Abstract
Adenosine is an important modulator of metabolic activity with powerful tissue and cell protective functions. Adenosine kinase (ADK), the major adenosine-regulating enzyme, is critical to adapt its intra- and extracellular levels in response to environmental changes. Lentiviral RNAi-mediated downregulation of ADK in human mesenchymal stem cells (hMSCs) has therefore been considered an effective tool for engineering therapeutically effective adenosine-releasing cell grafts that could constitute patient-identical autologous implants for clinical application. We constructed lentiviral vectors that co-express miRNA directed against ADK and an emerald green fluorescent protein (EmGFP) reporter gene. Following lentiviral transduction of hMSCs, we demonstrated up to 80% downregulation of ADK and 98% transduction efficiency. Transduced hMSCs continued to express EmGFP after four to six consecutive passages, and EmGFP-positive hMSC grafts survived in the hippocampal fissure of mouse brains and provided efficient adenosine-dependent neuroprotection in a mouse model of seizure-induced cell loss.
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Acknowledgements
This project was supported by grant R01 NS058780 from the National Institutes of Health, and by the Epilepsy Research Foundation through the generous support of Arlene and Arnold Goldstein Family Foundation.
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Ren, G., Boison, D. (2017). Engineering Human Mesenchymal Stem Cells to Release Adenosine Using miRNA Technology. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_16
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DOI: https://doi.org/10.1007/978-1-4939-7108-4_16
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