Generally speaking, tissue-specific stem cells are thought to do what their niches tell them to do. But new work from Hans Clevers at the Hubrecht Institute in the Netherlands shows that mouse intestinal stem cells can take control in a culture dish.

The intestinal lining where stem cells reside consists of valley-like crypts between projections called villi. These contain many cell types, including the mucus-secreting goblet cells, the nutrient-absorbing enterocytes and the mysterious Paneth cells. Working with mice, Clevers has previously shown that all these cell types are continuously generated by intestinal stem cells expressing the protein Lgr5. Now he shows that the same thing can happen in culture. In fact, a single intestinal stem cell can generate multiple crypt-villi systems that are indistinguishable in appearance from the organoids generated when entire crypts are placed in culture1.

A single cell grows into an inverted crypt-villus system. Left, in vivo. Right, in culture. Credit: Toshiro Sato and Hans Clever

After about 4 days, single cells formed structures of about 100 cells, a rate of division consistent with what happens in vivo. After two weeks, stem cells from the lab-grown organoids could be dissociated and replated to form new organoids, a process that could be repeated at least four times without any loss in replating efficiency.

One key to the study was just keeping the cells alive, says Clevers. “It has previously been impossible to maintain adult intestine, let alone epithelium in isolation for more than a few days in culture.” The intestinal stem cells died almost immediately when isolated from other cells, so the researchers added a Rho kinase inhibitor that is known to help human embryonic stem cells survive separation. To promote cell signalling between growing cells, they added a peptide that boosts activity of the signaling pathway called Notch. After that, they used a combination of R-spondin 1, epidermal growth factor, and Noggin in a Matrigel matrix. Under these conditions, a single cell could produce all the expected cell types in an organized structure. Lead author Toshiro Sato went through thousands of combinations to find the culture conditions for “eternal growth”, says Clevers. “He obtains 500-fold expansion every month.”

Gut linings wear out quickly, so it makes sense that the intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. The self-renewal process is generally believed to be supported by a layer of cells called myofibroblasts located at the base of the crypts, but this work shows that such support can be provided by simple culture conditions.

“These Lgr5 stem cells demonstrate a remarkable drive to form normal gut epithelium, considering that our artificial culture system provides nothing but a few growth factors,” says Clevers. “The application of adult stem cells in regenerative medicine may, in some cases, be simpler than we suspect.”

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