Urine-derived stem cells; A robust platform for generating native or engineered autologous Extracellular Vesicles

Background Mesenchymal stem cell (MSC) therapy holds great potential as a treatment form, yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome, including extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect the yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to; enhance potency, increase circulation time, or mediate targeting. However, efficient transfection of MSCs is challenging, and the process can affect MSC phenotype and chromosomal integrity, which are both imperative to preserve in a therapeutic setting. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production. Methods We isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to engineer USCs using transposon technology genetically. Results There were no observable differences at time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the different time points, but male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 hours without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell culture was established. Conclusions Isolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.