Engineering human mesenchymal stem cells to release adenosine using miRNA technology.

: 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.