Retinol Promotes In Vitro Growth of Proximal Colon Organoids through a Retinoic Acid-Independent Mechanism
Fig 2
Enzymes involved in ROL-RA pathway are differentially expressed along the length of the colon.
(A) Colonic Swiss rolls were assessed for expression of Aldh1a1 (top left) and Aldh1a3 (top right) genes by in situ hybridization. The proximal and distal portions of the colon are located inside and outside, respectively. Magnified views are shown for the proximal (middle) and distal colon (bottom). Representative images of three independent experiments are shown. Scale bars, 1 mm for images on the top and 50 μm for images on the middle and at the bottom. (B) Sections of the proximal (top) and distal (bottom) colon were assayed for protein expression of ALDH1a1 and ALDH1a3. Signals yielded by immunohistochemical staining (red) are shown in merged images with DAPI staining (blue). Representative data are shown for three independent experiments. Arrowheads show positive cells for ALDH1a1 or ALDH1a3 in the distal colon. Scale bars, 50 μm. (C) Colonic crypts were isolated from the entire length (whole colon), or from proximal (prox. colon) or distal (dist. colon) half of the colon. Crypts were then cultured for 6 days. Total RNA was extracted from the isolated crypts before (pre) or after the culture (post). Semi-quantitative PCR was performed for the genes indicated. Representative data are shown for three independent experiments. (D) Colonic crypts were separately isolated from proximal and distal colons and cultured as organoids. Frozen sections of organoids were subjected to immunohistochemistry for ALDH1a3 (left). Merged images with DAPI staining were also shown (right). Scale bars, 50 μm. Sections of 30 organoids were chosen from a series of sections obtained from each well (triplicate wells for each of three independent experiments) and the percentage of Ki-67+ cells to whole cell populations was quantitated (graph on the right). Values are presented as mean ± s.e.m. for those 270 sections.
