A new method for long-term culture of human primary colonic epithelium provides an important tool for studying colon stem cells, adenocarcinoma, colon cancer, and even Barrett’s esophagus, according to the November issue of Gastroenterology.
Self-renewal of the small intestinal and colonic epithelium is mediated by proliferation of stem cells and their progenitors, which are located in structures called crypts. However, it has been a challenge to isolate and culture these cells, or induce them to differentiate and form structures that are similar to those observed in human intestine or colon.
Sato et al. collected tissues from colons of healthy mice, and also from colon and small intestines of people that were healthy or had colon cancer, adenomas, or Barrett’s epithelium. They isolated the crypts and cultured them with different combinations of growth and survival factors (epidermal growth factor, noggin, R-spondin-1, Wnt3a, nicotinamide, gastrin, the Alk4/5/7 inhibitor A83-01, and/or the p38 inhibitor SB202190).
After some trial and error, the authors found they could induce the tissues to form organoids, which have features of the colonic epithelium, in terms of architecture, cell type composition, and self-renewal dynamics; the organoids also expressed stem cell genes. By altering the factors they added to the cells, they could induce these organoids to differentiate into different cell types of the intestine (such as after withdrawal of nicotinamide and SB202190 – see figure).
Importantly, Sato et al. did not observe cellular transformation or chromosome changes over time. However, cells in the organoids could undergo many more rounds of division than in other adult human epithelial culture systems; the replicative capacity in the optimized culture condition of Sato et al. allowed the cells to undergo at least 100 population doublings, which exceeded the Hayflick limit. The authors concluded that intestinal epithelial cells undergo senescence in other culture systems because of inadequate growth conditions, rather than inherent replicative aging.
The authors observed that after making a few adjustments to their system to culture healthy colon tissue, they could also culture adenoma and colorectal cancer organoids, which grew as irregular compact structures, rather than as simple cystic structures. These colorectal cancer organoids more closely represent the intestinal epithelium than colon cancer cell lines commonly used in research, such as CaCo2 or DLD1, and are therefore an important new tool for colon cancer studies.
Barrett’s esophagus is characterized by the development of intestinal goblet cells in the esophagus, so Sato et al. investigated whether their system could also be used to culture these diseased tissues. Again, with some modification, they found that they could culture Barrett’s epithelium organoids for more than 3 months. Importantly, addition of the γ-secretase inhibitor DBZ to the Barrett’s organoids for 4 days after the withdrawal of nicotinamide and SB202190 blocked proliferation and induced goblet cell differentiation, so these inhibitors might be used to treat patients with Barrett’s esophagus lesions, by differentiation therapy.
Sato et al. propose that their organoid culture system could be used in regenerative biology, through ex vivo expansion of the intestinal epithelia, and to study basic aspects of intestinal stem cell biology. The organoid culture might also be used for pharmacology, toxicology, or microbiology studies of gastrointestinal disorders. Because small biopsy specimens taken from adult donors can be expanded without any apparent limit or genetic damage, the authors say the technology might also be used to generate transplantable epithelium for regenerative purposes.
Read the article online.
Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and barrett’s epithelium. Gastroenterology 2011;141:1762–1772.