Children’s Hospital Los Angeles Researchers Grow Functional Tissue-Engineered Intestine from Human Cells

Children’s Hospital Los Angeles is the home of a remarkable new study that has succeeded in growing tissue-engineered small intestine from human cells. This tissue engineered intestine recapitulates several key functional characteristics of human intestine such as the ability to absorb sugars. It also has structural features of human small intestine, such as a mucosal lining, support structures tiny and ultra-structural components like cellular connections.

This work was published in the American Journal of Physiology; GI and Liver and brings surgeons one step closer to using tissue engineered intestines in human patients.

Tissue-engineered small intestines are grown from stem cells isolated from the intestine. These laboratory-grown tissues offer a promising treatment for clinical conditions such as short-bowel syndrome (SBS), which is a major cause of intestinal failure, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue engineered small intestines may also, perhaps in the future, offer a therapeutic alternative to intestinal transplantations, which is fraught with the problems of donor shortage and the need for lifelong immunosuppression.

Senior author Dr. Tracy Grikscheit, who is a principal investigator at the Saban Research Institute, which is housed at the Children’s Hospital of Los Angeles (CHLA), and the Developmental Biology and Regenerative Medicine program at the Children’s Hospital of Los Angeles. Dr. Grikscheit is also a pediatric surgeon at CHLA and assistant professor of surgery at the Keck School of Medicine of the University of Southern California.

Grikscheit main interest, as a clinician, is to find strategies to treat the most vulnerable young patients. For example, babies who are born prematurely can sometimes develop a devastating disease called necrotizing enterocolitis (NEC), in which life-threatening intestinal damage demands that large portions of the small intestine be surgically removed. Without a long enough intestine, NEC babies are dependent on intravenous feeding. This intravenous feeding is costly and may cause liver damage. NEC and other contributors to intestinal failure occur in 24.5 out of 100,000 live births, and the incidence of SBS is increasing and nearly a third of patients die within five years.

CHLA scientists had previously shown that tissue-engineered small intestine could be generated from human small intestine donor tissue implanted into immunocompromised mice. These initial studies were published in July 2011 in the biomedical journal Tissue Engineering, Part A, and while it was a hopeful study, only basic components of the intestine were identified in the implanted intestine. To be clinically relevant, it is necessary to make tissue engineered intestines that form a healthy barrier that can still absorb nutrition and regulate the exchange of electrolytes.

This new study, however, showed that mouse tissue engineered small intestines are quite similar to the tissue-engineered small intestines made from human intestinal stem cells. Both contain important building blocks such as the stem and progenitor cells that continue to regenerate the intestine throughout the lie of the organism. These cells are found within the engineered tissue in specific locations and are close to other specialized cells that are known to be necessary for the intestine to function as a fully functioning organ.

“We have shown that we can grow tissue-engineered small intestine that is more complex than other stem cell or progenitor cell models that are currently used to study intestinal regeneration and disease, and proven it to be fully functional as it develops from human cells,” said Grikscheit. “Demonstrating the functional capacity of this tissue-engineered intestine is a necessary milestone on our path toward one day helping patients with intestinal failure.”

Growing Intestinal Stem Cells

Researchers from MIT and Brigham and Women’s Hospital in Boston, MA have discovered a protocol that allows them to grow unlimited quantities of intestinal stem cells. These intestinal stem cells can then be induced to differentiate into pure populations of various types of mature intestinal cells. Scientists can used these cultured intestinal cells to develop new drugs and treat gastrointestinal diseases, such as Crohn’s disease or ulcerative colitis.,

The small intestine has a small repository of adult stem cells that differentiate into mature adult cells that have specialized functions. Until recently, there was no good way to grow large numbers of these intestinal stem cells in culture. Intestinal stem cells, you see, only retain their immature characteristics when they are in contact with supportive cells known as Paneth cells.

paneth cells

In order to grow intestinal stem cells in culture, researchers from the laboratories of Robert Langer at the MIT Koch Institute for Integrative Cancer Research and Jeffrey Karp from the Harvard Medical School and Brigham and Women’s Hospital, determined the specific molecules that Paneth cells make that keep the intestinal stem cells in their immature state. Then they designed small molecules that mimic the Paneth cell-specific molecules. When Langer and Karp’s groups grew the intestinal stem cells in culture with those small molecules, the cells remained immature and grew robustly in culture.

Langer said, “This opens the door to doing all kinds of thing, 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.”

The inner mucosal layer of the intestine has several vital functions: the absorption of nutrients, the secretion of mucus of create a barrier between our own cells and the bacteria and viruses and habitually inhabit our bowels, and alerting the immune system to the presence of potential disease-causing agents in the bowel.

The intestinal mucosa is organized into a collection of folds with small indentations called “intestinal crypts.”  At the bottom of each crypt is a small pool of intestinal stem cells that divide to routinely replace the specialized cells of the intestinal epithelium.  Because the cells of the intestinal epithelium show a high rate of turnover (they only last for about five days), these stem cells must constantly divide to replenish the intestine.


Once these intestinal stem cells divide, they can differentiate into any type of mature intestinal cell type.  Therefore, these intestinal stem cells provide a marvelous example of a “multipotent stem cell.”

Obtaining large quantities of intestinal stem cells could certainly help gastroenterologists  treat gastrointestinal diseases that damage the epithelial layer of the gut.  Fortunately, recent studies in laboratory animals have demonstrated that the delivery of intestinal stem cells can promote the healing of ulcers and regeneration of new tissue, which offers a new way to treat inflammatory bowel diseases like ulcerative colitis.

This, however, is only one of the many uses for cultured intestinal stem cells.  Researchers are literally salivating over the potential of studying things like goblet cells, which control the immune response to proteins in foods to which many people are allergic.  Alternatively, scientists would like to investigate the properties of enteroendocrine cells, which secrete hunger hormones and play a role in obesity.  I think you can see, that large numbers of intestinal stem cells could be a boon to gastrointestinal research.

Karp said, “If we had ways of performing high-throughput screens of 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.”

The laboratory of Hans Clevers in 2007 identified a molecule that is specifically made by intestinal stem cells called Lgr5.  Clevers is a professor at the Hubrecht Institute in the Netherlands and he and his co-workers have just identified particular molecules that enable intestinal stem cells to grow in synthetic culture.  In culture, these small clusters of intestinal stem cells differentiate and form small sphere-like structures called “organoids,” because they consist of a ball of intestinal cells that have many of the same organizational properties of our own intestines, but are made in culture.

Clevers and his colleagues tried to properly define the molecules that bind Paneth cells and intestinal stem cell together.  The purpose of this was to mimic the Paneth cells in culture so that the intestinal stem cells would grow robustly in culture.  Clevers’ team discovered that Paneth cells use two signal transduction pathways (biochemical pathways that cells use to talk to each other) to coordinate their “conversations” with the adjacent stem cells.  These two signal transduction pathways are the Notch and Wnt pathways.

Fortunately, two molecules could be used to induce intestinal stem cell proliferation and prevent their differentiation: valproic acid and CHIR-99021.  When Clevers and others grew mouse intestinal stem cells in the presence of these two compounds, they found that large clusters of cells grew that consisted of 70-90 percent pure stem cells.  When they used inhibitors of the Notch and Wnt pathway, they could drive the cells to form particular types of mature intestinal cells.

“We used different combinations of inhibitors and activators to drive stem cells to differentiate into specific populations of mature cells,” said Xiaolei Yin, first author of this paper.  Yin and others were able to get this strategy to work with mouse stomach and colon cells, and that these small molecules also drove the proliferation of human intestinal stem cells.

Presently, Clevers’ laboratory is trying to engineering intestinal tissues for potential transplantation in human patients and for rapidly testing the effects of drugs on intestinal cells.

Ramesh Shivdasani from Harvard Medical School and Dana-Farber Cancer Institute would like to use these cells to investigate what gives stem cells their ability to self-renew and differentiate into other cell types.  “There are a lot of things we don’t know about stem cells,” said Shivdasani.  “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.”

X. Yi, et al. “Niche-independent high-purity cultures of Lgr5 intestinal stem cells and their progeny.” Nature Methods 2013; DOI:10.1038/nmeth.2737.

Adult Stem Cells Isolated From Human Intestines

A laboratory at the University of North Carolina at Chapel Hill has, for the first time, isolated adult stem cells from human intestinal tissue. This achievement should provide a much-needed resource for stem cells researchers to examine the nuances of stem cell biology. Also, these new stem cells should provide stem cell researchers a new tool to treat inflammatory bowel diseases or to mitigate the side effects of chemotherapy and radiation, which often damage the gut.

Scott T. Magness, assistant professor in the departments of physiology at UNC, Chapel Hill, said, “Not having these cells to study has been a significant roadblock to research. Until now, we have not had the technology to isolate and study these stem cells – now we have the tools to start solving many of these problems.”

The study represents a leap forward for a field that for many years has had to resort to conducting experiments with mouse stem cells. While significant progress has been made using mouse models, differences in stem cell biology between mice and humans have kept researchers from investigating new therapeutics for human afflictions.

Adam Grace, a graduate student in Magness’ lab, and one of the first authors of this publication, noted, “While the information we get from mice is good foundational mechanistic data to explain how this tissue works, there are some opportunities that we might not be able to pursue until we do similar experiments with human tissue”

This study from the Magness laboratory was the first in the United States to isolate and grow single intestinal stem cells from mice. Therefore, Magness and his colleagues already had experience with the isolation and manipulation of intestinal stem cells. In their quest to isolate human intestinal stem cells, Magness and his colleagues also procured human small intestinal tissue for their experiments that had been discarded after gastric bypass surgery at UNC.

To develop their technique, Magness and others simply tried to recapitulate the technique they had developed in used to isolate mouse intestines to isolate stem cells from human intestinal stem cells. They used cell surface molecules found on in the membranes of mouse intestinal stem cells. These proteins, CD24 and CD44, were also found on the surfaces of human intestinal stem cells. Therefore, the antibodies that had been used to isolate mouse intestinal stem cells worked quite well to isolate human intestinal stem cells. Magness and his co-workers attached fluorescent tags to the stem cells and then isolated by means of fluorescence-activated cell sorting.

This technique worked so well, that Magness and his colleagues were able to not only isolated human intestinal stem cells, but also distinct types of intestinal stem cells. These two types of intestinal stem cells are either active stem cells or quiescent stem cells that are held in reserve. This is a fascinating finding, since the reserve cells can replenish the stem cell population after radiation, chemotherapy, or injury.

“Now that we have been able to do this, the next step is to carefully characterize these populations to assess their potential, said Magness. He continued: “Can we expand these cells outside the body to potentially provide a cell source for therapy? Can we use these for tissue regeneration? Or to take it to the extreme, can we genetically modify these cells to cure inborn disorders or inflammatory bowel disease? Those are some questions that we are going to explore in the future.”

Certainly more papers are forthcoming on this fascinating and important topic.