The efficacy and safety of Collagen-I and hypoxic conditions in urine-derived stem cell ex vivo culture

Upper urinary tract-derived urine stem cells (USCs) are considered a valuable mesenchymal stem cell source for autologous cell therapy. However, the reported culture condition for USCs is not appropriate for large-quantity production, because cells can show limited replicativity, senescence, and undesirable differentiation during cultivation. These drawbacks led us to reconstitute a culture condition that mimics the natural stem cell niche. We selected extracellular matrix protein and oxygen tension to optimize the ex vivo expansion of USCs, and compared cell adhesion, proliferation, gene expression, chromosomal stability, differentiation capacity, immunity and safety. Culture on collagen type I (ColI) supported highly enhanced USC proliferation and retention of stem cell properties. In the oxygen tension analysis (with ColI), 5% O₂ hypoxia showed a higher cell proliferation rate, a greater proportion of cells in the S phase of the cell cycle, and normal stem cell properties compared to those observed in cells cultured under 20% O₂ normoxia. The established reconstituted condition (ColI/hypoxia, USCs^recon) was compared to the control condition. The expanded USCs^recon showed highly increased cell proliferation and colony forming ability, maintained transcription factors, chromosomal stability, and multi-lineage differentiation capacity (neuron, osteoblast, and adipocyte) compared to the control. In addition, USCs^recon retained their immune-privileged potential and non-tumorigenicity with in vivo testing at week 8. Therefore, reconstituted condition allows for expanded uUSC cell preparations that are safe and useful for application in stem cell therapy.