Int J Stem Cells.  2022 Nov;15(4):347-358. 10.15283/ijsc21250.

Generation of Urothelial Cells from Mouse-Induced Pluripotent Stem Cells

• Zhang D
• Sun F
• Yao H
• Wang D
• Bao X
• Wang J
• Wu J

• KMID: 2536205
• DOI: http://doi.org/10.15283/ijsc21250

Abstract

Background and Objectives
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 in vitro 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.
Methods and Results
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-3β (GSK3β) inhibitor and Activin A. We investigated the optimal conditions for DE differentiation with GSK3β 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.
Conclusions
We built a model of directed differentiation of miPSCs into urothelial cells, which may provide the evi-dence for a regenerative potential of miPSCs in preclinical animal studies.

Keyword

Induced pluripotent stem cells; Differentiation; Urothelial cells; Tissue engineering

Figure

• 

  Fig. 1 Verify the pluripotent status of miPSCs. (A) qRT-PCR assays for expression of OCT4, NANOG, SOX2 during the induction of miPSCs to urothelial cells (mean±SD for six inde-pendent experiments, ****p<0.0001). (B) OCT4 and NANOG analysis by flow cytometry. All flow data represent n=3 experiments (C) Immu-nostaining of OCT4 and NANOG in miPSCs. Scale bar, 300 μm (these images are representative of four inde-pendent experiments). miPSCs: mouse-induced pluripotent stem cells, SD: standard deviation.

• 

  Fig. 2 Induction of posterior DE from miPSCs. (A) A schematic of the differentiation trajectory and the markers expressed at each stage of differentiation. (B) Expression analyses of CDX2, SOX17, and FOXA2 in the cells by qRT-PCR on day 3 (mean±SD for six independent experiments, ****p<0.0001). (C) Western blot analysis of CDX2 in miPSCs, miPSC-derived urothelial cells, mouse urothelial cells and miPSCs at day 3 after induction of differentiation. Scale bar, 130 μm (n=5). (D) Western blot analysis of CDX2 in miPSCs and differentiated miPSCs on day 3 (n=5). (E) Immunostaining of CDX2 in miPSCs, differentiated miPSCs on day 3 and mouse colon. Scale bar, 130 μm (these images are representative of four independent experiments). (F) Immunostaining of SOX17 and FOXA2 in differentiated miPSCs on day 3. Scale bar, 130 μm (these images are representative of four independent experiments). DE: definitive endoderm, miPSCs: mouse-induced pluripotent stem cells, SD: standard deviation, Uro: mouse urothelial cells.

• 

  Fig. 3 Effects of CHIR99021 treatment on posterior DE induction. (A) Expression analyses of CDX2, SOX17, and FOXA2 in differentiated cells treated with different doses of CHIR99021 by qRT-PCR (mean±SD for six independent experiments, ****p<0.0001). (B) Morphology of differentiated cells after treatment with 4 μM, 6 μM, or 8 μM CHIR99021 for 3 days. Scale bar, 130 μm. (C) Expression analyses of CDX2 SOX17, and FOXA2 in differentiated cells that underwent different durations of CHIR99021 treatment by qRT-PCR (mean±SD for six independent experiments, ****p<0.0001). DE: definitive endoderm, SD: standard deviation.

• 

  Fig. 4 Induction of caudal hindgut from posterior DE. (A) Expression analyses of HOXA13 and HOXD13 in the cells by qRT-PCR on day 7 (mean±SD for six independent experiments, ****p<0.0001). (B) Im-munostaining of HOXD13 in miPSCs and differentiated miPSCs on day 7. Scale bar, 130 μm (these images are representative of four indepen-dent experiments). DE: definitive endoderm, miPSCs: mouse-induced pluripotent stem cells, SD: standard deviation.

• 

  Fig. 5 Induction of urothelial cells from caudal hindgut. (A) Expression analyses of Uroplakin IA, Uroplakin IB, Uroplakin II, Uroplakin III, CK20, CK5, CK7, CK13, ZO-1, and E-cadherin in the cells by qRT-PCR on day 16 (mean±SD for six independent experiments, ****p<0.0001). (B) Western blot analysis of Uroplakin Ib and Uroplakin III in miPSCs, miPSC-derived urothelial cells, mouse urothelial cells and mouse colon (n=5). (C) Western blot analysis of CK20, CK7, CK13, ZO-1, and E-cadherin in miPSCs, miPSC-derived urothelial cells and mouse urothelial cells (n=5). (D) Immunostaining of Uroplakin II, CK20, CK7, CK13, ZO-1, and E-cadherin in differentiated miPSCs on day 16. Scale bar, 130 μm (these images are representative of four independent experiments). (E) Morphology of cells at different stages of differentiation. Scale bar, 130 μm. miPSCs: mouse-induced pluripotent stem cells, SD: standard deviation, Uro: mouse urothelial cells.

Reference

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