Genome engineering of human urine-derived stem cells to express lactoferrin and deoxyribonuclease.

Urine-derived stem cells (USCs) are adult kidney cells that have been isolated from a urine sample and propagated in tissue culture on gelatin-coated plates. Urine is a practical and completely painless source of cells for gene and cell therapy applications. We have isolated, expanded, and optimized transfection of USCs to develop regenerative therapies based on piggyBac transposon-modification. USCs from a healthy donor sample were isolated according to established protocols. Within two months, ten clones had been expanded, analyzed, and frozen. FACS analysis of individual clones revealed that all ten clones expressed characteristic USC markers (97-99% positive for CD44, CD73, CD90 and CD146; negative for CD31, CD34, and CD45).The isolated USCs were successfully differentiated along the osteogenic, adipogenic, and chondrogenic lineages, suggesting multipotent differentiation capacity. Additionally, the USCs were differentiated into podocytes positive for NEPHRIN, PODXL, and WT1. Transfection of USCs with a strongly-expressing GFP plasmid was optimized to achieve 61% efficiency in live cells using several commercially available lipophilic reagents. Transgene promoters were compared in five luciferase-expressing piggyBac transposons by IVIS imaging. The CMV promoter produced the highest luciferase signal, followed by EF1-α. Finally, HEK-293 and USCs were transfected with piggyBac transposons expressing lactoferrin and DNase1 for treatment of acute kidney injury associated with rhabdomyolysis. We found that both proteins were expressed in USCs and that lactoferrin was successfully secreted into the cell culture media. In conclusion, urine-derived stem cells represent a clinically relevant cell type that can express non-viral transgenes.