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bioRxiv · 10.1101/2025.08.31.673107

Urinary exfoliated cells exhibit mesodermal lineage differentiation potential in the absence of dedicated culture conditions

Kumagai, N.·Ando, T.·Kondoh, T.·Matsumoto, Y.·Abe, M.·Ikezumi, Y.
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Abstract

IntroductionUrine contains various cells derived from the kidney, including renal tubular epithelial cells, glomerular epithelial cells, and mesenchymal stem cells. Urinary exfoliated mesenchymal stem cells can be differentiated into adipocytes, osteoblasts, chondrocytes, muscle cells, or neuronal cells, if grown in specialized culture conditions. In this study, however, we identified the development of multiple mesodermal cell lineages from urinary exfoliated cells despite using a single culture system not designed for these cell differentiations. MethodsUrinary exfoliated cells used for culturing were obtained from pediatric patients with kidney disease. All cultures were maintained using an identical, non-specifically supplemented, rich, DMEM/F12-based growth medium, refreshed regularly throughout the experiments. Cells of different mesodermal lineages--adipocytes, osteoblasts, and chondrocytes--were identified by staining with Oil Red O, Alizarin Red, and Alcian Blue solutions. Moreover, the expression of mRNA markers specific for adipocytes, osteoblasts, and chondrocytes, as well as for stem cells and--more specifically--mesenchymal stem cells, was analyzed by RT-PCR. ResultsAlthough the abundances varied, in all 11 cultures of urinary exfoliated cells from six patients, dye staining revealed multiple cell clusters of mesodermal lineages. All cultures contained clusters of the adipocyte and chondrocyte lineages, and seven also contained clusters of the osteoblast lineage. Morphology differences were found even between clusters stained with the same dye. RT-PCR confirmed the presence of the three lineages, while also revealing expression of genes specific to pluripotent stem cells (Nanog, Oct3/4, SOX2, and LIF) and genes more specific for mesenchymal stem cells (CD73, CD90, and CD105). ConclusionsAmong urinary exfoliated cells cultured using our method, clusters of cells of mesodermal lineages were observed without needing to selectively adjust the culture medium. The cells seemed to differentiate and proliferate from mesenchymal stem cells in the urine. We envision that these initial findings may eventually contribute to a deeper understanding of urinary stem cell differentiation and to optimizing lineage-specific differentiation for research and therapeutic applications.

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Kumagai, N., Ando, T., Kondoh, T., Matsumoto, Y., Abe, M., Ikezumi, Y.. 2025-09-02. Urinary exfoliated cells exhibit mesodermal lineage differentiation potential in the absence of dedicated culture conditions. https://doi.org/10.1101/2025.08.31.673107

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