Fig. 3

Establishment of a xenograft model using HOXC8-imESCs and histological analysis. (A) Summary of the xenograft mouse model. Cell aggregates of Control-imESCs or HOXC8-imESCs (Fig. 3B) were transplanted under the renal capsules of immunodeficient NOD/Scid mice and harvested five weeks after transplantation for morphological evaluation. (B) Detection of GFP-expressing cells in cell aggregates of both cell lines. Cell viability within the aggregates, which were embedded in collagen gel for transplantation, was confirmed by GFP expression. Representative fluorescent images of Control-imESCs one day after culture are shown. Scale bar = 200 μm. (C) Whole and cross-sectional images of kidneys transplanted with Control-imESCs and HOXC8-imESCs, which were harvested five weeks after transplantation. Scale bar = 1 mm. (D) HE and trichrome stained images of xenograft lesions in Control-imESCs or HOXC8-imESCs. Scale bar = 100 μm. (E) Immunofluorescence staining of xenograft lesions for ACTA2 and COL1A1, as well as the corresponding HE and DAPI-staining. In ACTA2 and COL1A1 staining, the images on the right show magnified views of the regions outlined by dashed lines in the corresponding images on the left. Nuclei were counterstained with DAPI. Scale bar = 100 μm. (F) COL1A1-positive area and ACTA2-positive cell proportions. The collagen-positive area rate (%) was calculated by the average of the positive area ratios within the field of view at 200x magnification. The ACTA2-positive cells rate (%) was calculated as the ratio of ACTA2-positive cells to VIM-positive cells. Quantification was performed using three independent tissue sections (n = 3) for both Control-imESCs and HOXC8-imESCs xenografts. All quantified values are presented as the mean ± SD. **p < 0.01. (G) Immunofluorescence staining of xenograft lesions for VIM, human nuclear marker, and HOXC8, along with HE and DAPI staining of adjacent sections. In VIM, human nuclear marker, and HOXC8 staining, the images on the right are magnified views of the areas outlined by dashed lines in the corresponding images on the left. Nuclei were counterstained with DAPI. Scale bar = 100 μm.