Fig 2: CD133+ SP cells exhibit high self-renewal capacity. (A) A clone formation efficiency assay revealed that the total number of tumor spheres generated by the CD133+ SP cells were significantly higher, compared with the number generated by the MP cells. (B) Quantification of the results of reverse transcription-quantitative polymerase chain reaction analysis showed that the relative mRNA expression levels of Oct-4, EpCAM, Sox-2, Bmi-1 and Nestin were significantly upregulated in the CD133+ SP cells, compared with the MP cells. (C) Fluorescence microscopy revealed that the CD133+ SP cells exhibited more positive CD44 fluorescence and EpCAM stem cell proteins, whereas this fluorescence was not enriched in the MP cells. Magnification, ×400. Data are presented as the mean ± standard error of the mean. **P<0.01, vs. SP. Oct-4, octamer-binding transcription factor-4; EpCam, epithelial cell adhesion molecule; Sox-1, (sex determining region Y)-box 2; Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site-1; CD. cluster of differentiation; SP, side population; MP, main population.

Fig 3: CD133+ SP cells show high levels of differentiation. (A) In vitro proliferation assay revealed that the proliferation rate of the CD133+ SP cells were significantly higher, compared with the MP cells. (B) Morphology of the CD133+ SP cells changed rapidly on day 5 and later developed filaments, which resembled fibroblast. Magnification, ×100. Data are presented as the mean ± standard error of the mean. *P<0.05 and **P<0.01, between SP and MP cells. CD. cluster of differentiation; SP, side population; MP, main population; OD, optical density.

Fig 4: CD133+ SP cells are highly tumorigenic and invasive. (A) CD133+ SP cells initiate tumor growth in NON/SCID mice significantly faster, compared with MP cells. (B) Immunohistochemistry showed that SP cell-derived tumor tissues were more positive to the CD133 stem cell protein. Magnification, ×250. (C) CD133+ SP cell invasiveness was measured using a Matrigel assay. The number of CD133+ SP cells, which invaded across the membrane were significantly higher, compared with the number of MP cells. Data are presented as the mean ± standard error of the mean. **P<0.01, vs. SP. CD, cluster of differentiation; SP, side population; MP, main population.

Fig 5: Recapitulation of lumen formation during SMG development in a mouse SMG organoid culture. (A) Organoid growth from single cells was monitored at each time point. Black arrows indicate internal lumen formation as shown in the bright-field microscopy images. Scale bar indicates 100 µm. (B) SMG organoids were harvested at each time point and subjected to whole-mount immunofluorescence microscopy for the evaluation of KRT5 (white), KRT19 (green), and KRT7 (red) expressions from days 0 to 9. Up to day 5, images were obtained through maximum intensity projection. From days 7 to 9, single images were obtained from z-stack. Micro-lumens were denoted with white asterisks. Scale bars indicate 20 µm. Nuclei were counterstained with DAPI (blue). (C) KRT5 (white), CD133 (green), and KRT7 (red) expressions on day 4. Scale bars indicate 50 µm. Nuclei were counterstained with DAPI (blue). (D) The organoids were treated with 200 nM of VIP on day 2, and lumen enlargement was observed on day 4. Black arrows indicate internal lumen formation. Scale bars indicate 100 µm. (E) The proportion of organoids with lumen were determined (n = 3). The results are expressed as the mean ± SD. **p < 0.01. (F) SMG organoids were harvested on days 5 and 9, and 200 nM of VIP was treated on the last 1 day. Organoid were subjected to immunofluorescence staining for KRT5 (green) and KRT7 (red). Scale bars indicate 50 µm. Nuclei were counterstained with DAPI (blue). All experiments were performed three times independently.