Kondoh, T. | Search bioRxiv

bioRxiv2025

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

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.

cell biology↗

bioRxiv2019

Modeling the efficiency of filovirus entry into cells in vitro: Effects of SNP mutations in the receptor molecule

Interaction between filovirus glycoprotein (GP) and the Niemann-Pick C1 (NPC1) protein is essential for membrane fusion during virus entry. Some single-nucleotide polymorphism (SNPs) in two surface-exposed loops of NPC1 are known to reduce viral infectivity. However, the dependence of differences in entry efficiency on SNPs remains unclear. Using vesicular stomatitis virus pseudotyped with Ebola and Marburg virus GPs, we investigated the cell-to-cell spread of viruses in cultured cells expressing NPC1 or SNP derivatives. Eclipse and virus-producing phases were assessed by in vitro infection experiments, and we developed a mathematical model describing spatial-temporal virus spread. This mathematical model fit the plaque radius data well from day 2 to day 6. Based on the estimated parameters, we found that SNPs causing the P424A and D508N substitutions in NPC1 most effectively reduced the entry efficiency of Ebola and Marburg viruses, respectively. Our novel approach could be broadly applied to other virus plaque assays. Author SummaryEbola virus belongs to the family Filoviridae, together with Marburg virus and Cueva virus. In 2015, the World Health Organization included Ebola and Marburg viruses among the infectious diseases that should be globally prioritized because these filoviruses can cause severe hemorrhagic fever in humans and nonhuman primates, and antiviral agents to these viruses are very limited. Filovirus particles bear the envelope glycoprotein (GP), which is the only viral surface protein and thus responsible for receptor binding and membrane fusion. Interaction between filovirus GP and the Niemann-Pick C1 (NPC1) protein is essential for membrane fusion during virus entry. Some single-nucleotide polymorphism (SNPs) in two surface-exposed loops of NPC1 are known to reduce viral infectivity. However, the dependence of differences in entry efficiency on SNPs remains unclear. In this study, combining in vitro experiments and mathematical models, we evaluated on the interaction between GP and wildtype and mutant NPC1, enabling us to estimate the cellular entry efficiency during plaque formation.

microbiology↗