Nusse Laboratory at Sanford Identifies Elusive Mouse Liver Stem Cell


Stanford University life science researchers have managed to successfully identify a stem cell population that has eluded many laboratories for some time. Essentially, the Stanford team has discovered a previously unknown population of liver cells in mice that function as liver stem cells. Such a find could aid drug testing and increase our present understanding of liver biology and disease.

Researchers in the laboratory of Roel Nusse at the Stanford University School of Medicine have identified a cell type in the liver of laboratory mice that can both self-renew and make new liver cells. This discovery by Nusse and others settles a long-standing conundrum of how the liver maintains itself when liver cells grow old and die.

“There’s always been a question as to how the liver replaces dying hepatocytes,” said professor of developmental biology Roel Nusse, PhD. “Most other tissues have a dedicated population of cells that can divide to make a copy of themselves, which we call self-renewal, and can also give rise to the more-specialized cells that make up that tissue. But there never was any evidence for a stem cell in the liver.”

It was assumed for some time that mature hepatocytes (liver cells) would themselves divide to replace a dying neighbor. However, hepatocytes have an abnormal amount of DNA, which would make cell division extremely difficult.

Nusse and his team published this research in the Aug. 5 edition of the journal Nature. The first author of this paper, Bruce Wang, MD, an assistant professor of gastroenterology and hepatology at the University of California-San Francisco, led the research while serving as a visiting scholar in Nusse’s lab.

The liver is a large, multi-lobed organ that filters toxins from the blood, synthesizes blood proteins, and makes digestive enzymes and bile. It is involved in many important metabolic processes. The liver contains a central vein that carries blood through it. The stem cells identified by Wang and Nusse are found adjacent to this vein.

Classically, hepatocytes were largely thought to be all alike. Most mature hepatocytes are “polyploid,” which means that they have more than the normal two copies of each chromosome. With all this extra DNA, it makes it difficult or even impossible, for these cells to divide normally, but this extra DNA might confer other benefits.

“If it’s not necessary for a cell to maintain the capacity to divide, it can do whatever it wants with its genome,” said Nusse. “Red blood cells, for instance, have no DNA. Muscle cells have many copies of each chromosome.” Having extra copies of chromosomes might allow these cells to make large amounts of particular proteins quickly, for example.

However, the cell population identified by Wang and Nusse in the livers of laboratory mice is diploid, and have only two copies of each chromosome. These cells can divide to make carbon copies of themselves, or to make cells that begin their lives as diploid but then acquire additional copies of their chromosomes as they move away from the central vein into the main body of the liver.

“People in the field have always thought of hepatocytes as a single cell type,” said Wang. “And yet the cell we identified is clearly different from others in the liver. Maybe we should accept that there may be several subtypes of hepatocytes, potentially with different functions.”

To identify liver-specific stem cells, Wang and Nusse identified cells that express the protein Axin2. They found these axin2-expressing cells surrounding the central vein. Axin2 is produced by cells in response to the presence of members of the Wnt signaling protein family, and decades of research by Nusse and other laboratories have shown that the Wnt proteins play a critical role in embryonic development, and in the growth and maintenance of stem cells throughout the body.

Interestingly, Wang and Nusse, and others showed that the endothelial cells that line the interior surface of the central vein in the liver produce Wnt2 and Wnt9b. These secreted Wnt proteins confer stem cell properties on the neighboring hepatocytes that surround the central vein.

Finally, Nusse’s team discovered that a portion of the descendants of the Axin2-expressing cells move outward from the central vein over time, become polyploid and begin to express other, hepatocyte-specific genes. About one year after being born, these descendant cells had effectively replaced about 30 percent of the entire mouse liver, and made up about 40 percent of all hepatocytes in the liver.

These newly identified liver stem cells also express genes associated with very early embryonic development, which may give a clue as to when and where they arise.

“Perhaps these stem cells in the adult liver actually arise very early in development,” said Nusse, “when the embryo sets aside a certain population of cells to maintain the organ during adult life.”

Although the current research was conducted in mice, it is possible that there are more than just one kind of hepatocyte in humans as well, and this realization could transform the study of liver biology. For example, hepatocytes have proven notoriously difficult to grow in laboratory culture for study or for use in drug testing.

“The most common reason that promising new drugs for any type of condition fail is that they are found to be toxic to liver,” said Wang. “Researchers have been trying for decades to find a way to maintain hepatocytes in the laboratory on which to test the effects of potential medications before trying them in humans. Perhaps we haven’t been culturing the right subtype. These stem cells might be more likely to fare well in culture.”

There’s also an opportunity to better understand human disease.

“Does liver cancer arise from a specific subtype of cells?” said Wang. “This model also gives us a way to understand how chromosome number is controlled. Does the presence of the Wnt proteins keep the stem cells in a diploid state? These are fundamental biological questions we can now begin to address.”

Treating Colon Cancer By Activating Damaged Genes


What if doctors could turn cancer cells into healthy cells? It would change everything about how we treat cancer. Researchers may have discovered a way to do that in colorectal cancer.

What if we could turn the clock back on cancer cells and return them to their healthy status?   A new study in animals might have accomplished exactly that.

A research team from the Memorial Sloan Kettering Cancer Center has reactivated a defective gene in mice with colorectal cancer.  This gene, adenomatous polyposis coli, or Apc, is commonly defective in colorectal cancer cells.  Approximately 90 percent of colorectal tumors have a loss-of-function mutation of this gene.

At the onset of this research project, The Sloan Kettering group suppressed the expression of the Apc gene in mice.  The Apc gene encodes a protein that regulates an important cell signaling pathway known as the Wnt signal pathway.  Suppression of Apc activates the Wnt signaling pathway, which helps cancer cells grow and survive.

Afterwards, they reactivated the Apc gene, which returned Wnt signaling to its normal levels and the cancerous tumors stopped growing, and normal intestinal function was restored in four days. By two weeks after Apc was reactivated, the tumors were gone and there were no lingering signs of no signs of cancer relapse during the six-month follow-up.

The same approach turned out to be effective in mice with colorectal cancer tumors that result from activating mutations in the Kras gene and loss-of-function mutations in the p53 gene.  In humans, about half of colorectal tumors have these mutations

This study was published in the prestigious international journal, Cell, by Scott Lowe and his colleagues.  “Treatment regimens for advanced colorectal cancer involve combination chemotherapies that are toxic and largely ineffective, yet have remained the backbone of therapy over the last decade,” said Lowe.

Apc reactivation might very well be the way to improved treatment for colorectal cancer.  It is doubtful it will be helpful in other types of cancer, but in the future, it might become so.  “The concept of identifying tumor-specific driving mutations is a major focus of many laboratories around the world,” said Lukas Dow, Ph.D., of Weill Cornell Medical College, who is the first author of this study.

“If we can define which types of mutations and changes are the critical events driving tumor growth, we will be better equipped to identify the most appropriate treatments for individual cancers,” said Dow.

Colorectal cancer begins in the colon or rectum, and it remains the second-most prevalent cause of cancer death in developed countries.

According to the Surveillance, Epidemiology, and End Results Program, in 2012, there were 1,168,929 people living with colon and rectal cancer in the United States.

Estimates postulate that there will be 132,700 new cases of colorectal cancer in the United States in 2015, and about 49,700 people will lose their lives to this disease. Worldwide, colorectal cancer is the cause of approximately 700,000 deaths each year.

Internist and gastroenterologist Dr. Frank Malkin expressed optimism regarding genetic research into colorectal cancer.  He said in an interview with the medical news service, Healthline: “They’ve identified a suppressor gene that can turn a tumor on and off. It can suppress the cancer and destroy it rapidly. That’s very promising.”

Cancers are normally treated with a combination of surgery, chemotherapy, and radiation.  These rather harsh treatments can take a lasting toll.  Easier and more effective treatments could change the lives of cancer patients.

Michelle Gordon, D.O., FACOS, FACS, finds it encouraging. “If this treatment is to be believed, all current modalities will be obsolete.”

However, Malkin and Gordon both cautioned that it is simply too early to bring this strategy to the clinic to treat human patients.

“There are so many unknowns when taking a mouse model to humans,” Gordon told Healthline. “This may be the foundational step that will lead to curing most colorectal cancers. This study can provide hope to future generations of colorectal cancer [patients], but I believe a cure is decades away.”

Researchers know Apc mutations initiate colorectal cancer, but they are unsure if Apc mutations are involved in promoting tumor growth after the cancer has developed.

The next step in this work will examine the ability of Apc reactivation to affect tumors that have spread or metastasized to distant locations in the body.  Lowe and his colleagues are also hard at work to determine precisely how Apc works.  That will help scientists develop safe treatments that change cancer cells into normal cells. Such a drug could make colorectal cancer treatment easier, faster, and safer.

How this research will impact other types of cancer remains unclear.  “Cure rates for colorectal cancers are better than they used to be, especially when treated in the early stages,” said Malkin.  Nevertheless, it is still far better to stop tumors before they start.

According to Malkin, the number of colon cancer cases has dropped dramatically since routine colonoscopy screening began. A colonoscopy allows doctors to find and remove polyps before they turn cancerous.  Malkin also looks forward to genetic research that will identify those at greater risk for colorectal cancers.

“Right now, we’re using colonoscopy to screen people over 50, most who don’t have the genetic predisposition and will never get colorectal cancer,” he said. “We don’t yet have the genetic studies that would help us identify high-risk patients so we don’t have to screen everyone.”

I must admit that I remain skeptical as to whether or not this will work.  The reasons for my skepticism lie in the fact that tumor cells in the colon are the result of a series of mutations in cells that cause the cells to overgrow and eventually become invasive.  Colorectal carcinoma cells have mutations in several genes and not just Apc.  Apc reactivation worked in these mice because this was the only gene affected in these animals.  In a cancerous human colon, the cancer cells have a variety of mutations.  Kurt Vogelstein’s work at Johns Hopkins has shown this in great detail.  If Lowe could demonstrate the efficacy of his treatment in mice with humanized immune systems that have been infected with human colorectal carcinoma cells, then I will believe that this technique could work in human patients.  For now, I remain skeptical.

A More Efficient Way to Make Induced Pluripotent Stam cells


Mark Stadtfeld and his colleagues at the NYU Longone Medical Center has devised a new method for making induced pluripotent stem cells that greatly increases efficiency at which these cells are made.

Induced pluripotent stem cells or iPSCs are made from mature, adult cells by mean of a combination of genetic engineering and cell culture techniques. In short, the expression of four genes is forced in adult cells; Oct4, Sox2, Klf4, and c-Myc or OSKM. The proteins encoded by these four genes cooperatively work to drive a fraction of the cells into an immature state that resembles that of embryonic stem cells. These cells are them grown in cell culture systems that select for those cells that can grow continuously and form colonies of cells derived from progenitor cells. These cell colonies are them repeated isolated a re-cultured until an iPSC line has been established.

Unfortunately, this process is rather inefficient and tedious, since less than one percent or so of the reprogrammed cells actually undergo successful reprogramming. Additionally, it can take several weeks to properly establish an iPSC line. Thus, stem cell scientists have been looking at several different ways to boost the efficiency of this process.

Stadtfeld and his coworkers tried to add compounds to the cultured cells to determine if the culture conditions could actually augment the efficiency of the reprogramming process. “We especially wanted to know if these compounds could be combined to obtain stem cells at high-efficiency,” said Stadtfeld.

The compounds to which Stadtfeld was referring were two cell signaling proteins called Wnt and TFG-beta. Both of these compounds regulate a host of cell growth processes. Stadtfeld wanted to try regulating both of these pathways at the same time, in addition to providing cells with ascorbic acid, which is also known as vitamin C. Even vitamin C is more popularly known as an antioxidant, vitamin C also can remodel chromatin (that tight structure into which cells package their DNA).

When mouse skin fibroblasts were treated with OSKM and a compound that activates Wnt signaling, the efficiency of iPSC derivation increased slightly. The same thing was observed if fibroblasts were treated with OSKM and a compound that inhibits TGF-beta signaling or vitamin C. However, when all three of these compounds were combined, OSKM-engineered fibroblasts were reprogrammed at an efficiency of close to 80 percent. When different cell types were used as the starting cell, such as blood progenitor cells, the efficiency jumped to close to 100 percent; a result that was also observed if liver progenitor cells were used as the starting cell.

Stadtfeld is confident that these dramatic increases in iPSC derivation should improve future studies with iPSCs, since his protocol should make iPSC derivation more predictable. “It’s just a lot easier this way to study the mechanisms that govern reprogramming, as well as detect any undesired features that might develop in iPSCs,” he said.

Vitamin C and the two compounds used to manipulate the Wnt and TGF-β pathways have been widely used in research and have few unknown or hazardous effects. However, OKSM has in some cases caused undesired features in iPSCs, such as increased mutation rates. Stadtfeld believes that by making iPSC induction more rapid and efficient, his new technique might also make the resulting stem cells safer. “Conceivably it reduces the risk of abnormalities by smoothening out the reprogramming process,” Dr. Stadtfeld says. “That’s one of the issues we’re following up.”

Micro-Grooved Surfaces Influence Stem Cell Differentiation


Martin Knight and his colleagues from the Queen Mary’s School of Engineering and Materials Science and the Institute of Bioengineering in London, UK have shown that growing adult stem cells on micro-grooved surfaces disrupts a particular biochemical pathway that specified the length of a cellular structure called the “primary cilium.” Disruption of the primary cilium ultimately controls the subsequent behavior of these stem cells.

Primary cilia are about one thousand times narrower than a human hair. They are found in most cells and even though they were thought to be irrelevant at one time, this is clearly not the case.

Primary Cilium

The primary cilium acts as a sensory structure that responds to mechanical and chemical stimuli in the environment, and then communicates that external signal to the interior of the cell.  Most of the basic research on this structure was done using a single-celled alga called Chlamydomonas.

Martin Knight and his team, however, are certain that primary cilia in adult stem cells play a definite role in controlling cell differentiation.  Knight said, “Our research shows that they [primary cilia] play a key role in stem cell differentiation.  We found it’s possible to control stem cell specialization by manipulating primary cilia elongation, and that this occurs when stem cells are grown on these special grooved surfaces.”

When mesenchymal stromal cells were grown on grooved surfaces, the tension inside the cells was altered, and this remodeled the cytoskeleton of the cells.  Cytoskeleton refers to a rigid group of protein inside of cells that act as “rebar.” for the cell.  If you have ever worked with concrete, you will know that structural use of concrete requires the use of reinforcing metal bars to prevent the concrete from crumbling under the force of its own weight.  In the same way, cytoskeletal proteins reinforce the cell, give it shape, help it move, and help it resist shear forces.  Remodeling of the cytoskeleton can greatly change the behavior of the cell.

The primary cilium is important for stem cell differentiation.  Growing mesenchymal stromal cells on micro-grooved surfaces disrupts the primary cilium and prevents stem cell differentiation.  This simple culture technique can help maintain stem cells in an undifferentiated state until they have expanded enough for therapeutic purposes.

Once again we that there are ways to milk adult stem cells for all they are worth.  Destroying embryos is simply not necessary to save the lives of patients.

A Molecular Switch that Causes Stem Cell Aging


A study from the Cincinnati Children’s Hospital Medical Center, in collaboration with the University of Ulm in Germany has discovered a molecular switch that causes the aging of blood stem cells. This same work suggests a therapeutic strategy to delay stem cell aging.

Hematopoietic stem cells (HSCs) reside in the bone marrow and make all the red and white blood cells that populate the bloodstream. Proper HSC function is absolutely vital to the ongoing production of different types of blood cells that allow the immune system to fight infections and organs to receive adequate quantities of oxygen.

Hartmut Geiger from the Cincinnati Children’s Hospital Medical Center and the University of Ulm was the senior researcher on this project. Dr. Geiger said, “Although there is a large amount of data showing that blood stem cell function declines during aging, the molecular processes that cause this remain largely unknown. This prevents rational approaches to attenuate stem cell aging. This study puts us significantly closer to that goal through novel findings that show a distinct switch in a molecular pathway is very critical to the aging process.”

The pathway to which Dr. Geiger referred is the Wnt signaling pathway, which plays a foundational role in animal development, cell-cell communication, tissue generation, and is also involved in the pathology of various diseases.

Crystal structure of XWnt8
Crystal structure of XWnt8

Analysis of mouse models and cultured HSCs showed that under normal conditions, Wnt signaling in HSCs occurred through the so-called “canonical” Wnt signaling pathway. The canonical Wnt signaling pathway utilizes the typical components of Wnt signaling that were first identified in the fruit fly and then isolated and characterized in vertebrates (shown below).

Canonical Wnt signaling

However, Wnt proteins can also signaling through other, distinct signal transduction pathways, and these types of pathways are collectively known as “noncanonical” Wnt signaling pathway. In aging HSCs, a switch from canonical Wnt signaling to noncanonical Wnt signaling marked the onset of HSC aging.  See below for one example of non-canonical Wnt signaling.

Non-canonical Wnt signaling

To test this observation, Geiger’s group overexpressed Wnt5 in HSCs (a Wnt protein known to induced signaling through noncanonical Wnt signaling pathways), and immediately, the HSCs began to show the signs of aging.

One of the targets of Wnt5 signaling is a protein called Cdc42, which influences the cytoskeleton of cells.  Therefore, Geiger and his crew asked if Cdc42 was activated in those HSCs that overexpressed Wnt5.  The answer to this question was a clear “yes.”  Then they treated cultured HSCs with a molecule that inhibited Cdc42 activity.  This treatment reversed the aging process in HSCs.

To test their hypothesis in a living animal, Geiger and others removed a copy of the Wnt5 gene from HSCs in laboratory mice.  Mice that lacked functional Wnt5 protein in HSCs, showed rejuvenation of the aged HSCs.  Mice that lacked both copies of the Wnt5 gene showed a delayed aging process in their HSCs.

Even though this study has definitely made an important contribution to understanding HSC aging, more work is needed before a therapeutic strategy is in place.

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.

Isolation of Pancreatic Stem Cells


There has been a robust debate as to whether or not the pancreas has a stem cell population. Several studies suggested that the pancreatic duct cells could differentiate into hormone-secreting pancreatic cells. Unfortunately, when the cells of the pancreatic duct are marked, they clearly never contribute to regeneration of the pancreas. According to an article that appeared in the journal Developmental Cell by Oren Ziv, Benjamin Glaser, and Yuval Dor entitled “The Plastic Pancreas,” tying off the pancreatic duct kills off the acinar cells, but it leads to a large increase in the number of hormone-secreting beta cells. Something seems to be contributing cells to the adult pancreas. However when lineage studies tried to confirm that the pancreatic duct cells formed the new cells, it failed to find any connection between the new cells in the pancreas and the duct.

Pancreas

Recent experiments from Chris Wright’s lab suggest that the acinar cells are a population of progenitor cells that divide and differentiate into different kinds of pancreatic cell types after injury to the pancreas. A similar result was observed in work by Desai and others. If that’s not odd enough for you, another set of experiments from Pedro Herrera research group has shown once all the insulin-secreting beta cells are killed off, the adjacent glucagon-secreting cells transdifferentiate into insulin-secreting beta cells. Therefore, something interesting is afoot in the pancreas.

All these experiments were done with rodents. Whether or not they are transferable to human remains uncertain. Nevertheless, a fascinating paper in EMBO Journal from Hans Clevers lab at the Hubrecht Institute, Utrecht, Netherlands haws succeeded in culturing pancreatic precursor cells.

Here’s how they did it. Clevers and his crew took the pancreatic duct of mice and partially tied it off. In order to stem cells from the digestive tract to grow, they must upregulate a signaling pathway called the “Wnt” pathway. The Wnt pathway is quiet in the pancreas, but one the pancreas is injured, the Wnt pathway swings into gear and the cells begin to divide.

When Clevers and company dropped pancreatic duct tissue into culture, Wnt signaling activity soared and the cells grew into a mini-organ (organoid) that resembled and tiny pancreas in a culture dish. In fact, a single cell taken from the pancreatic duct could be cultured into an organoid.

Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding. (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.
Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding.  (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.

This experiment shows that there are techniques for growing unlimited quantities of pancreatic cells.  The therapeutic possibilities of this technology is tremendous.  In Clever’s own words, “We have found a way to activate the Wnt pathway to produce an unlimited expansion of pancreatic stem cells isolated from mice.  By changing the growth conditions we can select two different fates for the stem cells and generate large numbers of either hormone-producing beta cells or pancreatic duct cells.”

Can this work with human pancreatic duct cells?  That is the $64,000 question.   Clevers and his groups will almost certainly try to answer this questions next.  If Clevers and his crew can get this to work, then the possibilities are vast indeed.

Researchers Create Inner Ear Structures From Stem Cells


Indiana University scientists have used mouse embryonic stem cells to make key structures of the inner ear. This accomplishment provides new insights into the sensory organ’s developmental process and sets the stage for laboratory models of disease, drug discovery and potential treatments for hearing loss, and balance disorders.

Eri Hashino, professor of otolaryngology at the University of Indiana School of Medicine, and his co-workers, were able to use a three-dimensional cell culture method that directed the stem cells to form inner-ear sensory epithelia that contained hair cells and supporting cells and neurons that detect sound, head movements and gravity.

In the past, other attempts to grow inner-ear hair cells in standard culture systems have not succeeded. Apparently the cues required to form inner-ear hair bundles, which are essential for detecting auditory or vestibular signals, are absent in cell-culture dishes.

Inner ear hair cells
Inner ear hair cells

To conquer this barrier, Hashino and his team changed their culture system. The suspended the cells as aggregates in a specialized culture medium and this mimicked conditions normally found in the body as the inner ear develops.

Another strategy that paid off was to precisely time the application of several small molecules that coaxed the stem cells to differentiate from one stage to the next into precursors for the inner ear.

a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, c, Pax2 (b) and Calb2 (c) are expressed in all Myo7a+ stem-cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d–g, The structural organization of vesicles with Calb2+ Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, nonsensory epithelium. h, Tuj1+ neurons extending processes to hair cells. i, The synaptic protein Snap25 is localized to the basal end of hair cells. j, The postsynaptic marker Syp colocalizes with Ctbp2 (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n > 100 cells, *P < 0.05, ***P < 0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 µm (i), 5 µm (j).  Also, BMP = Bone morphogen protein, FGF = fibroblast growth factor, LGN = Small molecule that inhibits BMP signaling, Wnt = small secreted glycoprotein involved in cell signaling.
a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, c, Pax2 (b) and Calb2 (c) are expressed in all Myo7a+ stem-cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d–g, The structural organization of vesicles with Calb2+ Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, nonsensory epithelium. h, Tuj1+ neurons extending processes to hair cells. i, The synaptic protein Snap25 is localized to the basal end of hair cells. j, The postsynaptic marker Syp colocalizes with Ctbp2 (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n > 100 cells, *P < 0.05, ***P < 0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 µm (i), 5 µm (j). Also, BMP = Bone morphogen protein, FGF = fibroblast growth factor, LGN = Small molecule that inhibits BMP signaling, Wnt = small secreted glycoprotein involved in cell signaling.

Even though the added growth factors made a big difference to the success of this experiment, it was the three-dimensional suspension culture system that provided many important mechanical cues. The tension caused by the pull of the cells on each other played a very important role in directing the differentiation of the cells to become inner-ear precursors.

Karl A Koehler, first author of this paper and a graduate student in the medical neuroscience program at IU School of Medicine said: “The three-dimensional culture allows the cells to self-organize into complex tissues using mechanical cues that are found during embryonic development.”

Hashino added that they were “surprised to see that once stem cells are placed in 3-D culture, these cells behave as if they knew not only how to self-organize into a pattern remarkably similar to the native inner ear.” Hashino continued: “Our initial goal was to make inner-ear precursors in culture, but when we did testing we found thousands of hair cells in a culture dish.”

Electrophysiological testing of these stem cell-derived hair cells showed that they were, in fact, functional, and were similar to those that sense gravity and motion. Moreover, neurons like those that normally link the inner-ear cells to the brain had also developed in their cell culture system, and were connected to the hair cells.

Hashino thinks that additional research is needed to determine how to derived inner-ear cells involved in auditory sensation might be made from stem cells, and how such techniques might be adapted to make human inner ear cells.

Using Bone Marrow Stem Cells to Reprogram Neurons and Regenerate the Retina


Spanish researchers from the Center for Genomic Regulation (CGR) have regenerated the retina in mice by reprogramming neurons with bone marrow stem cells.

Cell reprogramming normally uses genetic engineering techniques that introduces genes into cells that push them into another cell fate without taking them through an embryonic-like state. One strategy for reprogramming cells fuses those cells with other cells that express genes that drive the fused cell into a different cell fate.

Pia Cosma and her team have used cell fusion to reprogram retinal neurons in mice. The mechanism consisted of introducing bone marrow stem cells into the damaged retina. The transplanted stem cells fused with existing retinal neurons, which conveyed to these retinal neurons the ability to regenerate the retina.

“For the first time we have managed to regenerate the retina and reprogram its neurons through in vivo cell fusion. We have identified a signaling pathway that, once activated, allows the neurons to be reprogrammed through their fusion with bone marrow cells,” said Pia Cosma, who is the head of the Reprogramming and Regeneration group at the CGR and ICREA (Institució Catalana de Recerca i Estudis Avançats) research professor.

Daniela Sanges, first author or the work and postdoctoral researcher in Pia Cosma’s laboratory, said, “This discovery is important not only because of the possible medical applications for retinal regeneration but also for the possible regeneration of other nervous tissues.”

The study demonstrates that the regeneration of nervous tissue by means of cell fusion is possible in mammals and describes this new technique as a potential mechanism for the regeneration of more complex nervous tissue.

This research is in the very early stages but already there are laboratories interested in being able to continue the work and take it to a more applied level.

Daniela Sanges, Neus Romo, Giacoma Simonte, Umberto Di Vicino, Ariadna Diaz Tahoces, Eduardo Fernández, Maria Pia Cosma. Wnt/β-Catenin Signaling Triggers Neuron Reprogramming and Regeneration in the Mouse Retina . Cell Reports – 25 July 2013 (Vol. 4, Issue 2, pp. 271-286)

Learning About Limb Regeneration from Fingernails


Fingertip amputation in mammals results in regeneration of the nail, the attendant nerves, and even the damaged bone. Humans can also regenerate a fingertip in as little as two months. This seemingly simple regenerative event remains poorly understood.

However work from the NYU Langone Medical Center has provided a greater understanding of this somewhat opaque event. By using genetically engineered mice, the NYU team was able to elucidate a chain of events that unfolds after finger amputation.

This may seem like a terribly small thing, but understanding the regeneration of a finger tip can lead to augmentation of this process so that eventually entire fingers can be regenerated and even entire limbs.

“Everyone knows that fingernails keep growing, but no one really knows why,” said lead author Mayumi Ito, assistant professor of dermatology in the Ronald O. Perelman Department of Dermatology at NYU School of Medicine. Also, the connection between the regenerative ability of the bone and surrounding to the growth and/or regeneration of the nail is equally poorly understood.

Ito and others have discovered an important clue, and that is a population of stem cells in the nail matrix. The nail matrix contains a bed that is rich in nerve termini and blood vessels that stimulate nail growth.

To review the structure of the nail, the nail plate consists of the hard visible part of the nail. The nail plate is composed of hard, keratinized, squamous cells that are loosely attached to the germinal matrix but strongly attached to the sterile matrix. The nail matrix is the tissue that a nail [nail plate] protects. It lies beneath the nail and contains nerves, lymph and blood vessels. The matrix is responsible for producing cells that become the nail plate. It has two parts: the sterile matrix and the germinal matrix.

anatomy_nail

The stem cell population lies within the nail matrix, and these stem cells depend on a family of signaling proteins known as “Wnt” proteins. Wnt proteins are secreted glycoproteins that bind to Frizzled receptors. The Frizzled receptors bind Wnts and cause the polymerization of the Dsh or Disheveled protein at the cell membrane, and this inhibits GSK-3, a protein kinase. GSK-3 places phosphate groups on beta-catenin, and this marks beta-catenin for destruction. Once GSK-3 is inhibited, beta-catenin levels increase and it moves into the nucleus where it combines with Tcf proteins to activate the transcription of target genes.

Wnt signaling pathway

Wnt proteins play a crucial role in hair and tissue regeneration, and now they appear to play a truly vital role in bone regeneration as well.

Ito recounted her experiments: “When we blocked the Wnt-signaling pathway in mice with amputate fingertips, the nail and bone did not grow back as they normally would.”

a, Experimental scheme. Three-week-old K14–Cre-ER;β-catenin conditional knockout (cKO) mice and littermates were treated with Tam for 7 days immediately after distal-tip amputation, and analysed at the indicated time points. b, Whole-mount transparent specimen of a regenerated digit 5 weeks after amputation. c, Whole-mount alizarin red analysis. d, Trichrome staining. e, f, Quantification analyses of the nail length and the bone length 5 weeks after amputation. g, Analysis of Wnt activation in regenerating nail epithelium using TOPGAL at 3 weeks after amputation. The lower panel is a schematic illustration of the upper panel. h, Quantitative analyses of the distance between nerve tip and wound epidermis and the innervations at 3 weeks after amputation. i, Proliferation analyses by Ki67 immunohystochemistry at 3 weeks after amputation. Red bars in h, right panel, indicate the averages. Dashed lines indicate the border between nail epithelium and connective tissue. Asterisks in part h, bottom panel, indicate autofluorescence from blood cells. Data are presented as the mean ± s.d. Scale bars, 500 μm (b–d); and 100 μm (h).
a, Experimental scheme. Three-week-old K14–Cre-ER;β-catenin conditional knockout (cKO) mice and littermates were treated with Tam for 7 days immediately after distal-tip amputation, and analysed at the indicated time points. b, Whole-mount transparent specimen of a regenerated digit 5 weeks after amputation. c, Whole-mount alizarin red analysis. d, Trichrome staining. e, f, Quantification analyses of the nail length and the bone length 5 weeks after amputation. g, Analysis of Wnt activation in regenerating nail epithelium using TOPGAL at 3 weeks after amputation. The lower panel is a schematic illustration of the upper panel. h, Quantitative analyses of the distance between nerve tip and wound epidermis and the innervations at 3 weeks after amputation. i, Proliferation analyses by Ki67 immunohystochemistry at 3 weeks after amputation. Red bars in h, right panel, indicate the averages. Dashed lines indicate the border between nail epithelium and connective tissue. Asterisks in part h, bottom panel, indicate autofluorescence from blood cells. Data are presented as the mean ± s.d. Scale bars, 500 μm (b–d); and 100 μm (h).

When Ito and her team manipulated the Wnt pathway they discovered that they could stimulate regeneration of bone and tissue just beyond the fingernail. “Amputations of this magnitude ordinarily do not grow back,” noted Ito.

a, Experimental scheme. Three-week-old K14–Cre-ER;β-cateninfl/ex3 (mutant) mice and littermate controls were treated with Tam for 7 days starting from 2 weeks after amputation at the proximal level. b–f, Immunohistochemical analyses with indicated markers 3 weeks after amputation. g, Whole-mount transparent specimen of regenerated digits. h, Whole-mount alizarin red analysis. i, j, Quantification analyses of the nail (i) and bone length (j) 4 weeks after amputation. Red bars in d show the averages. Arrowheads in c and e, bottom panels, indicate TCF1− proximal matrix and FGF2+ epidermis, respectively. Arrowheads in d point to nerves. Fine dotted lines in b and h indicate the amputation plane. Dashed lines indicate the border between epidermis and connective tissue. Quantified data are presented as the mean ± s.d. Scale bars, 100 μm (b–f); and 500 μm (g and h).
a, Experimental scheme. Three-week-old K14–Cre-ER;β-cateninfl/ex3 (mutant) mice and littermate controls were treated with Tam for 7 days starting from 2 weeks after amputation at the proximal level. b–f, Immunohistochemical analyses with indicated markers 3 weeks after amputation. g, Whole-mount transparent specimen of regenerated digits. h, Whole-mount alizarin red analysis. i, j, Quantification analyses of the nail (i) and bone length (j) 4 weeks after amputation. Red bars in d show the averages. Arrowheads in c and e, bottom panels, indicate TCF1− proximal matrix and FGF2+ epidermis, respectively. Arrowheads in d point to nerves. Fine dotted lines in b and h indicate the amputation plane. Dashed lines indicate the border between epidermis and connective tissue. Quantified data are presented as the mean ± s.d. Scale bars, 100 μm (b–f); and 500 μm (g and h).

These findings suggest that Wnt signaling is essential for fingertip regeneration, and indicate that the way to develop therapies for regenerating lost limbs is to more deeply understand Wnt signaling and its role in limb regeneration. Some 1.7 million people in the US alone live with amputations. Therefore, research of this type could prove remarkably useful.

Developmental Regression: Making Placental Cells from Embryonic Stem Cells


A research group from Copenhagen, Denmark has discovered a way to make placental cells from embryonic stem cells. In order to do this, the embryonic stem cells must be developmentally regressed so that they can become wither placenta-making cells rather than inner cell mass cells.

This study is significant for two reasons. First of all, it was thought to be impossible to make placental cells from embryonic stem cells because embryonic stem cells (ESCs) are derived from the inner cell mass cells of 4-5-day old human blastocysts. These early embryos begin as single-celled embryos that divide to form 12-16-cell embryos that undergo compaction. At this time, the cells on the outside become trophoblast cells, which will form the trophectoderm and form the placenta and the cells on the inside will form the inner cell mass, which will form the embryo proper and a few extraembryonic structures. Since ESCs are derived from inner cell mass cells that have been isolated and successfully cultured, they have already committed to a cell fate that is not placental. Therefore, to differentiate ESCs into placental cells would require that ESCs developmentally regress, which is very difficult to do in culture.

Secondly, if this could be achieved, several placental abnormalities could be more easily investigated, For example, pre-eclampsia is a very serious prenatal condition that is potentially fatal to the mother, and is linked to abnormalities of the placenta. Studying a condition such as pre-eclampsia in a culture system would definitely be a boon to gynecological research.

Because human ESCs can express genes that are characteristic of trophoblast cells if they are treated with a growth factor called Bone Morphogen Protein 4 (BMP4), it seems possible to make placental cells from them (see Xu R.H., Chen X., Li D.S., Li R., Addicks G.C., Glennon C., Zwaka T.P., Thomson J.A. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 2002;20:1261–1264, and Xu RH. Methods Mol Med. 2006;121:189-202). However, a study by Andreia S. Bernardo and others from the laboratory of Roger Pedersen at the Cambridge Stem Cell Institute strongly suggested that BMP4 treatment, even in the absence of FGF signaling (another growth factor that has to be absent for BMP4 to induce trophoblast-like gene expression from ESCs) the particular genes induced by BMP4 are not exclusive to trophoblast cells and more closely resemble mesodermal gene profiles (see AS Bernardo, et al., Cell Stem Cell. 2011 Aug 5;9(2):144-55).

Into the fray of this debate comes a paper by stem cells scientists at the Danish Stem Cell Center at the University of Copenhagen that shows that it is possible to rewind the developmental state of ESCs.

In this paper, Josh Brickman and his team discovered that if they maintained mouse ESCs under specific conditions, they could cause the cells to regress into very early pre-blastocyst embryonic cells that can form trophoblast cells or ICM cells.

“It was a very exciting moment when we tested the theory, said Brinkman. “We found that not only can we make adult cells but also placenta, in fact we got precursors of placenta, yolk sac as well as embryo from just one cell.”

“This new discovery is crucial for the basic understanding of the nature of embryonic stem cells and could provide a way to model the development of the organism as a whole, rather than just the embryonic portion,” said Sophie Morgani, graduate student and first author of this paper. “In this way we may gain greater insight into conditions where extraembryonic development is impaired, as in the case of miscarriages.”

To de-differentiate the ESCs, Brinkman and his colleagues grew them in a solution called “2i.”  This 2i culture medium contained inhibitors of MEK and GSK3.  MEK is a protein kinase that is a central participant in the “MAP kinase signaling pathway, which is a signaling pathway that is central to cell growth and survival.  This particular signaling pathway is the target of the anthrax toxin, which illustrates its importance,  GSK3 stands for “glycogen synthase kinase 3,” which is a signaling protein in the Wnt pathway.

When the mouse ESCs were grown in 2i medium they expressed genes normally found only in pre-blastocyst embryos (Hex, for example).  Therefore, the 2i medium directs mouse ESCs to de-differentiate.  When ESCs grown in 2i were implanted into mouse embryos, they divided and differentiated into cells that were found in placental and embryonic fates.  This strongly argues that the ESCs grown in 2i became pre-blastocyst embryonic cells.  When the ESCs grown in 2i were also grown with LIF, which stands for “leukemia inhibitory factor” (LIF is a protein required for the maintenance of mouse ESCs in culture), the 2i cells were maintained in culture and grew while maintaining their pre-blastocyst status.  These cells differentiated into placental cells, embryonic or fetal cells.  Essentially, the 2i-cultured cells when from being pluripotent to being “totipotent,” or able to form ALL cell types in the embryo, fetus, or the adult.

ESC de-differentiation in totipotence

“In our study we have been able to see the full picture unifying LIF’s functions: what LIF really does, is to support the very early embryo state, where the cells can make both embryonic cells and placenta. This fits with LIFs’ role in supporting implantation,” said Brinkman.

This study definitively shows that ESCs are NOT embryos.  ESCs can regress in their development but embryos develop forward, becoming more committed as they develop and more restricted in the cell fates they can form.  This should effectively put the nail in the coffin of Lee Silver’s argument against Robert P. George that embryonic stem cells are embryos.  They are definitely and unequivocally, since embryos do NOT develop in reverse, but ESCs can and do.

Robert P. George argues that early human embryos, like the kind used to make ESCs are very young  members of the human race and deserve, at the minimum, the right not to be harmed.  Silver counters that George’s argument is inconsistent because George would not extend the same right to an ESC cell line, which is the same as an embryo.  His reasoning is that mouse ESCs can be transplanted into other mouse embryos that have four copies of each chromosome.  The messed up mouse embryo will make the placenta and the ESCs will make the inner cell mass and the mouse will develop and even come to term.  This is called tetraploid rescue, and Silver thinks that this procedure is a minor manipulation, but that it shows that ESCs are functionally the same as embryos.

I find Silver’s argument wanting on just about all fronts.  This is not a minor manipulation.  The tetraploid embryo is bound for certain death, but the implanted ESCs use the developmental context of the tetraploid embryo to find their place in it and make the inner cell mass.  The ESCs do not do it all on their own, but instead work with the tetraploid embryo in a complex developmental give-and-take to make an embryo with the placenta from one animal and the embryo proper from another.

Thus Silver’s first argument does not demonstrate what he says it does.  All it demonstrates is that ESCs can contribute to an embryo, which is something we already knew and expected.  This new data completes blows Silver’s assertion out of the water, since ESCs can take developmental steps backward and embryos by their very nature and programming, do not.  Thus these two entities are distinct entities and are not identical.  The early embryo is a very young human person, full stop.  We should stop dismembering them in laboratories just to stem our scientific curiosity.

Alligator Stem Cells and Tooth Replacement


Mammals usually have one set of baby teeth (also known as milk teeth) and after those are lost, we have one set of adult teeth and these are not replaced if they are lost. This condition is called “monophyodont.” Reptiles and sharks, however constantly replace their teeth. This condition is called “polyphyodont.” Alligators and crocodiles are among one group of reptiles that replace their teeth throughout their lives, and because the development of these creatures has been studied to some extent, it is known that the ability of these creatures to replace their teeth on a regular basis results from a resident stem cell population. Studying that stem cell population more closely might provide clues for tooth replacement in humans.

American Alligator
American Alligator

A research team led by scientists at the Keck School of Medicine professor of pathology Cheng-Ming Chuong at the University of Southern California. Dr. Chuong and his collaborators from around the world have identified unique cellular and molecular mechanisms behind tooth renewals in American alligators.

Chuong explained, “Humans naturally have only two sets of teeth – baby teeth and adult teeth. Ultimately, we want to identify stem cells that can be used as a resource to stimulate tooth renewal in adult humans who have lost teeth. But, to do that, we must first understand how they renew in other animals and why they stop in people.”

Even though humans cannot replace their adult teeth, a tissue called the dental lamina remains, which is known to be crucial for tooth development.

Why are alligators potentially a good model system for tooth replacement in mammals? First author of this study, Ping Wu, explained it this way, “Alligator teeth are implanted in sockets of the dental bone, like human teeth. They have 80 teeth, each of which can be replaced up to 50 times over their lifetime, making them the ideal model for comparison to human teeth.”

Through the use of microscopic imaging techniques, Chuong and others found that each alligator tooth is a complex unit of three components: a functional tooth, a replacement tooth, and the dental lamina, all other which are at different developmental stages.

The tooth units are built to enable a smooth transition from dislodgement of the functional, mature tooth to replacement with a new tooth. Further imaging studies strongly suggested that the dental lamina contains a stem cell population from which new replacement teeth develop.

“Stem cells divide more slowly than other cells, said co-author Randall B. Widelitz, who serves as an associate professor of pathology at USC. Widelitz continued, “The cells in the alligator’s dental lamina behaved like we would expect stem cells to behave. In the future, we hope to isolate those cells from the dental lamina to see whether we can use them to regenerate teeth in the lab.”

The researchers also intend to learn what molecular networks are involved in repetitive renewal and hope to apply the principles to regenerative medicine in the future.

The authors also noted that novel cellular mechanisms are used during the development of the tooth unit. Also, unique molecular signaling speeds growth of replacement teeth when functional teeth are lost.

See P. Wu PNAS 2013; DOI: 10.1073/pnas.12132110.