How Skeletal Stem Cells form the Blueprint of the Face


A new study from the laboratory of University of Southern California (USC) Stem Cell researcher J Gage Crump, who is at the Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, has identified the key molecular signals that control the critical timing of the development of the vertebrate face.

Previous work has demonstrated that two molecular signals, in particular the JaggedNotch and Endothelin 1 signaling, are integral for shaping the face. Loss of either of these signals results in facial deformities in zebrafish and humans. This illustrates the essential contribution these signaling pathways make to the development of the face.

Lindsey Barske, a researcher in Crump’s laboratory and her colleagues utilized sophisticated genetic, genomic, and imaging tools to study face formation in zebrafish and showed that the Jagged-Notch and Endothelin 1 pathways work in tandem to control when and where the facial stem cells form face-specific cartilage.

In the lower part of the face, the Endothelin 1 signal accelerates cartilage formation early in development, but in the upper face, the Jagged-Notch signal transduction pathway produces signals that prevent stem cells from making cartilage until later in development.

Barske and her colleagues discovered that these timing differences in facial stem cell activity and facial cartilage production play a major role in making the upper and lower cartilage regions of the face.

The earliest blueprint of the facial skeleton is established by intersecting signals that control when stem cells transform cartilage into bone. It also appears that small tweaks to the timing of these events accounts for the different skull shapes observed in vertebrate animals. Also, small, nuanced changes in facial cartilage production and ossification can also account for the diverse array of facial shapes observed in humans.

This work was published in PLOS Genetics 12(4): e1005967. doi:10.1371/journal.pgen.1005967.

Rare Stem Cell Heals Damaged Lungs; Notch Signaling May Hold the Key to Lung Fibrosis


Patients who survive an acute lung injury are able to recover their lung function, which suggests that adult lungs regenerate to a certain extent. Depending on the cause and severity of the injury, multiple progenitor cells, including alveolar type II cells and distal airway stem cells, have been shown to drive lung tissue regeneration in mice. Now, Andrew Vaughan and others have described another cell type in the lungs involved in the repair process in mice when mouse lungs are damaged from influenza virus infection or inhalation of the anticancer drug bleomycin.  This cell type is called the rare lineage-negative epithelial progenitor (LNEP).

LNEP cells are quiescently present within normal distal mouse lung and do not express mature lineage markers (for example, a protein called club cell 10 or CC10 or surfactant protein C, otherwise known as SPC).  However, Vaughan and others demonstrate that LNEPs are activated to proliferate and migrate to damaged sites and mediate lung remodeling following major injury.

Vaughan and others used lineage tracing approaches and cell transplantation strategies and showed that LNEP cells, but not mature epithelial lineage cells, are multipotent in their ability to give rise to both club cells and alveolar cells.  Interestingly, activation of the Notch signaling pathway in LNEP cells initially activated them, but persistent Notch activation inhibited subsequent alveolar differentiation, resulting in failed tissue regeneration (characterized by the formation of abnormal honeycomb cysts in the mouse lung).  Thus Notch signaling is only required at the beginning of their activation, and then must be down-regulated if the LNEP cells are to reconstruct normal lung tissue.  Interestingly, scarred over or fibrotic lungs from patients with idiopathic pulmonary fibrosis or a disease called scleroderma show evidence of hyperactive Notch signaling and their lungs also contain very similar-looking honeycomb cysts.  This strongly suggests that dynamic Notch signaling also regulates the function and differentiation of LNEP-analogous human lung progenitor cells.  Thus designing treatments that properly regulate Notch signaling and, consequently, LNEP activity may potentially halt the development of lung fibrosis in humans.

Making Cardiac Stem Cells That are a Notch Above the Rest


The human heart has a stem cell population all its own. This stem cell population replaces heart cells at a leisurely rate throughout the life of the heart. Unfortunately, a heart attack overwhelms this repair system, and the heart simply lacks the capacity to heal itself to beyond particular limits.

However, there is the hope that physicians will one day be able to augment the healing capacity of the heart, and a few clinical trials and several animal experiments strongly suggest that this is the possible.

A new paper by Yoshiki Sawa at and his team from Osaka University has examined a way to increase the healing capabilities of human cardiac stem cells (CSCs).

In this paper, which was published in the journal Circulation, isolated CSCs from a 12-year old patient and grown in culture. However, the cells were grown in several different types of culture conditions. The density at which cells are grown can affect their biological characteristics. Therefore, Sawa and his group plated these cells at four different densities; single, low, mid and high densities. The single, low and med density-grown cells divided faster than the cells grown as high density. Also, the cells grown at lower cell densities retained their ability to form either heart muscle or blood vessels whereas the cells grown at high densities stated to make blood vessels en mass.

When scientists from Sawa’s group examined why the cells grown at high densities turned into blood vessel cells, they discovered that these cells activated a signaling pathway called the NOTCH pathway. Activation of the NOTCH pathway turned the cells into blood vessel-making cells and slowed their growth in culture.

JCS slide template

Presumably, the faster-growing, more plastic cells would be better for regenerative treatments that the slower-growing, less plastic cells. To test this hypothesis, Sawa and others transplanted cultured CSCs grown as different densities into the hearts of rats that had suffered a recent heart attack. They are used CSCs grown at high densities, but had been treated with a drug that inhibits the NOTCH pathway.

The results were remarkable. The lower the densities at which the cells were grown, the better they repaired the heart. However, the high-density cells grown in the presence of a NOTCH inhibitor (called GSI), were just as good at repairing the heart as the cells grown at low density. While the cells grown in the presence of GSI at high density still grew slowly, they showed an enhanced capacity to induce the formation of new blood vessels in the damaged heart tissue and form new heart muscle.

In conclusion these authors state: “Therapeutic effects of CSC-transplantation for heart disease may be enhanced by reducing NOTCH signaling in CSCs.”

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.

New 3D Method Used to Grow Miniature Pancreas


Researchers from the University of Copenhagen, in collaboration with an international team of investigators, have successfully developed an innovative three-dimensional method to grow miniature pancreas from progenitor cells. The future goal of this research is to utilize this model system to fight against diabetes. This research was recently published in the journal Development.

The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.
The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.

The new method takes cell material from mice and grows them in vividly picturesque tree-like structures.  The cells used were mouse embryonic pancreatic progenitors, and they were grown in a compound called Matrigel with accompanying cocktails of growth factors.  In vitro maintenance and expansion of these pancreatic progenitors requires active Notch and FGF signaling, and therefore, this culture system recapitulated the in vivo conditions that give rise to the pancreas in the embryo.

Professor Anne Grapin-Botton and her team at the Danish Stem Cell Centre, in collaboration with colleagues from the Ecole Polytechnique Fédérale de Lausanne in Switzerland, have developed a three-dimensional culture method that takes pancreatic cells and vigorously expands them. This new method allows the cell material from mice to grow vividly into several distinct picturesque, tree-like structures. The method offers tremendous long-term potential in producing miniature human pancreas from human stem cells. Human miniature pancreas organoids would be valuable as models to test new drugs fast and effectively, without the use of animal models.

“The new method allows the cell material to take a three-dimensional shape enabling them to multiply more freely. It’s like a plant where you use effective fertilizer, think of the laboratory like a garden and the scientist being the gardener,” says Anne Grapin-Botton.

In culture, pancreatic cells neither thrive nor develop if they are alone. A minimum of four pancreatic cells, growing close together is required for these cells to undergo organoid development.

“We found that the cells of the pancreas develop better in a gel in three-dimensions than when they are attached and flattened at the bottom of a culture plate. Under optimal conditions, the initial clusters of a few cells have proliferated into 40,000 cells within a week. After growing a lot, they transform into cells that make either digestive enzymes or hormones like insulin and they self-organize into branched pancreatic organoids that are amazingly similar to the pancreas,” adds Anne Grapin-Botton.

The scientists used this system to discover that the cells of the pancreas are sensitive to their physical environment, and are influenced by such seemingly insignificant factors as the stiffness of the gel and contact with other cells.

An effective cellular therapy for diabetes is dependent on the production of sufficient quantities of functional beta-cells. Recent studies have enabled the production of pancreatic precursors but efforts to expand these cells and differentiate them into insulin-producing beta-cells have proved a challenge.

“We think this is an important step towards the production of cells for diabetes therapy, both to produce mini-organs for drug testing and insulin-producing cells as spare parts. We show that the pancreatic cells care not only about how you feed them but need to be grown in the right physical environment. We are now trying to adapt this method to human stem cells,” adds Anne Grapin-Botton.

Stem Cells Build “Biobridges” to Aid Brain Repair


University of South Florida (USF) scientists have suggested a new strategy for stem cell-mediated brain repair following trauma.

In several preclinical experiments, the USF group found that transplanted stem cells build a “biobridge” that links an injured site in the brain to a site where neural stem cells form.

Principal investigator, Cesar Borlongan, professor and director of the USF Center for Aging and Brain Repair, said: “The transplanted stem cells serve as migratory cues for the brain’s own neurogenic cells, guiding the exodus of these formed host cells from their neurogenic niche towards the injured brain.”

Cesar Borlongan
Cesar Borlongan

On the strength of these preclinial studies in laboratory animals, the US Food and Drug Administration recently approved a limited clinical trial to transplant SanBio Inc.’s SB632 cells into patients with traumatic brain injuries. SB632 cells are a proprietary product of SanBio, Inc., and SB632 cells are derived from mesenchymal stem cells but they have been genetically engineered to express the intracellular domain of the Notch protein (NICD; see C. Tate, et al., Cell Transplantation, Vol. 19, pp. 973–984, 2010). If the Notch protein, which functions as a signaling protein and normally sits in the cell membrane, has its outer piece removed, the protein is constitutively activated. This full-time activation of the Notch protein and its downstream targets drive SB632 cells to form neural cells; something that mesenchymal stem cells typically do not readily make.

The Notch pathway. Notch is synthesised as a precursor protein that is processed by a furin-like convertase (S1 cleavage) in the Golgi before being transported to the cell surface, where it resides as a heterodimer. Interaction of Notch receptors with Notch ligands, such as Delta-like or Jagged, between two bordering cells leads to a cascade of proteolytic cleavages. The first cleavage (S2 cleavage) is mediated by ADAM-family metalloproteases such as ADAM10 or TNF-alpha-converting enzyme (TACE, also known as ADAM17), generating a substrate for S3 cleavage by the gamma-secretase complex. This cleavage releases the Notch intracellular domain (NICD) from the cell membrane. NICD then translocates to the nucleus, where it interacts with the DNA-binding protein RBP-Jkappa (also known as CBF1) and cooperates with Mastermind to displace corepressor proteins, thus activating the transcription of Notch target genes. The basic helix-loop-helix proteins hairy/enhancer of split (such as Hes1, 5 and 7) and Hes-related proteins (Hey1, 2 and L) and EphrinB2 are the best characterised downstream targets. Blockade of Notch signalling has been achieved by using different strategies, including (A) anti-DLL4 monoclonal antibodies, (B) gamma-secretase inhibitors such as DBZ and DAPT, (C) soluble DLL4-Fc, (D) anti-Notch1 neutralising antibodies, and (E) Notch1-trap.
The Notch pathway. Notch is synthesised as a precursor protein that is processed by a furin-like convertase (S1 cleavage) in the Golgi before being transported to the cell surface, where it resides as a heterodimer. Interaction of Notch receptors with Notch ligands, such as Delta-like or Jagged, between two bordering cells leads to a cascade of proteolytic cleavages. The first cleavage (S2 cleavage) is mediated by ADAM-family metalloproteases such as ADAM10 or TNF-alpha-converting enzyme (TACE, also known as ADAM17), generating a substrate for S3 cleavage by the gamma-secretase complex. This cleavage releases the Notch intracellular domain (NICD) from the cell membrane. NICD then translocates to the nucleus, where it interacts with the DNA-binding protein RBP-Jkappa (also known as CBF1) and cooperates with Mastermind to displace corepressor proteins, thus activating the transcription of Notch target genes. The basic helix-loop-helix proteins hairy/enhancer of split (such as Hes1, 5 and 7) and Hes-related proteins (Hey1, 2 and L) and EphrinB2 are the best characterised downstream targets. Blockade of Notch signalling has been achieved by using different strategies, including (A) anti-DLL4 monoclonal antibodies, (B) gamma-secretase inhibitors such as DBZ and DAPT, (C) soluble DLL4-Fc, (D) anti-Notch1 neutralising antibodies, and (E) Notch1-trap.

While this over-simplifies the field to some extent, there are two views on how stem cells heal brain damage caused by injury or neurodegenerative disorders. One view postulates that stem cells implanted into the brain directly replace dead or dying cells by differentiating into neurons and glial cells. The other view is that transplanted stem cells secrete growth factors that indirectly rescue the injured tissue. This present USF study argues for a third view, namely that implanted stem cells for a causeway in the brain between damaged areas and those anatomical structures that give birth to neural stem cells.

In this USF study, Borlongan and his group randomly assigned rats with traumatic brain injury and confirmed neurological impairment to one of two groups. The first group received transplants of SB632 cells into the region of the brain affected by traumatic injury. The second group received a sham procedure in which solution alone was infused into the brain with no implantation of stem cells.

At one and three months post-TBI (traumatic brain injury), the rats that had received SB632 transplants showed significantly better motor and neurological function and reduced brain tissue damage when compared to rats that had received no stem cells. These robust improvements despite the fact that the transplanted stem cells showed fair to poor survival that diminished over time.

Next, Borlongan’s laboratory workers examined the brain tissue of these rats. At three months post-TBI, the brains of transplanted rats showed massive cell proliferation and differentiation of stem cells into neuron-like cells in the area of injury. This was accompanied by a solid stream of stem cells that had migrated from the brain’s uninjured subventricular zone (where many new stem cells are formed) to the brain’s site of injury.

In contrast, those rats that had received solution alone showed limited proliferation and neural-commitment of stem cells, and only showed scattered migration to the site of brain injury and almost no expression of newly formed cells in the subventricular zone. Thus, without the addition of transplanted stem cells, the brain’s self-repair process appeared insufficient to mount a defense against the cascade of TBI-induced cell death.

Borlongan concluded that the transplanted stem cells create a neurovascular matrix that bridges the gap between the region in the brain where host neural stem cells arise and the site of injury. This pathway, or “biobridge,” ferries the newly emerging host cells to the specific place in the brain in need of repair, and helps them to promote functional recovery from traumatic brain injury.

Converting Mesenchymal Stem Cells to Bone Makers


Within human bone, cells called osteoblasts make new bone and without the constant activity of osteoblasts, bone becomes thin and fragile. Osteoblasts are derived from mesenchymal stem cells in the bone marrow. When bones break, orthopedic surgeons try to use growth factors to push more mesenchymal stems and their progeny to become osteoblasts. The growth factor in question is bone morphogen protein (BMP). BMP, however, does not work consistently, and it has some rather nasty side effects (cancer, The specific complications that are drawing the most concern include swelling in the neck and throat, radiating leg pain, and male sterility). Therefore, an alternative method for converting mesenchymal stem cells into osteoblasts is highly desirable.

Kurt Hankenson from the University of Pennsylvania School of Veterinary Medicine has worked on this very problem and described the situation this way, “In the field, we’re always searching for new ways for progenitor cells to become osteoblasts so we became interested in the Notch signaling pathway.” When it comes to BMP, Hankenson said, “it has become clear that BMPs have some issues with safety and efficacy.”

Is there a better way to make bone? There seems to be. A protein called Jagged-1 has been shown by Hankenson’s team to be highly expressed in bone. Jagged-1 is a component of the widely used Notch signaling pathway, which is found in the nervous system and in many other cells as well.

In mouse stem cells, introducing Jagged-1 blocks the progression of mesenchymal stem cells to osteoblasts. This finding has actually hampered osteoblast research for the last two years. Hankenson again, “That had been our operating dogma for a year or two.”

However, as is so often the case in science, you never truly know the result of an experiment until you actually do it. When Jagged-1 was added to human mesenchymal stem cells, the results were very different. Hankenson said, “It was remarkable to find that just putting the cells onto the Jagged-1 ligand seemed sufficient for driving the formation of bone-producing cells.”

From a developmental genetics perspective, this makes perfect sense, since mutations in the Jagged-1 gene cause an inherited disease known as Alagille syndrome which causes liver problems, abnormal metabolisms, and fragile bones that break easily. Also, genome-wide association studies have shown that particular versions of the Jagged-1 gene cause low bone density.

Hankenson and his collaborators are examining ways to manipulate the levels of the Jagged-1 protein in patients with bone problems. To that end, Hankenson is collaborating with Kathleen Loomes of Penn’s Perelman School of Medicine and the Children’s Hospital of Pennsylvania to study pediatric patients with Alagille syndrome to determine if bone abnormalities in these patients are indeed connected to Jagged-1 malfunctions.

Hankenson and his former graduate student Mike Dishowitz started a company called Skelegen through the University of Pennsylvania’s Center for Technology Transfer (CTT) UPstart program. The goal of Skelegen is to develop and improve a system for delivering Jagged-1 to sites that require new bone growth in the hopes of treating bone fractures and other skeletal problems.

See Fengchang Zhu et al., “Pkcdelta is required for Jagged-1 induction of hMSC osteogenic differentiation.” Stem Cells 2013; DOI 10.1002/stem.1353.

Drug Induces Hearing Restoration in Rodents


Fish and birds are able to regenerate their hearing after damage, but mammals are not able to do so, and hearing loss is irreversible in mammals like human beings. However, a new study has shown that the application of a particular drug can activate genes normally expressed during hair cell development. This work resulted from collaboration between researchers at Harvard Medical School, the Massachusetts Eye and Ear Infirmary, and Keio University School of Medicine in Japan. This finding is a first in the field or regenerative medicine.

Hair Cell Regeneration

In the cochlea, small cells known as hair cells convert sound waves into electrical signals that are interpreted by the brain into sounds. If these hair cells are damaged or destroyed by acoustic injury, then a permanent loss of hearing ensues. Such damage is treated with cochlear implants, which are surgically implanted devices that convert sounds to electrical signals.

“Cochlear implants are very successful and have helped a lot of people, but there’s a general feeling among clinicians, scientists, and patients that a biological repair would be preferable,” said Albert Edge, an otologist at Harvard University and the Massachusetts Eye and Ear Infirmary and lead author of the Neuron paper that reports these findings.

In previous work, Edge and his colleagues had shown that inhibiting the Notch signaling pathway was important for hair cells to form properly during fetal development (Jeon, S.J., Fujioka, M., Kim, S.C., and Edge, A.S.B. (2011). Notch signaling alters sensory or neuronal cell fate specification of inner ear stem cells. J. Neurosci. 31, 8351–8358). In their new study, Edge and his colleagues inhibited the Notch signaling pathway to determine, if such inhibition could initiate hair cell regeneration in adult mammals. They used a variety of approaches. In their first experiments, they used different inhibitors to determine their effects on isolated ear tissues. This allowed them to isolate one inhibitor in particular, the ɣ-secretase inhibitor LY411575, that led to increased expression of several molecular markers found in developing hair cells.

LY411575
LY411575

“It was quite a surprise,” said Edge. “We were very excited when we saw that a secretase inhibitor would have any effect at all in an adult animal.”

Next, Edge and his co-workers tested the inhibitor in mice that had hearing damage and reduced hair cell populations as a result of exposure to a loud noise. They tagged cells in the inner ear to follow their fate and discovered that the inhibitor, when applied to the inner ears of the mice, caused supporting cells to differentiate into replacement hair cells. These newly formed hair cells partially restored hearing at low sound frequencies, but not at higher frequencies. This effect lasted for at least three months.

This study examined the effect of the inhibitor when it was given one day after noise damage, which is a time when Notch signaling is naturally increased. This it is possible that a small window of time exists after an acoustic injury during which the drug is effective.

Edge concluded: “The improvement we saw is modest. So we’re now looking at variations of the approach and whether we can use the same drug to treat other types of hearing loss.”

See: Mizutari K, Fujioka M, Hosoya M, Bramhall N, et al. (2013) Notch Inhibition Induces Cochlear Hair Cell Regeneration and Recovery of Hearing after Acoustic Trauma. Neuron 77, 58-69.

Leukemia Gene is a Key Factor for Nerve Cell Differentiation


Research from the laboratory of Pierre Vanderhaeghen from the Universite’ Libre de Bruxelles has provided a new perspective on brain development and neural stem cell biology.

The cerebral cortex is the most complex structure in the brain. It is the seat of such higher cortical functions as consciousness, learning and memory, emotion, motor control, and language. To execute these functions, the cerebral cortex is composed of an array of cortical neurons, and these cells are adversely affected in cases of neurological or even psychiatric disorders.

According to work from Vanderhaeghen’s laboratory, a gene known as BCL6 is a key element in the development of cortical neurons during development. BCL6 acts as a transcription factor, which is to say that it plays a role in gene expression. In the case of BCL6, this gene product prevents gene expression (functions as a repressor). In the immune system, BCL6 is made in antibody-producing cells (B cells) and it controls the response of B cells to a signaling protein called Interleukin 4 (IL-4). IL-4 drives the differentiation of B cells into antibody-making plasma cells and drives the maturation of plasma cells into those that make distinct types of antibodies. Even more interestingly, BCL6 is frequently abnormal in a blood cancer known as diffuse large B cell lymphoma (DLBCL),

Two members of Vanderhaeghen’s lab discovered BCL6 in a search for genes that modulate the production of new nerve cells from mouse embryonic stem cells. If they overexpressed BCL6 in neural stem cells made from mouse embryonic stem cells, these stem cells transformed en mass into cortical neurons. Because BCL6 is normally known for its role in blood cancers (lymphomas), this BCL6-medicated function was a complete surprise.

Because data from overexpression studies is always suspect without verification, Vanderhaeghen and his colleagues used mouse genetics to confirm the role of BCL6 in the production of cortical neurons. Vanderhaeghen’s team made mutant mice embryos that had lost a functional copy of the BCL6 gene. When these mice developed to the fetal stage, it was clear that they had small cerebral cortexes that consisted of far fewer cortical neurons. Therefore, BCL6 overexpression increases cortical neuron production and the absence of it decreases cortical neuron production. This certainly confirms the role of BCL6 in cortical neuron development.

Next, Vanderhaeghen’s lab determined how BCL6 was influencing the development of cortical neurons. A protein that is encoded by the Notch gene are essential in the self-renewal of neural stem cells. BCL6 works with another protein called SIRT1 to repress the Notch pathway, and this repression moves the progeny of neural stem cells to differentiate into cortical neurons.

Because cortical neurons are the main entities affected by neurological and psychiatric disorders, this understanding of cortical neuron development might provide insights into inherited forms of dementia, behavioral problems or other types of neurological problems. Also, Vanderhaeghen’s work bring together three major players involved in cancer BCL6), aging, Alzheimer’s disease, metabolism and diabetes (SIRT1), and brain and heart development and cancer (Notch). Because these three genes were not know to interact with each other prior to this work, Vanderhaeghen’s findings have opened up a new avenue of possible targets for therapies and model systems for understanding stem cell renewal and differentiation.

The Notch Signaling Pathway


Because stem cell differentiation is controlled by signal transduction pathways, some of my readers have suggested that I discuss particular signal transduction pathways. In the previous post, the Notch signaling pathway was mentioned, and this provides a good reason to introduce my readers to it.

To think of signal transduction, one should consider the popular board game, Mouse Trap. When your game icon lands on the mouse trap spot on the game board, you turn a crank, and this crank rotates a vertical gear that is connected to a gear. Once that gear turns, it pushes lever that is braced with a rubber band until it snaps back and hits a swinging boot. The boot kicks over a bucket, which sends a marble down a rickety staircase. At the bottom of the staircase, the marble enters a chute and eventually taps a vertical pole. At the top of this pole is an open hand (palm-up) that supports another marble. The movement of the pole, caused by the tapping of its base by the first marble knocks the second marble free and it falls through a hole in its platform into a bathtub, and then through a hole in the tub onto one end of a seesaw. The propulsion of the seesaw launches a plastic diver on the other end into a round tub that is on the same base as the barbed pole that supports the mouse cage. The tub’s movement shakes the cage free from the top of the pole and the cage falls to trap the mouse.

This machine that traps the mouse is very similar to signal transduction in cells. The signal to catch the mouse (turning the crank), is far removed from the cage that eventually catches the mouse. Also, the act of catching the mouse (the dropping of the mouse cage), requires the prior execution of many other causally linked steps.

Notch signaling begins with a cell surface protein called Notch. Notch has a large region of the protein outside the cell and a small part of it that intersects the cell membrane, and another region that extends into the cell interior. All three of these domains of the Notch protein play an essential role in the function of Notch.

To turn the crank of this mouse trap, Notch must bind to its receptor. The Notch receptor can be a member of the DSL (Delta, Serrate and Lag-2) gene family.  The receptor is found on the surface of another cell. The binding of Notch to its receptor is the action that “turns the crank” on this mouse trap. Notch binding changes the structure of Notch, and it is clipped into two unequal halves by an enzyme that clips proteins at specific sites (the gamma-secretase). The Notch protein is now broken into a portion that remains anchored in the cell membrane, and another regions that remains inside the cell. This portion of the Notch protein is called “Intracellular Notch” or ICN (Wang MM.Int J Biochem Cell Biol.2011 Nov;43(11):1550-62 & D’Souza B, Miyamoto A, Weinmaster G. Oncogene. 2008 Sep 1;27(38):5148-67).

With the cleavage of Notch, the boot has knocked over the bucket and the marble has moved down the rickety staircase to the chute. ICN is able to enter the cell nucleus. There are proteins in the cytoplasm that can bind to ICN and prevent it from doing so, but we will not discuss them at this time (see van Tetering G, Vooijs M. Curr Mol Med. 2011 Jun;11(4):255-69).

Once in the nucleus, ICN teams up with another protein to activate the express of particular genes. Therefore, what began at the cell surface with the binding of the Notch protein by its receptor had culminated in the changes in gene expression in the nucleus. The other proteins that work together with ICN are members of the “CSL” gene family. CSL stands for “CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1.” When ICN combines with CSL the two proteins are converted from inactive proteins into a complex that actives the synthesis of messenger RNAs for specific genes. This rattles the pole that brings the cage down on the mouse’s head (see Kovall RA. Oncogene. 2008 Sep 1;27(38):5099-109).

What are the target genes of Notch signaling? Great question, but the answer is frustrating, since it depends on the cell type. In developing pancreas, once of the target genes of Notch signaling is PTF1a, but in other cell types and tissues, other genes are activated.

In embryonic stem cells and other stem cells as well, the Notch signaling pathway plays a vital role in the differentiation of these cells into various cell types. Notch signaling is also an important component of the pathology of organ failure in many organs and is also a central pathway involved in the onset and maintenance of several different types of cancers.  Understanding its function and how to regulate it is crucial.