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Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation
Published on: October 20, 2014
Mouse embryonic stem cells have underdeveloped antiviral mechanisms that can be exploited for the development of
Ruoxing Wang1, Chengwen Teng, Joseph Spangler
11 Department of Biological Sciences, The University of Southern Mississippi , Hattiesburg, Mississippi.
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.
Insights
Mouse embryonic stem cells (mESCs) lack antiviral responses, making them ideal for mRNA gene expression. This discovery enables a novel cell model and potential regenerative medicine strategies.
Area of Science:
- Immunology
- Stem Cell Biology
- Molecular Biology
Background:
- Mouse embryonic stem cells (mESCs) exhibit a deficiency in type I interferon (IFN) production upon exposure to viral infections and double-stranded RNA.
- Fibroblasts and epithelial cells mount robust antiviral responses, including IFN expression and cytotoxicity, when exposed to single-stranded RNA and mRNA.
Purpose of the Study:
- To investigate the intrinsic antiviral response capabilities of mESCs.
- To leverage the mESC's unique lack of antiviral response for developing a novel mRNA-mediated gene expression system.
- To explore the potential of mRNA-based gene delivery in mESCs for regenerative medicine applications.
Main Methods:
- Exposure of mESCs, fibroblasts, and epithelial cells to various RNA stimuli (viral infection, double-stranded RNA, single-stranded RNA, synthetic mRNA).
- Assessment of type I interferon (IFN) expression and cytotoxicity.
- Transfection of mESCs with synthetic mRNA encoding reporter proteins (e.g., green fluorescent protein) and transcription factors (e.g., Etv2).
Main Results:
- mESCs demonstrated a significant deficiency in IFN expression and cytotoxicity when challenged with viral RNA analogs, unlike fibroblasts and epithelial cells.
- Synthetic mRNA introduction into mESCs did not trigger adverse antiviral responses.
- mESCs successfully expressed functional proteins from synthetic mRNA, tolerating repeated transfections.
Conclusions:
- mESCs possess an intrinsic deficiency in antiviral responses, representing a novel characteristic important for innate immunity research.
- The lack of antiviral responses in mESCs provides a unique platform for efficient and safe mRNA-mediated gene expression.
- This approach offers a promising strategy for mRNA-based cell differentiation and gene therapy in regenerative medicine, circumventing safety issues associated with viral/DNA vectors.
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