By Bruce Goldman
Illustration by Greg Clarke
Calvin Kuo’s got a gut in a dish. He’s introducing it to a lot of his fellow researchers at Stanford to help him get up to speed on what to do with it. “I’m a hematologist. My specialization is blood,” says Kuo, who’s been at Stanford since 2001. “The intestine is a little far afield for me.”
About five years ago, Kuo, an MD/PhD and associate professor of hematology, tripped on the gut when he crossed paths with Akifumi Ootani, MD, PhD, a young researcher in Japan. Between them, they’re making rapid strides in growing intestinal tissue in culture. Having succeeded at making mini-mouse intestines, they’re moving on to re-creating human innards.
Scientists have cultivated other tissues — skin, prostate, lung, thyroid, liver, stomach and more. Being able to do this with intestines would bring big payoffs, says Ootani, who now works as a postdoctoral research scientist in Kuo’s lab. For example, scientists could much more readily test drugs, study bowel cancer and learn more about the mechanisms of infectious diseases (many of whose causal pathogens access our innards via our entrails), he says. Kuo, Ootani and their collaborators are already using the mouse intestine cultures to study intestinal stem cells. In September, the Kuo lab received $2.5 million from the National Institutes of Health to start a parallel effort with human intestinal tissue, with a lofty long-term ambition of engineering artificial intestines to replace injured or defective ones.
What’s inside of those dishes Kuo and Ootani are holding looks like extremely tiny gumballs — “gutballs,” you could call them — that have assembled themselves from bits of a mouse’s intestinal tissue embedded in a protein matrix, or gel. Under the right conditions, those fragments grow into round structures, which, aside from their spherical shape and petite proportions, mimic the intestine in both form and function.
“These cultures are little mini-organs, up to a quarter-inch big. They’re large enough that you can pick up the dish every couple of days and see them growing,” Kuo says.
“If you were to take a thin section of one of them and stain it and put it in front of a pathologist, they’d look at it and say, ‘Oh, that’s a section of an intestine,’” says Justin Sonnenburg, PhD, an assistant professor of microbiology and immunology, one of several researchers who’ve begun putting the Kuo lab’s mouse intestine cultures through their paces.
They even contract, just like the real McCoy. The intestine is, in its essence, a tube surrounded by rings of smooth muscle that periodically undergo rhythmic, sequential contractions followed by relaxations. This squeezes food through the tube — a process called peristalsis. Along the way, the food gradually gets absorbed.
Getting anything even remotely similar to happen in a petri dish — or for that matter, just getting intestinal tissue to survive in culture for longer than a week or 10 days — hasn’t been easy. In fact, until just a few years ago it had never been done, although people have been trying for more than 30 years.
Until now, the only alternative to studying the intestine in a living organism was to study it in cultures generated from one or another intestinal cell type. But most of these single-cell cultures begin with cancer cells, which are by definition cells gone haywire. Anyway, cultures of a single intestinal cell type can’t possibly mimic the environment the real intestine experiences.
It takes a village to make an intestine. Just to get the ball rolling, there are four different major cell types that collectively comprise the intestinal epithelium — the gut’s surface lining, which does what most of us think of when we think about what an intestine does, and more. These four basic epithelial cell types absorb nutrients — in the small intestine, carbohydrates, amino acids (the constituents of protein) and fats; in the large intestine, chiefly water. They secrete mucus that lubricates the intestine, and hormones that regulate feeding behavior and peristalsis. And they promote immune functions including fighting off pathogens. On top of all that, the gut epithelium manages to play host to trillions of friendly bacteria, without which we’d be toast because they do everything from helping us digest our food to fending off incursions by nastier bugs.
All that crunching and punching and lunching, plus all that constant direct exposure to the toxins and villains of the outer world (the intestinal lumen is considered the outside, not the inside, of the body), take a toll on the intestine’s hard-working components. The organ has evolved to accommodate the burnout rate by replacing its constituent cells very rapidly — most of them turn over every five to seven days. This calls for ship-shape, active-duty stem cells, and lots of them.
But intestinal stem cells don’t grow on trees. To thrive, they need a support system composed of a network of numerous cell types that, together, provide just the right microenvironment, or “niche.” Within that niche, stem cells benefit from a complicated cascade of largely uncharted biochemical signals that tell them to chill out, or proliferate, or start differentiating into one or another mature epithelial cell type.
“The fact that these little spheroids can contract indicates the presence of working muscle cells and neurons.”
Early attempts by numerous researchers to grow intestinal cells in culture using disaggregated cells typically ended in tears when the cells started to die within hours of being removed from their normal position in a living tissue. Being separated from neighboring cells, with which they must constantly interact, was not OK with them. Even with researchers’ best efforts, they remained viable for less than two weeks. It’s a vicious circle: Healthy stem cells are key to a healthy intestine, but for lack of a real intestine’s three-dimensional architecture, stem cells couldn’t do their job of replacing differentiated cells.
Recently, researchers in the Netherlands (with a lot of help from a postdoctoral researcher trained by Ootani in their native Japan some years ago) figured out how to grow an intestinal cell culture from a single stem cell, using a complex mix of exotic external growth factors. But this method is pricey and inefficient. Moreover, the intestinal stem cell can mature into only the four basic intestinal epithelial cell types, so it can’t produce supporting tissues such as blood vessels, muscles and nerves.
The Kuo lab’s tissue-culture methodology gets around all that by starting with bits of minced tissue that are large enough to retain the original intestinal architecture, including the stem-cell niche. “We have a lot of the supporting cells — not just the intestinal epithelium, but supporting cell types such as fibroblasts,” Kuo says. “The fact that these little spheroids can contract indicates the presence of working muscle cells and neurons.” Jay Pasricha, MD, chief of the division of adult gastroenterology and hepatology in the Department of Medicine, has now confirmed this presence.
Kuo gives all the credit to Ootani: “It was Akifumi who single-handedly came up with this intestinal culture technique and introduced us to its potential.”
Five years ago, in 2004, Kuo found himself in correspondence with a stranger from Saga, a city of close to 1 million on Japan’s Kyushu Island. Akifumi Ootani had spent his life in Saga, gone to high school and college and finished medical school there. Now he was a PhD student in the school of medicine at Saga University, trying to figure out how to get intestinal cells to grow in culture in the laboratory of internal medicine professor Kazuma Fujimoto, MD, PhD. “My mentor asked me to develop a culture system for intestine. In Japan, when the boss asks me something I have to say yes.” It was “a good challenge for me,” he adds with some understatement.
From another mentor, pathology professor Hajime Sugihara, MD, PhD, he learned a new technique for culturing skin, cornea, thyroid, lung and fat tissue, which relied on preserving the stem-cell niche. “I imagined that this microenvironment would also be important for maintaining intestine in culture,” Ootani says. In order to mimic it by preserving the stem-cell niche, he tried using fairly intact tissue fragments, not single cells, as starter materials.
That was the first key ingredient in Ootani’s recipe. A second component was to encase those bits of minced tissue within a porous protein matrix, or gel, that preserves the unique three-dimensional gut architecture, sustaining the microenvironment in which stem cells grow best.
On top of the gel layer is air, while underneath is a nutrient-rich liquid medium. Air and liquids easily diffuse into the matrix and reach the cells directly. The idea is to reproduce what the cells lining a living intestine are seeing — which includes plenty of oxygen supplied by red blood cells. “When tissues are cultured, they don’t get all that much oxygen, since they are not connected to the heart,” says Kuo. The air layer above the porous gel makes oxygen freely available. Likewise, the liquid phase below puts all the cultured cells in contact with the nutrients they need.
By mid-2004, Ootani had figured out how to get intestinal fragments to grow in culture. This meant, by definition, that his little gutballs were providing the all-important biochemical signals their resident intestinal stem cells needed. But he wondered whether externally supplied biochemical signals could spur the cells on to greater feats.
And externally supplied biochemical signals, or growth factors, happened to be a specialty of Kuo’s. Earlier that year, Ootani had read a paper Kuo had published in Proceedings of the National Academy of Sciences describing intestinal cells’ requirement for an external growth factor called Wnt. When such a growth factor binds to receptors on a cell’s surface, it can ignite a furious pinball game of rapid-fire interactions among different molecules within the cell, causing it to proliferate.
Kuo’s laboratory had stumbled on the intestine somewhat accidentally. “We had been studying blood-vessel and bone growth, and were evaluating a lot of growth factors and their inhibitors,” Kuo says. “When we gave mice one of the proteins we were testing, an inhibitor of the Wnt pathway, we were very surprised to find they rapidly lost weight and died within days with gastrointestinal symptoms.” Autopsies showed the guts of these mice had shriveled up.
Then a postdoctoral scholar in the Kuo lab, Frank Kuhnert, PhD, found that the Wnt-inhibiting protein caused stem cells in the mice’s intestines to disappear — pronto. Clearly, Wnt was critical to intestinal stem-cell health. Kuo’s group immediately spun their attention to intestinal stem cells and regeneration.
In October 2004, Ootani e-mailed Kuo and let him know he had an intestinal culture system up and running, and asked if they might benefit from one another’s expertise. The timing was propitious, as Kuo was scheduled to give a presentation in Tokyo in December. Ootani came up from Saga. They met, and exchanged observations in a restaurant. Each recognized their common interest. The sake didn’t hurt, either. Kuo offered Ootani a job on the spot.
Ootani showed up and started working at Stanford in August 2005. It was a good move. First of all, Ootani’s cultures felt right at home in dry, sunny California. “It’s much easier to grow these cultures without contamination here than in Japan,” says Ootani.
Second, the foreseen synergies between Ootani’s and Kuo’s work paid off. While Ootani’s cultures could expand on their own by providing themselves with growth factors, stimulating the Wnt pathway using a variety of techniques developed by Ootani and Kuo has dramatically increased the growth of Ootani’s cultures, which now can thrive for a year or longer — a huge leap from the mere minutes or hours intestinal tissue could survive in culture prior to the innovation.
“Many of the spheres start to rhythmically contract, just like live intestines do, within about 10 days or two weeks,” Ootani says. “Apparently they’re pretty comfortable here.”
Just ask Manuel Amieva about that. Amieva, who went through Stanford’s MD/PhD program simultaneously with Kuo, is an assistant professor of pediatrics and of microbiology and immunology. Amieva’s research interest lies with pathogens that infect the intestine and cause diseases such as diarrhea, the biggest cause of infant death in the developing world. He is also a widely acclaimed microscopist.
Hoping to do some imaging of his intestine-in-a-dish cultures, Kuo asked Amieva if he could help.
“I said, ‘Oh, that sounds interesting, bring it over,’” Amieva recalls. When Ootani showed up, petri dish in hand, Amieva stuck it under a microscope and started focusing on one of the little spheroids, trying to get the best view for shooting some video footage of it. As he was looking through the lens, the gutball impulsively perpetrated peristalsis.
“I jumped back! This thing was alive. It scared me. Usually, cells move at a sluggish pace,” says Amieva. But not this thing. “Now I was hooked, because I realized that we were dealing with this little Frankenstein monster living in a petri dish.”
This new technology promises to diffuse rapidly — because it’s such a valuable research tool and because Kuo is reaching out to establish collaborations with key researchers.
“Our ability to grow mouse gut in culture helps us to study stem cells in a way not possible using any other methodology,” Kuo says. “Now we can begin to learn how intestine works by introducing target genes into cultured cells one at a time and seeing how each affects the tissue. No one’s been able to do this before using bona fide intestinal cells.” (Previous attempts had to rely on cancerous cells.) “We can study intestinal stem cells, as well as their relationship to, for instance, colon cancer initiation and proliferation. We can also test the effects of cancer therapeutics in the culture system, and perhaps even use it as a screening methodology.”
He’s already working with Amieva and Sonnenburg to study bacterial interactions with the intestine.
In one of Amieva’s projects, the spherical organoids allow him to follow the process of infection as it happens. With a microneedle, he injects pathogens into the hollow center of the spheres, which corresponds to the open tube inside an intestine; the mouse tissues have been bioengineered so their cells glow green, the bacteria have been designed to glow red.
“That way we can follow the infected cells under a microscope,” he says. “And we can start looking for exactly which bacterial factors and which interactions between the bugs and the intestinal cells are important for the bacteria to succeed in causing the infection.”
Amieva and Sonnenburg are also teaming up to pit good against evil inside the gutballs. Sonnenburg has been introducing friendly bacteria, one strain at a time, into germ-free mice so he can study their actions with the mouse’s gut and with one another. “We’re putting first Justin’s good bugs and then my bad bugs into these cultures, to see whether the good bugs can treat or prevent disease,” Amieva says.
“The rest of us are very lucky that it’s Calvin who’s developed this, because he’s so interested in having this model widely applied,” says Sonnenburg. “He understands that this system is powerful beyond any single person’s vision. So he’s trying to put it in the hands of as many people as possible.” No guts, no glory.
When you’re on a roll, why stop with mice?
“We’d like to extend our examination of mouse intestinal culture system to human tissue,” says Calvin Kuo, MD, PhD. “That will allow even more accurate recapitulation of different disease processes that affect people. We also see potential for one day being able to grow artificial human intestines that can be transplanted into patients,” says Kuo, an associate professor of hematology. The long-term vision: replacement parts for people with Crohn’s disease or other severe intestinal abnormalities.
Not that we’re going to see a full-sized, full-fledged, flexible, fleshy tube rearing up from a petri dish like a cobra charmed by a flute anytime soon. Realistically, the first steps will be to simply get human intestinal fragments to grow in culture.
Trying to grow human intestine in culture comes with plenty of challenges. The mouse cultures grow best if you start with very young (neonatal) tissue. “You can’t do that with humans,” says Kuo. “We’re trying to acquire tissue samples surgically removed from patients here at Stanford Hospital, and to use that as starting material.”
So far, human cultures haven’t lasted nearly as long as their murine counterparts, says research scientist Akifumi Ootani, MD, PhD. Kuo thinks human gut may prove tougher to grow in culture than mouse gut. “It’s a bigger organ, with thicker walls composed of more-cohesive tissue that may be more difficult to disaggregate,” he notes.
As their collaborator, Sarah Heilshorn, PhD, assistant professor of materials science in the School of Engineering, explains: “In living organisms, cells exist in a three-dimensional world, touching materials of one sort or another all around them. In the lab, we put them on flat petri dishes and expect them to behave the same way. Sometimes they do, and sometimes they don’t.”
Kuo and Ootani use a commercial collagen gel for their intestine cultures. Heilshorn’s lab spends a lot of time thinking about how to design materials that mimic the body. “Calvin and I wondered if maybe the gel composition and oxygen availability might need to be adjusted to make these human tissues feel more at home,” she says.
Kuo has enlisted Heilshorn to develop a superior scaffolding material for human tissue and perhaps, down the road, a more fully developed organ. The scaffold would be composed of specially engineered proteins whose carefully spaced components can mimic growth factors identified by Kuo as critical to sustaining Ootani’s cultures.
Is there any possibility of coaxing these spheroid intestinal cultures into going tubular? Heilshorn’s lab is developing scaffolds to encourage tubular structures such as blood vessels and has already developed scaffolding for application and delivery of neural cells. “It’s quite conceivable that you could encourage these intestinal cells to grow as tubes, too,” she says.
“A complete, full-scale intestine would be the eventual dream goal,” says Kuo.