is noted that transfection efficiency of polyIC is similar among D3, D3-FBs, and 10T1/2 cells as determined by the expression of eGFP from its synthetic mRNA transfected to these cells (data not shown) with the method that we have previously described [11]. Therefore, the different expression levels of IFN in response to polyIC transfection among the three cell types are attributed to their intrinsic properties.…”

“…1A). Since mESCs are unable to express IFNb in response to synthetic viral RNA analogs and live viral infection [11,12], we examined whether differentiation would change this deficiency. As shown in Fig.…”

“…Similarly, mESCs are susceptible to the cytopathic effect of bacterial and viral infection, but they do not show immune responses typically seen in differentiated cells [9,10]. We recently reported that mESCs are unable to express type I interferon (IFN) [11,12] and have attenuated responses to these cytokines [13]. Therefore, the IFN system, which is the central part of innate immunity in differentiated somatic cells [14], is not fully functional in ESCs.…”