Additional file 7 of Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells
Creators: name: Chen, JunLong
name: Horiuchi, Shinichiro
name: Kuramochi, So
name: Kawasaki, Tomoyuki
name: Kawasumi, Hayato
name: Akiyama, Saeko
name: Arai, Tomoki
name: Morinaga, Kenichi
name: Kimura, Tohru
name: Kiyono, Tohru
name: Akutsu, Hidenori
name: Ishida, Seiichi
name: Umezawa, Akihiro
Publisher: name: figshare
Year: 2024
Subjects: subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Computational Biology

description: Additional file7: Puromycin-based selection of intestinal epithelial cells (A) Phase-contrast photomicrographs of RYU, LONG and human ESCs upon exposure to puromycin. Puromycin (1 µg/mL) was added (Day 0). Scale bars: 500 µm. (B) Experimental schematic for RYU on LONG. (C) Phase-contrast micrograph and immunocytochemistry of RYU with exposure to puromycin. Immunocytochemistry was performed with antibodies to proteins involved in barrier functions (ECAD, CDH17, ZO-1, Claudin-2, Claudin-7) and CDX2. Nuclei were stained with DAPI. Scale bars: 500 (black) and 50 (yellow) µm. (D) Trans-epithelial electrical resistance measurements for RYU. LONG and Caco-2 cells served as negative and positive controls, respectively. White bars are puromycin-untreated RYU on LONG, and black bars are puromycin-treated RYU on LONG. Results are expressed as mean ± SD (n = 3 triplicate biological experiments). Statistical significance was determined using Student's t-test. (E) Lucifer Yellow permeability tests for RYU on LONG. Caco-2 cells served as a control. Black and white bars are treated and non-treated with puromycin, respectively. Results are expressed as mean ± SD (n = 3 triplicate biological experiments). Statistical significance was determined using Student's t-test. *&lt;0.05, **&lt;0.01. (F) Lucifer Yellow permeability test was performed for barrier performance. Barrier performance of RYU (Puromycin-treated (+) and non-treated (-)) was altered in culture. Blue line, Puromycin-treated; orange line, Puromycin-untreated. Results are expressed as mean ± SD (n = 3 triplicate biological experiments). Statistical significance was determined using Student's t-test; *&lt;0.05, **&lt;0.01. (G) Lucifer yellow concentration over time in lucifer yellow permeability tests 16 and 20 days after confluence. Puromycin-untreated (-, orange), Puromycin-treated (+, blue). (H) Phase-contrast photomicrographs of RYU on LONG (Puromycin-treated (+) and non-treated (-)) 16 and 20 days after confluence. Pre- and post-Lucifer Yellow permeability test is shown on the left and right panels, respectively. Scale bars: 500 µm. (I) Permeability test with Rhodamine 123. The efflux ratio (ER) for Rhodamine 123 was derived from the Papp values associated with basal-to-apical transport (B to A) and apical-to-basal transport (A to B). iPSC-RYU and ESC-RYU on LONG. Analysis was performed in the Puromycin-treated (+) or non-treated (-). ESC-RYU: ESC-derived intestinal epithelial cells, iPSC-RYU: iPSC-derived intestinal epithelial cells. Caco-2 cells served a positive control. Results are expressed as mean ± SD (n = 3 triplicate biological experiments). (J) Expression of the genes for transporters (P-gp, BCRP, PEPT1, OATP2B1, MRP2, and MRP3). The expression level of adult small intestine whole tissue was set to 1.0. Expression levels were calculated from the results of independent (biological) triplicate experiments. Black bars are puromycin-untreated, and gray bars are puromycin-treated RYU on LONG. Results are expressed as mean ± SD (n = 3). ES-RYU: human ESC-derived intestinal epithelial cells, iPS-RYU: human iPSC-derived intestinal epithelial cells.