Fig. 4: NOX1-expressing cells demonstrate stem cell potential in vitro.
From: NOX1 and NPY1R mark regional colon stem cell populations that serve as cancer origins in vivo
a, Nox1-2A-eGFP single cells from caecum and distal colon crypts were isolated by FACS according to their GFP expression level. The lines on the plot indicate the boundaries of the gates set for demarcating a GFP-neg and GFP-high population. The x-axis indicates that the GFP signal was measured by excitation with a 488 nm laser and emission collected by a 530/40 nm filter. b, Caecum (n = 3; 3.2633 ± 0.1335%) and colon (n = 3; 3.9233 ± 0.3398%) organoid outgrowth efficiencies were calculated as the number of organoids formed at passage 0 (P0) relative to the total number of cells seeded. Only GFP+ cells had the potential to grow organoids. c, Representative images of newly established caecum (top) and distal colon organoids (bottom) at P0 and P10. GFP+ cell-derived organoids were passaged for more than ten passages. d, Npy1r-eGFP-IRES-CreERT2 single cells from the distal colon were isolated by FACS according to their GFP expression level. The lines on the plot indicate the boundaries of the gates set for demarcating a GFP-neg, GFP-mid and GFP-high population. The x-axis indicates that the GFP signal was measured by excitation with a 488 nm laser and emission collected by a 530/40 nm filter. e, The distal colon organoid outgrowth efficiencies of GFP− and GFP+ cells showed that only GFP+ cells had the potential to grow organoids (n = 6; 5.6166 ± 0.8734%). f, Representative images of distal colon organoids at P0 and P10. GFP+ cell-derived organoids were passaged for more than ten passages. g, Schematic of the organoid differentiation protocol used to culture Matrigel-seeded cells, first for 2 d in organoid growth medium (OGM) before switching to organoid differentiation medium (ODM) once the organoids had been established for 3 (distal colon) or 5 d (caecum). h,i, qPCR analysis of Nox1-2A-eGFP-derived caecum (h) and distal colon (i) organoids, showing a decrease in the expression of Nox1 (caecum = 0.1253 ± 7.623 × 10−3; colon = 0.02760 ± 6.952 × 10−4) and Lgr5 (caecum = 0.1336 ± 5.044 × 10−3; colon = 0.03647 ± 9.903 × 10−4) and an increase in the expression of the lineage markers Dra (caecum = 97.575 ± 15.55; colon = 7.020 ± 0,5976), Muc2 (caecum = 3.3936 ± 0.1463; colon = 5.765 ± 0.5768) and ChgA (caecum = 4.073 ± 0.2047; colon = 5.147 ± 0.1707) after differentiation (n = 3). j, qPCR analysis of Npy1r-eGFP-IRES-CreERT2-derived distal colon organoids showing a decrease in the expression of Lgr5 (0.112 ± 9.644 × 10−3) and an increase in the expression of the lineage markers Dra (23.9223 ± 1.908), Muc2 (2.8366 ± 0.1787) and ChgA (3.4676 ± 0.1252) after differentiation (n = 3). Npy1r expression was unchanged after differentiation (0.8083 ± 0.1501). Statistical significance in b, e and h–j was determined by two-tailed t-test and the data are presented as means ± s.e.m. Scale bars, 150 μm. FSC, forward scatter.
