The availability of autologous adult stem cells is one of the essential prerequisites for human stem cell therapy. Urine-derived stem cells (USCs) are considered as desirable cell sources for cell therapy because donor-specific USCs are easily and non-invasively obtained from urine. Efficient isolation, expansion, and differentiation methods of USCs are necessary to increase their availability. Here, we developed a method for efficient isolation and expansion of USCs using Matrigel, and the rho-associated protein kinase (ROCK) inhibitor, Y-27632. The prepared USCs showed significantly enhanced migration, colony forming capacity, and differentiation into osteogenic or chondrogenic lineage. The USCs were successfully reprogramed into induced pluripotent stem cells (USC-iPSCs) and further differentiated into kidney organoid and hematopoietic progenitor cells (HPCs). Using flavonoid molecules, the isolation efficiency of USCs and the production of HPCs from the USC-iPSCs was increased. Taken together, we present an improved isolation method of USCs utilizing Matrigel, a ROCK inhibitor and flavonoids, and enhanced differentiation of USC-iPSC to HPC by flavonoids. These novel findings could significantly enhance the use of USCs and USC-iPSCs for stem cell research and further application in regenerative stem cell-based therapies.

Flavonoids enhance the self-renewal and differentiation potential of mesenchymal stem cells (MSCs) and have therapeutic activities, including regenerative, anti-oxidative, and anti-inflammatory effects. Recent studies have revealed that MSC-derived extracellular vesicles (MSC-EVs) have therapeutic effects on tissue regeneration and inflammation. To facilitate further research on the therapeutic potential of MSC-EVs derived from flavonoid-treated MSCs, we surveyed the production of EVs and their therapeutic applications in wound regeneration. MSCs treated with flavonoids enhanced EV production twofold compared with naïve MSCs. EVs produced by MSCs treated with flavonoids (Fla-EVs) displayed significant anti-inflammatory and wound-healing effects in vitro. The wound-healing capacity of EVs was mediated by the upregulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Interestingly, the protein level of p-ERK under inhibition of MEK signals was maintained in Fla-EV-treated fibroblasts, suggesting that Fla-EVs have a higher therapeutic potential than naïve MSC-EVs (Cont-EVs) in wound healing. Moreover, the in vivo wound closure effect of the Fla-EVs showed significant improvement compared with that of the flavonoid-only treatment group and the Cont-EVs. This study provides a strategy for the efficient production of EVs with superior therapeutic potential using flavonoids.