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RNAi and microRNA-Mediated Gene Regulation in Stem Cells

Volume 650 of the series Methods in Molecular Biology pp 225-240

Date:

Engineering Human Mesenchymal Stem Cells to Release Adenosine Using miRNA Technology

  • Gaoying RenAffiliated withDepartment of Medicine, University of Washington
  • , Detlev BoisonAffiliated withRobert S. Dow Neurobiology Laboratories, Legacy Research

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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 extra-cellular levels in response to environmental changes. Lentiviral RNAi-mediated down-regulation 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 coexpress miRNA directed against ADK and an emerald green fluorescent protein (EmGFP) reporter gene. Following lentiviral transduction of hMSCs, we demonstrated up to 80% down-regulation of ADK and 98% transduction efficiency. Transduced hMSCs continued to express EmGFP after 4–6 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.

Key words

Adenosine adenosine kinase epilepsy kainic acid RNAi lentivirus human mesenchymal stem cells cell therapy