[Biological characteristics of urine-derived stem cells during in vitro expansion]

To explore the biological characteristics of urine-derived stem cells (USCs) during in vitro expansion to provide a reference for seed cell selection.

USCs were isolated from fresh midstream urine samples from healthy volunteers by using the centrifugation combined with adherent cultivation method, followed by continuous passaging at a 1∶3 ratio. The morphological changes of cells were observed under inverted microscopy and analyzed using Image J software. After osteogenic and chondrogenic induction and identification of cells at passage 3 (P3), the biological characteristics of cells at different passages (P3, P5, and P7) were compared. Cell proliferation, cell cycle, senescence, and apoptosis were assessed via cell counting kit 8 assays, flow cytometry, β-galactosidase staining, and Annexin Ⅴ-fluorescein isothiocyanate/propidium iodide staining. RT-PCR was used to detect the expression of stemness genes (Nanog, Oct4, c-Myc, and Sox2). Tube formation assays were conducted to evaluate the in vitro tube-forming ability of cells at different passages.

After osteogenic and chondrogenic induction, USCs were positive for alkaline phosphatase‌, Alizarin red, and Alcian blue staining, respectively. During expansion, the morphology of USCs gradually shifted from rice-grain-like to spindle-shaped, with increasing trends in long diameter, short diameter, area, and aspect ratio ( P<0.05). Surface antigen expression was similar across passages. The proliferation capacity declined with increasing passages, and the proportions of S and G ₂/M phases decreased. The proportion of senescent cells gradually increased ( P<0.05), but the proportion of apoptotic cells showed no significant change ( P>0.05). Compared to USCs at P3, the expression of Oct4 was significantly lower in USCs at P7 ( P<0.05), whereas no significant difference was found in the expression of other stemness genes among different passages ( P>0.05). Tube formation assays demonstrated that USCs at P3 and P5 exhibited superior in vitro tube-forming ability compared to cells at P7 ( P<0.05). When co-cultured with human umbilical vein endothelial cells (HUVECs), USCs at P3 showed higher total tube length than the HUVECs only group ( P<0.05).

During in vitro expansion, USCs was accompanied by cell enlargement, reduced proliferation, and accumulation of senescence. Compared to USCs at P7, those at P3 and P5 retained better proliferative status and in vitro tube formation ability.