Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium

• Published in: Gastroenterology (New York, N.Y. 1943) Vol. 141; no. 5; pp. 1762 - 1772
• Main Authors: Sato, Toshiro, Stange, Daniel E, Ferrante, Marc, Vries, Robert G.J, van Es, Johan H, van den Brink, Stieneke, van Houdt, Winan J, Pronk, Apollo, van Gorp, Joost, Siersema, Peter D, Clevers, Hans
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2011
• Subjects: Adenocarcinoma - pathology; Adenoma - pathology; Adult; Aged; Aged, 80 and over; Animals; Barrett Esophagus - pathology; Biopsy; Cell Culture Techniques - methods; Cell Proliferation - drug effects; Cells, Cultured; Colon - pathology; Colonic Neoplasms - pathology; Dysplasia; Epithelium - pathology; Ex Vivo Culture Technology; Gastroenterology and Hepatology; Hayflick Model; Humans; Intestine, Small - pathology; Mice; Mice, Inbred C57BL; Middle Aged; Neoplasia; Niacinamide - pharmacology; Organoids - pathology; Signaling; Wnt3A Protein - pharmacology; Dysplasia; recombinant human Wnt-3A or Wnt-3A conditioned medium plus murine epidermal growth factor, murine noggin, human R-spondin-1; EGF; murine epidermal growth factor, murine noggin, human R-spondin-1; g; ENR; DAPI; 4′,6-diamidino-2-phenylindole; human intestinal stem cell; Ex Vivo Culture Technology; green fluorescent protein; gastrin; GFP; HISC; Signaling; Hayflick Model; WENR; Neoplasia; epidermal growth factor; DBZ; dibenzazepine
• ISSN: 0016-5085; 1528-0012
• DOI: 10.1053/j.gastro.2011.07.050

Background & Aims We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Results Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. Conclusions We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.