biostudies-literatureZhang DNational Natural Science Foundation of ChinaTaishan Scholars Program of Shandong Province347-358https://www.ebi.ac.uk/biostudies/studies/S-EPMC9705153biostudies-literatureUnknown15(4)<h4>Background and objectives</h4>The search for a suitable alternative for urethral defect is a challenge in the field of urethral tissue engineering. Induced pluripotent stem cells (iPSCs) possess multipotential for differentiation. The <i>in vitro</i> derivation of urothelial cells from mouse-iPSCs (miPSCs) has thus far not been reported. The purpose of this study was to establish an efficient and robust differentiation protocol for the differentiation of miPSCs into urothelial cells.<h4>Methods and results</h4>Our protocol made the visualization of differentiation processes of a 2-step approach possible. We firstly induced miPSCs into posterior definitive endoderm (DE) with glycogen synthase kinase-3<i>β</i> (GSK3<i>β</i>) inhibitor and Activin A. We investigated the optimal conditions for DE differentiation with GSK3<i>β</i> inhibitor treatment by varying the treatment time and concentration. Differentiation into urothelial cells, was directed with all-trans retinoic acid (ATRA) and recombinant mouse fibroblast growth factor-10 (FGF-10). Specific markers expressed at each stage of differentiation were validated by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) assay, immunofluorescence staining, and western blotting Assay. The miPSC-derived urothelial cells were successfully in expressed urothelial cell marker genes, proteins, and normal microscopic architecture.<h4>Conclusions</h4>We built a model of directed differentiation of miPSCs into urothelial cells, which may provide the evidence for a regenerative potential of miPSCs in preclinical animal studies.International journal of stem cellsGeneration of Urothelial Cells from Mouse-Induced Pluripotent Stem Cells.PMC97051538187052581572835tsqn201909199Wu JZhang DWang DBao XSun FYao HWang JfalseGeneration of Urothelial Cells from Mouse-Induced Pluripotent Stem Cells.<h4>Background and objectives</h4>The search for a suitable alternative for urethral defect is a challenge in the field of urethral tissue engineering. Induced pluripotent stem cells (iPSCs) possess multipotential for differentiation. The <i>in vitro</i> derivation of urothelial cells from mouse-iPSCs (miPSCs) has thus far not been reported. The purpose of this study was to establish an efficient and robust differentiation protocol for the differentiation of miPSCs into urothelial cells.<h4>Methods and results</h4>Our protocol made the visualization of differentiation processes of a 2-step approach possible. We firstly induced miPSCs into posterior definitive endoderm (DE) with glycogen synthase kinase-3<i>β</i> (GSK3<i>β</i>) inhibitor and Activin A. We investigated the optimal conditions for DE differentiation with GSK3<i>β</i> inhibitor treatment by varying the treatment time and concentration. Differentiation into urothelial cells, was directed with all-trans retinoic acid (ATRA) and recombinant mouse fibroblast growth factor-10 (FGF-10). Specific markers expressed at each stage of differentiation were validated by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) assay, immunofluorescence staining, and western blotting Assay. The miPSC-derived urothelial cells were successfully in expressed urothelial cell marker genes, proteins, and normal microscopic architecture.<h4>Conclusions</h4>We built a model of directed differentiation of miPSCs into urothelial cells, which may provide the evidence for a regenerative potential of miPSCs in preclinical animal studies.2022-01-01T00:00:00Z2022 Nov2025-04-04T23:44:38.695Z2025-04-04T23:44:38.695ZS-EPMC97051533576905610.15283/ijsc21250