David H. Koch Institute for Integrative Cancer Research

New means of growing intestinal stem cells.

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The small intestine, like most other body tissues, has a small store of immature adult stem cells that can differentiate into more mature, specialized cell types. Until now, there has been no good way to grow large numbers of these stem cells, because they only remain immature while in contact with a type of supportive cells called Paneth cells.

New means of growing intestinal stem cells

In a new study appearing in the Dec. 1 online edition of Nature Methods, the researchers found a way to replace Paneth cells with two small molecules that maintain stem cells and promote their proliferation. Stem cells grown in a lab dish containing these molecules can stay immature indefinitely; by adding other molecules, including inhibitors and activators, the researchers can control what types of cells they eventually become.

“This opens the door to doing all kinds of things, ranging from someday engineering a new gut for patients with intestinal diseases to doing drug screening for safety and efficacy. It’s really the first time this has been done,” says Robert Langer, the David H. Koch Institute Professor, a member of MIT‘s Koch Institute for Integrative Cancer Research, and one of the paper’s senior authors.

Jeffrey Karp, an associate professor of medicine at Harvard Medical School and Brigham and Women’s Hospital, is also a senior author of the paper. The paper’s lead author is Xiaolei Yin, a postdoc at the Koch Institute and Brigham and Women’s Hospital.

From one cell, many

The inner layer of the intestines has several critical functions. Some cells are specialized to absorb nutrients from digested food, while others form a barrier that secretes mucus and prevents viruses and bacteria from entering cells. Still others alert the immune system when a foreign pathogen is present.

This layer, known as the intestinal epithelium, is coated with many small indentations known as crypts. At the bottom of each crypt is a small pool of epithelial stem cells, which constantly replenish the specialized cells of the intestinal epithelium, which only live for about five days. These stem cells can become any type of intestinal epithelial cell, but don’t have the pluripotency of , which can become any cell type in the body.

If scientists could obtain large quantities of intestinal epithelial stem cells, they could be used to help treat gastrointestinal disorders that damage the epithelial layer. Recent studies in animals have shown that intestinal stem cells delivered to the gut can attach to ulcers and help regenerate healthy tissue, offering a potential new way to treat ulcerative colitis.

Using those stem cells to produce large populations of specialized cells would also be useful for drug development and testing, the researchers say. With large quantities of goblet cells, which help control the immune response to proteins found in food, scientists could study food allergies; with enteroendocrine cells, which release hunger hormones, they could test new treatments for obesity.

“If we had ways of performing high-throughput screens on large numbers of these very specific cell types, we could potentially identify new targets and develop completely new drugs for diseases ranging from inflammatory bowel disease to diabetes,” Karp says.

Controlling cell fate

In 2007, Hans Clevers, a professor at the Hubrecht Institute in the Netherlands, identified a marker for intestinal epithelial stem cells—a protein called Lgr5. Clevers, who is an author of the new Nature Methods paper, also identified growth factors that enable these stem cells to reproduce in small quantities in a lab dish and spontaneously differentiate into , forming small structures called organoids that mimic the natural architecture of the intestinal lining.

In the new study, the researchers wanted to figure out how to keep stem cells proliferating but stop them from differentiating, creating a nearly pure population of stem cells. This has been difficult to do because stem cells start to differentiate as soon as they lose contact with a Paneth cell.

Paneth cells control two signaling pathways, known as Notch and Wnt, which coordinate cell proliferation, especially during embryonic development. The researchers identified two small molecules, valproic acid and CHIR-99021, that work together to induce stem cells to proliferate and prevent them from differentiating into mature cells.

When the researchers grew mouse intestinal stem cells in a dish containing these two small molecules, they obtained large clusters made of 70 to 90 percent stem cells.

Once the researchers had nearly pure populations of stem cells, they showed that they could drive them to develop into particular types of  by adding other factors that influence the Wnt and Notch pathways. “We used different combinations of inhibitors and activators to drive stem cells to differentiate into specific populations of mature cells,” Yin says.

This approach also works in mouse stomach and colon cells, the researchers found. They also showed that the small molecules improved the proliferation of human intestinal stem cells. They are now working on engineering intestinal tissues for patient transplant and developing new ways to rapidly test the effects of drugs on intestinal cells.

Another potential use for these cells is studying the biology that underlies stem cells’ special ability to self-renew and to develop into other cell types, says Ramesh Shivdasani, an associate professor of medicine at Harvard Medical School and Dana-Farber Cancer Institute.

“There are a lot of things we don’t know about ,” says Shivdasani, who was not part of the research team. “Without access to large quantities of these cells, it’s very difficult to do any experiments. This opens the door to a systematic, incisive, reliable way of interrogating intestinal stem cell biology.”

Simple urine test uses nanotechnology to detect dangerous blood clotting

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Life-threatening blood clots can form in anyone who sits on a plane for a long time, is confined to bed while recovering from surgery, or takes certain medications.

There is no fast and easy way to diagnose these clots, which often remain undetected until they break free and cause a stroke or heart attack. However, new technology from MIT may soon change that: A team of engineers has developed a way to detect blood clots using a simple urine test.

The noninvasive diagnostic, described in a recent issue of the journal ACS Nano, relies on  that detect the presence of , a key  factor.

Such a system could be used to monitor patients who are at high risk for blood clots, says Sangeeta Bhatia, senior author of the paper and the John and Dorothy Wilson Professor of Biochemistry.

“Some patients are at more risk for clotting, but existing blood tests are not consistently able to detect the formation of new clots,” says Bhatia, who is also a senior associate member of the Broad Institute and a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES).

Lead authors of the paper are Kevin Lin, a graduate student in chemical engineering, and Gabriel Kwong, a postdoc in IMES. Other authors are Andrew Warren, a graduate student in Health Sciences and Technology (HST), and former HST postdoc David Wood.

Sensing thrombin

Blood clotting is produced by a complex cascade of protein interactions, culminating in the formation of fibrin, a fibrous protein that seals wounds. The last step of this process—the conversion of fibrinogen to fibrin—is controlled by an enzyme called thrombin.

Current tests for blood clotting are very indirect, Bhatia says. One, known as the D-dimer test, looks for the presence of fibrin byproducts, which indicates that a clot is being broken down, but will not detect its initial formation.

Bhatia and her colleagues developed their new test based on a technology they first reported last year for early detection of colorectal cancer. “We realized the same exact technology would work for blood clots,” she says. “So we took the test we had developed before, which is an injectable nanoparticle, and made it a thrombin sensor.”

Finding blood clots before they wreak havoc

The system consists of , which the Food and Drug Administration has approved for human use, coated with peptides (short proteins) that are specialized to interact with thrombin. After being injected into mice, the nanoparticles travel throughout the body. When the particles encounter thrombin, the thrombin cleaves the peptides at a specific location, releasing fragments that are then excreted in the animals’ urine.

Once the urine is collected, the protein fragments can be identified by treating the sample with antibodies specific to peptide tags included in the fragments. The researchers showed that the amount of these tags found in the urine is directly proportional to the level of blood clotting in the mice’s lungs.

In the previous version of the system, reported last December in Nature Biotechnology, the researchers used mass spectrometry to distinguish the fragments by their mass. However, testing samples with antibodies is much simpler and cheaper, the researchers say.

Rapid screening

Bhatia says she envisions two possible applications for this kind of test. One is to screen patients who come to the emergency room complaining of symptoms that might indicate a blood clot, allowing doctors to rapidly triage such patients and determine if more tests are needed.

“Right now they just don’t know how to efficiently define who to do the more extensive workup on. It’s one of those things that you can’t afford to miss, so patients can get an unnecessarily expensive workup,” Bhatia says.

Another application is monitoring patients who are at high risk for a clot—for example, people who have to spend a lot of time in bed recovering from surgery. Bhatia is working on a urine dipstick test, similar to a pregnancy , that doctors could give  when they go home after surgery.

“If a patient is at risk for thrombosis, you could send them home with a 10-pack of these sticks and say, ‘Pee on this every other day and call me if it turns blue,'” she says.

The technology could also be useful for predicting recurrence of clots, says Henri Spronk, an assistant professor of biochemistry at Maastricht University in the Netherlands.

“High levels of activation markers have been related to recurrent thrombosis, but they don’t have good sensitivity or specificity. Through application of the nanoparticles, if proven well-tolerated and nontoxic, alterations in the normal low levels of physiological thrombin generation might be easily detected,” says Spronk, who was not part of the research team.

Bhatia plans to launch a company to commercialize the technology, with funding from MIT’s Deshpande Center for Technological Innovation. Other applications for the nanoparticle system could include monitoring and diagnosing cancer. It could also be adapted to track liver, pulmonary, and kidney fibrosis, Bhatia says.