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Story of Discovery—Intestinal Stem Cells

Every 5 days, the inner lining of the gastrointestinal tract, called the intestinal epithelium, completely renews itself. Understanding exactly how it accomplishes this feat would help researchers develop new therapies to rejuvenate damaged intestinal tissue and explore new approaches to treating diseases such as colon cancer, where regulation of this process is lost. Studies over the past several decades have pointed to a relatively small number of cells that lie at the heart of intestinal epithelium renewal. The discovery of these cells—the intestinal stem cells, the ancestors to all other cells in the intestinal epithelium—has led to prolific research by the NIDDK’s Intestinal Stem Cell Consortium (ISCC) into the complex dynamics that regulate the development and turnover of the intestinal lining, the most rapidly regenerating tissue in the body.

The Cellular Structure of the Intestine

At first glance, the intestinal epithelium may appear to be a simple barrier that separates the contents of the gut from the rest of the body, but research over the past century has revealed its extremely diverse and dynamic makeup.

The wall of the small intestine is lined with fingerlike structures, called villi, that project into the intestinal space (lumen) and help the gut absorb materials by increasing its internal surface area. The most numerous epithelial cells on the villi are enterocytes, which absorb water and nutrients from ingested food. Scattered among the enterocytes are goblet cells, which secrete protective mucus; enteroendocrine cells, which release hormones that regulate digestive functions such as appetite control and the muscle contractions that move food through the gut; and tuft cells, which play a role in sensing intestinal contents and initiating immune responses.

Interspersed among the villi in the small intestine are areas of lymphatic tissue called Peyer’s patches, which play an important role in the immune system by preventing the growth of pathogenic bacteria. Specialized epithelial cells called microfold cells, or “M cells,” coat the Peyer’s patches and continuously sample the contents of the gut for signs of pathogenic microbes.

The intestinal wall is also dotted with pit-like structures called “crypts.” Nestled at the bottom of the crypts are Paneth cells, which are epithelial cells that secrete antibacterial compounds. Researchers in the late 1800s noticed that crypts also had a distinct attribute: while the cells in the villi appeared dormant, many cells in the crypts seemed to be constantly dividing. This gave rise to the idea that all cells in the intestinal lining are, rather ironically, “born” in crypts. The cells would then migrate up into the villi, where eventually they would be shed from the villi tips into the intestinal lumen.

Using a mouse model, researchers in the 1940s not only found evidence that this conveyer belt-type process takes place, but that it happens in a matter of mere days. It also raised several important questions. How do the crypts produce such a diverse variety of intestinal cells, each with their own specialized role in digestion? And how is this process of cell production and death regulated, especially during injury and disease, so there will always be an appropriate number of cells to maintain a functional intestinal wall? By the 1960s, researchers were beginning to suspect that the underlying answers to these questions related to the existence of stem cells—a pool of ancestral cells that continuously duplicate themselves while also producing progeny that morph into all the various types of intestinal cells, all in a tightly regulated process called differentiation. They also realized that these stem cells, if they did exist, must lie somewhere in the intestinal crypts.

Intestinal Stem Cells

In the 1960s and 70s, scientists zeroed in on a group of cells wedged among the Paneth cells at the bottom of intestinal crypts in mice. These small cells, called crypt base columnar (CBC) cells, were conspicuous because they were continuously dividing, unlike their larger Paneth cell neighbors. Labelling the CBC cells in mice with a radioactive compound, the scientists were able to track their progeny as they divided and moved up the walls of the crypts. The label was eventually found in several different types of intestinal cells in the villi, providing the first direct evidence that the CBC cells were stem cells that give rise to all other intestinal epithelial cells.

Another type of crypt cell that has characteristics of stem cells was discovered at around the same time as CBC cells. These cells were called “+4 cells” because they were typically found at a position about four cells above the bottom of the crypt. Unlike CBC cells, +4 cells were observed to divide slowly, or not at all. Later studies found that these +4 cells are able to undergo rapid divisions and take on CBC cell characteristics when the bottom of the crypts are damaged, suggesting that they act as a reserve to replace stem cells that are lost to disease or injury.

Above the +4 cells is a stretch of the crypt wall where the stem cell progeny rapidly divide as they migrate away from the crypt’s base. It was believed that the cells start to differentiate in this area, called the transient amplifying zone, taking on the characteristics of enterocytes, goblet cells, or any one of the other intestinal cell types. This model gained more support when advances in technology allowed researchers to identify the different types of intestinal cells (including stem cells) using molecular markers that are specific for each cell type. This enabled scientists to identify progenitor cells—the stem cell progeny that are on their way to becoming specialized intestinal cells.

The intestinal stem cell model has helped researchers understand the basis of digestive diseases during which the intestine is damaged and needs to heal (such as in celiac disease or inflammatory bowel disease) or in cases when regulation of cell proliferation goes awry (such as in colon cancer). Yet much work remains to further understand the many steps involved in the production of specialized intestinal cells and how this knowledge might be applied for therapeutic interventions.

The Intestinal Stem Cell Consortium

By the beginning of the 21st century, it was becoming clear that intestinal stem cells held great promise for understanding and treating digestive diseases. In 2009, the NIDDK formed the ISCC to grasp a better understanding of the biology of the intestinal stem cells during development, homeostasis, regeneration, and disease. The Consortium, consisting of a data coordinating center and nine study centers across the United States, enables participating researchers to share ideas and resources, including data, research materials, methods, and expertise. The immediate goals of the ISCC were and are to isolate, characterize, culture and, validate populations of intestinal stem cells; answer major questions in stem cell biology of the intestinal epithelium; and accelerate research by making information and resources available to the research community.

Since its inception, the ISCC has produced a wealth of information on the biology and therapeutic potential of intestinal stem cells. The ISCC’s earlier years focused on the respective roles of active and quiescent stem cells, along with the genetic mechanisms that control differentiation of stem cells into specialized intestinal cells. Consortium members also identified molecular markers that are unique to stem cells and their various stages of differentiation. This was an extremely important step in intestinal stem cell research, as it gave researchers more tools to identify and track specific cell populations in the gut. The ISCC also began efforts to recapitulate intestinal development outside of an animal model, which would enable researchers to examine events surrounding crypt formation more closely and to test the possibility of using cultured cells for therapeutic purposes.

Based on these early successes, the NIDDK renewed support for the ISCC in 2014. Consortium members continue to be extremely productive, demonstrating the synergy and efficiency of the ISCC. The following examples are just several of the many advances that the Consortium has contributed to intestinal stem cell research.

Making Mini-intestines

While most of the pioneering research on intestinal stem cells was accomplished in mouse models, the identification and characterization of intestinal stem cells allowed researchers to separate them from the surrounding tissue and culture them outside of an animal. This presented scientists with new opportunities to study more closely the molecular changes that occur in these cells. It also allowed investigators to coax cultured human intestinal stem cells to differentiate into various intestinal cell types in the laboratory, providing models to study types of human intestinal cells that are otherwise not easily accessible. For example, one group of ISCC scientists cultured stem cells from human intestinal crypts and induced them to differentiate into M cells. These cells even behaved like functional M cells: they took in pathogenic bacteria, much like they would in an intestine when they are delivering pathogens to the immune system in an underlying Peyer’s patch. This provided an important model system for studying how these cells protect the intestine from pathogenic microbes in the gut.

The ISCC has also used human pluripotent stem cells (PSCs) to create microscopic three-dimensional models of the intestine. PSCs are stem cells that can differentiate into many other different types of cells in the body, including cells that act as intestinal stem cells. The studies used induced pluripotent stems cells (which are derived from cells that were not originally stem cells but were induced to be pluripotent in the laboratory) and embryonic stem cell lines, used within NIH guidelines for human stem cell research. For example, the ISCC has cultured PSC-derived intestinal stem cells in a three-dimensional setting, allowing them to proliferate and differentiate, resulting in a conglomeration of cells that look and behave like a miniature portion of a human intestine (e.g., they contained functional villi-like and crypt-like structures). The ISCC has used these cellular arrangements, called “organoids,” as models of the human small intestine, and, more recently, of the human colon.

These laboratory-grown organoids can be used to study human intestinal and colonic diseases in a laboratory setting. For example, ISCC researchers recently succeeded in generating intestinal organoids containing functional nerve cells. The scientists then used these organoids as a model of a functional enteric nervous system—the mesh-like arrangement of nerves that governs the function of the gastrointestinal tract. They applied this model to study the molecular events in human diseases that involve the enteric system, such as Hirschsprung’s disease, where stool moves slowly (or stops completely) because the nerves near the end of the colon do not function properly.

Mini-intestines may also eventually be used to grow tissue to replace damaged intestinal tissue. In fact, ISCC researchers recently demonstrated that PSC-derived intestinal stem cells can be induced to differentiate into tissue that resembles different parts of the intestine, such as the ileum (the lower end of the small intestine) and duodenum (the section of the small intestine closest to the stomach). This is important from a therapeutic standpoint because distinct regions of the intestine have different functional roles in digestion.

Studies on Stem Cell Renewal and Plasticity

The ISCC has also concentrated efforts on understanding how intestinal stem cell proliferation and differentiation are regulated. Many studies have focused on the intestinal stem cell “niche,” or the environment in and surrounding the crypt that produces signals controlling when cells multiply and which type of mature epithelial cell they will become.

One of the most important of these signals is a family of molecules called Wnt proteins. Wnt proteins maintain the intestinal stem cell niche by helping to stimulate proliferation of intestinal stem cells at the bottom of the crypt and the partially differentiated cells in the transient amplifying zone. The ISCC has uncovered several key features of Wnt signaling that may be developed for therapeutic purposes. For example, a recent study by Consortium scientists showed that Wnt proteins are not required for cell proliferation in the early stages of intestinal development in the mouse (i.e., before villi are formed in the mouse embryo). This would be important to consider when developing therapies because tissue repair following injury is believed to rely on such embryonic pathways. Another recent study by ISCC researchers found that Wnt proteins are secreted by immune cells called macrophages in the connective tissue surrounding crypts in mice, and this is critical for intestinal cell proliferation and tissue repair following radiation-induced injury. Another ISCC study uncovered the surprising finding that Wnt proteins alone do not drive stem-cell proliferation in adult mice. Rather, they prime the stem cells for proliferation by making the stem cells more receptive to additional signaling proteins called R-spondins. The researchers found that R-spondins directly stimulate proliferation, which should be taken into consideration when developing therapeutics that aim to boost growth in the intestinal epithelium.

To gain a better understanding of the stages of intestinal epithelial cell differentiation, the ISCC has taken advantage of state-of-the-art techniques such as single-cell RNA sequencing, which allows researchers to compare gene activation in individual cells from mouse intestinal crypts. Scientists used this technique to detect progenitor cells that have begun to differentiate into enteroendocrine cells. These early enteroendocrine cells show significant plasticity—that is, they are able to revert back to an intestinal stem cell state when there is an injury. Single-cell RNA sequencing also allowed researchers to discover what appears to be one of the earliest stages of differentiation of intestinal stem cells, when they are simultaneously expressing genes for stem cells, secretory cells (such as goblet and enteroendocrine cells), and enterocytes. This provided valuable insight into how and when stem cells “decide” to become mature intestinal cells. Another study sought to provide a better characterization of the +4 cells that lie between the stem cell compartment and the transient amplifying zone. The researchers found that +4 cells have actually started down the path toward differentiation into secretory cells, although they can revert back to stem cells when the original supply of stem cells is lost. They also found that this shift between +4 cells and stem cells is at least partially driven by changes in the three-dimensional structure of the cells’ DNA, which controls the activity of genes required for the transition.

These advances greatly expand the understanding of how the intestinal epithelium develops into such an active, multifunctional, and critical component of the body. This helps set the stage for future studies that focus on treating damaged (or maintaining healthy) intestinal tissue.

The Future of Intestinal Stem-cell Research

The great progress in understanding the development and turnover of the intestinal epithelium has opened many doors that could lead to new therapies for treating digestive diseases. As scientists continue to investigate the intricate steps involved in the proliferation and differentiation of intestinal cells, efforts are underway to apply this knowledge toward ways to protect and heal the gastrointestinal tract. This is reflected in the ISCC’s long-term goals: to contribute to the greater understanding of stem cell biology and to lay the ground work for therapeutic manipulation of the intestinal epithelium. The coming years may eventually see intestinal stem cell-based therapies for a wide range of gastrointestinal diseases like inflammatory bowel disease, genetic disorders, disease-causing infections, radiation injury, and colon cancer.