Stem Cells Replace Hair Cells in Cochlea of Mice


In mammals, hearing loss is usually due to damage to the sound-sensing hair cells in the inner ear.

Originally, the hair cells were thought to be irreplaceable, but research in mice has shown that the supporting cells that provide structural support to the hair cells can turn into hair cells. If this technology can be applied in older animals, then it might provide a way to stimulate hair cell replacement in adults and treatments for deafness as a result of hair cell loss.

According to Albert Edge of the Harvard Medical School and Massachusetts Eye and Ear Infirmary, hair cell replacement definitely occurs, but does so as rather low levels. According to Edge: “The finding that newborn hair cells regenerate spontaneously is novel.”

 New Hair Cells in the Pillar Cell Region after Gentamicin Damage (A) Illustration of organ of Corti structure showing the Pou4f3-positive hair cells (blue), the Lgr5-positive supporting cells (red), and the remaining supporting cells in gray. Both the red and gray supporting cells are Sox2 positive. The green line indicates the xy plane from which the confocal slices in (B)–(G) are taken. (B–G) Confocal slices and cross sections from the midapex of neonatal organ of Corti explant cultures, treated with gentamicin and lineage-traced using the CAG-tdTomato reporter, were stained for DsRed (red). A white line on the whole-mount image shows the location of the cross section, and yellow and white brackets indicate IHCs and OHCs, respectively. Arrows point to new reporter-positive (or reporter-negative for Pou4f3) hair cells in the pillar cell region. Scale bar, 10 mm. (B) A reporter-positive hair cell from the Lgr5 lineage (such as those counted in H) was visible in the pillar cell region. (C and D) Reporter staining identified the hair cells marked by the white arrows as derived fromLgr5-positive cells; costaining for SOX2 (C) and location in the pillar cell region indicated that they were newly differentiated, and an OHC phenotype was suggested by the expression of PRESTIN (D). (D0 ) PRESTIN channel from (D) shows staining in the membrane and cuticular plate of the new hair cell. (E and F) Staining for the Sox2 lineage reporter identified the hair cells marked by the white arrows as derived from supporting cells; their location (pillar cell region) and costaining for SOX2 (E) identified them as newly differentiated cells, and costaining for PRESTIN (F) indicated an OHC identity. (G) The lack of Pou4f3 lineage reporter staining and the location in the pillar region identified the hair cell marked by the white arrow as a new hair cell, and costaining for PRESTIN indicated an OHC identity. (H) Increased numbers of Lgr5(blue bars) andSox2(red bars) reporter-positive hair cells were observed in the pillar cell region of the organ of Corti after gentamicin treatment (mean ± SEM per 100 mm; *p < 0.05, ***p < 0.001).
New Hair Cells in the Pillar Cell Region after Gentamicin Damage
(A) Illustration of organ of Corti structure showing the Pou4f3-positive hair cells (blue), the Lgr5-positive supporting cells (red), and the remaining supporting cells in gray. Both the red and gray supporting cells are Sox2 positive. The green line indicates the xy plane from which the confocal slices in (B)–(G) are taken.  (B–G) Confocal slices and cross sections from the midapex of neonatal organ of Corti explant cultures, treated with gentamicin and lineage-traced using the CAG-tdTomato reporter, were stained for DsRed (red). A white line on the whole-mount image shows the location
of the cross section, and yellow and white brackets indicate IHCs and OHCs, respectively. Arrows point to new reporter-positive (or reporter-negative for Pou4f3) hair cells in the pillar cell region. Scale bar, 10 mm.  (B) A reporter-positive hair cell from the Lgr5 lineage (such as those counted in H) was visible in the pillar cell region.  (C and D) Reporter staining identified the hair cells marked by the white arrows as derived fromLgr5-positive cells; costaining for SOX2 (C) and location in the pillar cell region indicated that they were newly differentiated, and an OHC phenotype was suggested by the expression of PRESTIN (D). (D0 ) PRESTIN channel from (D) shows staining in the membrane and cuticular plate of the new hair cell.  (E and F) Staining for the Sox2 lineage reporter identified the hair cells marked by the white arrows as derived from supporting cells; their location (pillar cell region) and costaining for SOX2 (E) identified them as newly differentiated cells, and costaining for PRESTIN (F) indicated an OHC identity.  (G) The lack of Pou4f3 lineage reporter staining and the location in the pillar region identified the hair cell marked by the white arrow as a new hair cell, and costaining for PRESTIN indicated an OHC identity.  (H) Increased numbers of Lgr5(blue bars) andSox2(red bars) reporter-positive hair cells were observed in the pillar cell region of the organ
of Corti after gentamicin treatment (mean ± SEM per 100 mm; *p < 0.05, ***p < 0.001).

Earlier work has shown that inhibition of the Notch signaling pathway increases the formation of new hair cells not from remaining hair cells but from nearby supporting cells that express a cell-surface protein called Lgr5.

When Edge and his team used small molecules to inhibit the Notch signaling pathway, even more support cells differentiated into hair cells, and the Lgr-5-expressing cells were the only supporting cells that differentiated under these conditions.

By combining these new findings about Lgr-5-expressing cells with the previous finding that Notch inhibition can regenerate hair cells, scientists should be able to design new hair cell regeneration strategies to treat hearing loss and deafness.

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.

INTESTINES COMPARED

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.

Stem Cell Transplant Repairs the Damage that Results from Inflammatory Bowel Disease


A source of stem cells from the digestive tract can repair a type of inflammatory bowel disease when transplanted into mice has been identified by British and Danish scientists.

This work resulted from a collaboration between stem cell scientists at the Wellcome Trust-Medical Research Council/Cambridge Stem Cell Institute at Cambridge University, and the Biotech Research and Innovation Centre (BRIC) at the University of Copenhagen, Denmark. This research paves the way for patient-specific regenerative therapies for inflammatory bowel diseases such as ulcerative colitis.

All tissues in out body probably contain a stem cell population of some sort, and these tissue-specific stem cells are responsible for the lifelong maintenance of these tissues, and, ultimately, organs. Organ-specific stem cells tend to be restricted in their differentiation abilities to the cell types within that organ. Therefore, stem cells from the digestive tract will tend to differentiate into cell types typically found in the digestive tract, and skin-based stem cells will usually form cell types found in the skin.

When this research team examined developing intestinal tissue in mouse fetuses, they discovered a stem cell population that differed from the adult stem cells that have already been described in the gastrointestinal tract. These new-identified cells actively divided and could be grown in the laboratory over a long period of time without terminally differentiating into adult cell types. When exposed to the right conditions, however, these cells could differentiate into mature intestinal tissue.

Fordham_CellStemCell_GraphicalAbstract

Could these cells be used to repair a damaged bowel? To address this question, this team transplanted these cells into mice that suffered from a type of inflammatory bowel disease, and within three hours the stem cells has attached to the damaged areas of the mouse intestine. integrated into the intestine, and contributed to the repair of the damaged tissue.

“We found that the cells formed a living plaster (British English for a bandage) over the damaged gut,” said Jim Jensen, a Wellcome Trust researcher and Lundbeck Foundation fellow, who led the study. “They seemed to response to the environment they had been placed in and matured accordingly to repair the damage. One of the risks of stem cell transplants like this is that the cells will continue to expand and form a tumor, but we didn’t see any evidence of that with this immature stem cell population from the gut.”

Because these cells were derived from fetal intestines, Jensen and his team sought to establish a new source of intestinal progenitor cells.  Therefore, Jensen and others isolated cells with similar characteristics from both mice and humans, and  made similar cells similar cells by reprogramming adult human cells in to induced pluripotent stem cells (iPSCs) and growing them in the appropriate conditions.  Because these cells grew into small spheres that consisted of intestinal tissue, they called these cells Fetal Enterospheres (FEnS).

Established cultures of FEnS expressed lower levels of Lgr5 than mature progenitors and grew in the presence of the Wnt antagonist Dkk1 (Dickkopf).  New cultures can be induced to form mature intestinal organoids by exposure to the signaling molecule Wnt3a. Following transplantation in a model for colon injury, FEnS contributed to regeneration of the epithelial lining of the colon by forming epithelial crypt-like structures that expressed region-specific differentiation markers.

“We’ve identified a source of gut stem cells that can be easily expanded in the laboratory, which could have huge implications for treating human inflammatory bowel diseases. The next step will be to see whether the human cells behave in the same way in the mouse transplant system and then we can consider investigating their use in patients,” Jensen said.

Taste Stem Cells Identified


Researchers at the Monell Center in Philadelphia, PA have successfully identified the location and markers for taste stem cells on the tongue. These findings will almost certainly allow scientists to grow and manipulate taste cells for clinical and research purposes.

Neurobiologist Robert Margolskee with the Monell Center who was also one of the authors of this study said: “Cancer patients who have taste loss following radiation to the head and neck and elderly individuals with diminished taste function are just two populations who could benefit from the ability to activate adult taste stem cells.”

Taste cells are located in rosette-like clusters known as taste buds in bumpy structures called papillae on the upper surface of the tongue. Two types of taste cells contain chemical receptors that initiate the perception of sweet, bitter, unami salty, and sour taste qualities. A third type of taste cell appears to serve as a support cell for these taste cells.

Gustatory_receptor_cell

A truly remarkable characteristic of these sensory cells is that they regularly regenerate, and all three taste cells undergo frequent turnover. The average lifespan of these cells is 10-16 days, which means that constant regeneration must occur in order for these cells to replace the cells that constantly die.

For decades, scientists who study taste have tried to identify the stem cell population that form these different taste cells. Scientists were also completely uncertain as to the location of these taste cell progenitors. Where they in the taste buds, near the taste buds, or someplace entirely different?

Monell scientists drew upon the strong association between oral taste cells and endocrine cells in the intestine. They reasoned that the cell-surface markers for stem cells in the intestine might also serve as markers for stem cells in the tongue. By using antibodies to a surface protein called Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5), the Monrell team observed two strong expression patterns for this marker in the tongue. One signal was underneath taste papillae at the back of the tongue and the second signal was an even weaker signal underneath taste buds in those papillae.

The Monell group hypothesized that the two levels of expression could indicate two different populations of cells that expressed Lgr5 at different levels. The stronger-expressing cells are probably the actual stem cells and those that more weakly express Lgr5 are those progeny of these stem cells that are beginning to differentiate. Therefore, the expression of the stem cell marker in these cells is fading.

Additional work showed that Lgr5-expressing cells were capable of differentiating into any of the three different types of taste cells.

Peihua Jiang. who is also a neurobiologist at the Monell Center, said: “THis is just the tip of the iceberg. Identification of these cells opens up a whole new area for studying taste cell renewal, and contributes to stem cell biology in general.”

In the future, the Monell group plans to program the Lgr5-expressing cells to differentiate into the different taste cell types, and explore how to grow these cells in culture. This will create a renewable source of taste receptor cells for research and perhaps even clinical use.

See Karen Yee, et al., “Lgr5-EGFP Marks Taste Bud Stem/Progenitor Cells in Posterior Tongue.” Stem Cells 2013 DOI: 10.1002/stem.1338.