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.

Results of STAP Cell Paper Questioned

Reports of Stimulus-Triggered Acquisition of Pluripotency or STAP cells has rocked the stem cell world. If adult cells can be converted into pluripotent stem cells so easily, then perhaps personalized, custom stem cells for each patient are just around the corner.

However, the RIKEN institute, which was heavily involved in the research that brought STAP cells to the world has now opened an investigation into this research, since leading scientists have voiced discrepancies about some of the figures in the paper and others have failed to reproduce the results in the paper.

Last week, Friday (February 14, 2014, spokespersons for the RIKEN centre, which is in Kobe, Japan, announced that the institute is looking into alleged irregularities in the work of biologist Haruko Obokata, who works at the institution. Obokata was the lead author listed on two papers that were published in the international journal Nature. These papers (Obokata, H. et al. Nature 505, 641–647 (2014), and Obokata, H. et al. Nature 505, 676–680 (2014) described a rather simple protocol for deriving pluripotent stem cells from adult mouse cells by exposing them to acidic conditions, other types of stresses such as physical pressure on cell membranes. The cells, according to these two publications, had virtually all the characteristics of mouse embryonic stem cells, but had the added ability to form placental structures, which is an ability that embryonic stem cells do not have. The investigation initiated by the RIKEN centre comes at the behest of scientists who have noticed that some of the images used in these papers might have been duplicated from other papers. Also, several scientists have notes that they have been unable, to date, to replicate her results.

These concerns came to a head last week when the science blog PubPeer, and others, noted some problems in these two Nature papers and in an earlier paper from 2011. Obokata is also the first author of this 2011 paper (Obokata, H. et al. Tissue Eng. Part A 17, 607–15 (2011), and this paper contains a figure that seems to have been used for one of the figures in the 2014 paper. Also, there is another figure duplication.

Harvard Medical School anesthesiologist Charles Vacanti who was the corresponding author of one of the Nature papers has said that has learned last week about a data mix up in the paper and has contacted the journal to request a correction. “It certainly appears to have been an honest mistake [that] did not affect any of the data, the conclusions or any other component of the paper,” says Vacanti. Note that Vacanti is a co-author on both papers and a corresponding author on one of them.

In the other paper, Obokata serves as the corresponding author and this paper contains an image of two placentas that appear to be very similar. Teruhiko Wakayama works at Yamanashi University in Yamanashi prefecture, and he is a co-author on both of these papers. According to Wakayama, he sent more than a hundred images to Obokata and suggests that there was confusion over which to use. He says he is now looking into the problem.

Additionally, ten prominent stem-cell scientists have been unable to repeat Obokata’s results. One particular blog listed eight failures from scientists in the field. However, most of those attempts did not use the same types of cells that Obokata used.

Some scientists think that this could simply be a case of experienced scientists working with a system that they know very well and can manipulate easily, unlike outsiders to this same laboratory. For example, Qi Zhou, a cloning expert at the Institute of Zoology in Beijing, who says most of his mouse cells died after treatment with acid, says that “setting up the system is tricky; as an easy experiment in an experienced lab can be extremely difficult to others, I won’t comment on the authenticity of the work only based on the reproducibility of the technique in my lab,” says Zhou.

However, others are more deeply concerned. For example, Jacob Hanna, a stem-cell biologist at the Weizmann Institute of Science in Rehovot, Israel, however, says “we should all be cautious not to persecute novel findings” but that he is “extremely concerned and sceptical”. He plans to try for about two months before giving up.

It could be that the protocol is far more complicated that thought. For example, even Wakayama has been having trouble reproducing the results. To be sure, Wakayama and a student of his were able to replicate the experiment independently before publication, but only after being coached by Obokata. But since he moved to Yamanashi, he has had no luck. “It looks like an easy technique — just add acid — but it’s not that easy,” he says.

Wakayama says that his own success in replicating Obokata’s results has convinced him that her technique works. “I did it and found it myself,” he says. “I know the results are absolutely true.”

Clearly one way to clear this up is for the authors of this groundbreaking paper to publish a detailed protocol on how to make STAP cells. This should clear up any problems with the papers. Vacanti says he has had no problem repeating the experiment and says he will let Obokata supply the protocol “to avoid any potential for variation that could lead to confusion”.

The journal Nature has said that there are aware of the problems with the papers and looking into the matter.

For now, that’s where the issue sits. Frustrating I know, but until we know more we will have to just “wait and see.”

Human STAP cells – Troubling Possibilities

Soon after the publication of this paper that adult mouse cells could be reprogrammed into embryonic-like stem cells simply by exposing them to acidic environments or other stresses , Charles Vacanti at Harvard Medical School has reported that he and his colleagues have demonstrated that this procedure works with human cells.

STAP cells or stimulus-triggered acquisition of pluripotency cells were derived by Vacanti and his Japanese collaborators last year. These new findings show that adult cells can be reprogrammed into embryonic-like stem cells without genetic engineering. However, this technique worked well in mouse cells, but it was not clear that it would work with human adult cells.

Vacanti and others shocked the world when they published their paper in the journal Nature earlier this year when they announced that adult cells in mice could be reprogrammed through exposure to stresses and proper culture conditions.

Now Vacanti has made good on his promise to test his protocol on human adult cells. In the photo below, provided by Vacanti, human adult cells were reprogrammed to a pluripotent state by exposing them to stresses, followed by growth in culture under specific conditions.

Human STAP cells
Human STAP cells

“If they can do this in human cells, it changes everything, said Robert Lanza of Advanced Cell Technologies in Marlborough, Massachusetts. Such a procedure promises cheaper, faster, and potentially more flexible cells for regenerative medicine, cancer therapy and cell and tissue cloning.

Vacanti and his colleagues say they have taken human fibroblast cells and tested several environmental stressors on them to recreate human STAP cells. He will not presently disclose which particular stressors were applied, he says the resulting cells appear similar in form to the mouse STAP cells. His team is in the process of testing to see just how stem-cell-like these cells are.

According to Vacanti, the human cells took about a week to resemble STAP cells, and formed spherical clusters just like their mouse counterparts. Vacanti and his Harvard colleague Koji Kojima emphasized that these results are only preliminary and further analysis and validation is required.

Bioethical problems potentially emerge with STAP cells despite their obvious potential. The mouse cells that were derived and characterized by Vacanti’s group and his collaborators were capable of making placenta as well as adult cell types. This is different from embryonic stem cells, which can potentially form all adult cell types, but typically do not form placenta. Embryonic stem cells, therefore, are pluripotent, which means that they can form all adult cell types. However, the mouse STAP cells can form all embryonic and adult cell types and are, therefore, totipotent. Mouse STAP cells could form an entirely new mouse. While it is now clear if human STAP cells, if they in fact exist, have this capability, but if they do, they could potentially lead to human cloning.

Sally Cowley, who heads the James Martin Stem Cell Facility at the University of Oxford, said of Vacanti’s present experiments: “Even if these are STAP cells they may not necessarily have the same potential as mouse ones – they may not have the totipotency – which is one of the most interesting features of the mouse cells.”

However the only cells known to be naturally totipotent are in embryos that have only undergone the first couple of cell divisions immediately after fertilization. According to Cowley, any research that utilizes totipotent cells would have to be under very strict regulatory surveillance. “It would actually be ideal if the human cells could be pluripotent and not totipotent – it would make everyone’s life a lot easier,” she opined.

Cowley continued: “However, the whole idea that adult cells are so plastic is incredibly fascinating,” she says. “Using stem cells has been technically incredibly challenging up to now and if this is feasible in human cells it would make working with them cheaper, faster and technically a lot more feasible.”

This is all true, but Robert Lanza from Advanced Cell Technology in Marlborough, Massachusetts, a scientist with whom I have often deeply disagreed, noted: “The word totipotent brings up all kinds of issues,” says Robert Lanza of Advanced Cell Technology in Marlborough, Massachusetts. “If these cells are truly totipotent, and they are reproducible in humans then they can implant in a uterus and have the potential to be turned into a human being. At that point you’re entering into a right-to-life quagmire”

A quagmire indeed, for Vacanti has already talked about using these STAP cells to clone human embryos. Think of it: the creation of very young human beings just for the purpose of ripping them apart and using their cells for research or medicine. Would we allow this if the embryo were older; say the age of a toddler? No we would rightly condemn it as murder, but because the embryo is very young, that somehow counts against it. This is little more than morally grading the embryo according to astrology.

Therefore, whole Vacanti’s experiments are exciting and novel, they hold chilling possibilities. Lanza is right, and it is doubtful that scientists would show the same deference or sensitivities to the moral exigencies he has shown.

Growing Intestinal Stem Cells

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

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

paneth cells

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

Langer said, “This opens the door to doing all kinds of thing, ranging from someday engineering a new gut for patients with intestinal diseases to doing drug screening for safety and efficacy. It’s really the first time this has been done.”

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

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


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

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

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

Karp said, “If we had ways of performing high-throughput screens of large numbers of these very specific cell types, we could potentially identify new targets and develop completely new drugs for diseases ranging from inflammatory bowel disease to diabetes.”

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

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

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

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

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

Ramesh Shivdasani from Harvard Medical School and Dana-Farber Cancer Institute would like to use these cells to investigate what gives stem cells their ability to self-renew and differentiate into other cell types.  “There are a lot of things we don’t know about stem cells,” said Shivdasani.  “Without access to large quantities of these cells, it’s very difficult to do any experiments.  This opens the door to a systematic, incisive, reliable way of interrogating intestinal stem cell biology.”

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

Circulating Factor Rejuvenates the Hearts of Older Mice

Two researchers associated with the Harvard Stem Cell Institute, one of whom is a practicing cardiologist at Brigham and Women’s Hospital, and a cell biologist have identified a protein in the blood of mice and humans decreases during aging and may prove to be the first effective treatment for the form of age-related heart failure that affects millions of Americans.

Growth Differentiation Factor 11 or GDF11 circulates throughout the bloodstream of humans and mice. Injections of GDF11 into old mice that have developed thickened heart walls in a manner similar to aging humans, the hearts were reduced in size and thickness, and resembled the healthy hearts of younger mice.

Even more important than the implications for the treatment of diastolic heart failure, the finding by Richard T. Lee, from Harvard Medical School and Amy Wagers, who is a professor in Harvard’s Department of Stem Cell and Regenerative Medicine, might rewrite our understanding of aging. These findings were published in the prestigious, international journal Cell.

“The most common form of heart failure [in the elderly] is actually a form that’s not caused by heart attacks but is very much related to the heart aging,” said Lee, who, like Wagers, is a principal faculty member at HSCI.

“In this study, we were able to show that a protein that circulates in the blood is related to this aging process, and if we gave older mice this protein, we could reverse the heart aging in a very short period of time,” Lee said. “We are very excited about it because it opens a new window on the most common form of heart failure.”
He added, “This is the coolest thing I’ve ever been a part of.”

Doug Melton, HSCI co-director, called the discovery “huge. It’s going to change the way we think about aging.”

Lee, who practices medicine as a cardiologist, noted that he has approximately 300 patients, and of these “I think I have about 20 who are suffering from this type of heart failure, which we sometimes call diastolic heart failure,” said Lee. “They come into the hospital, have a lot of fluid taken off, then they’ll go home. Then they come back again. It’s really frustrating because we don’t have any drugs to treat this. We need to work as hard as we can to figure out if this discovery can be turned into a treatment for heart failure in our aging patients.”

The Lee and Wagers labs would like to move GDF-11 toward clinical trials. Lee predicts that they might be able to begin these studies in four to five years. However, they need to determine the other tissues that are affected by GDF11.

Wagers was a postdoctoral research fellow at Stanford University, where she learned how to work with the “parabiotic” mouse system. Parabiosis refers to two animals that share a common circulatory system. Wagers used this system to link the circulatory system of young mice with that of older mice. When she did that, she and her colleagues discovered that soluble factors in the blood of young animals have a rejuvenating effect on various tissues in older animals. In particular, the spinal cord and the musculature of the older animals showed marked improvements.

“As we age, there are many changes that occur in different parts of the body,” Wagers said, “and those changes are often associated with a decline in the function of our bodies. One of the interests of my laboratory is in understanding why this happens and whether it is an inevitable consequence of aging, or if it might be reversible.

“In this study, we compared young and old animals and identified a substance in the blood that is present at high levels when you’re young and lower levels when you’re old. We further found that when we supplemented the low levels of this substance that were present in old animals to the levels normally seen in youth, this could have a dramatic effect on the heart.

“It’s been observed for many, many years that when aging occurs it affects multiple body systems sort of in a semi-synchronous way,” Wagers said, “and this suggests that there may be some common signal that drives the body’s response to getting older. We hypothesized that this common signal might be a substance that was traveling in the bloodstream, because the bloodstream accesses organs throughout the body.”

“I think Amy and I started thinking about something like this almost five years ago,” said Lee, who added that he and Wagers were brought together by HSCI. “Without the Harvard Stem Cell Institute, this never would have happened,” he said.

Lee and Wagers conducted their first experiment about four years ago, and the results were startling, Lee said. “A fellow named Francesco Loffredo was examining the hearts of the aging mice. He came to me and said, ‘You don’t have to analyze it; you can see it with the naked eye.’ I couldn’t believe that, and I said ‘Go back, analyze it, and do it blinded.’ Then I looked at the hearts, and I could see he was correct,” Lee recalled.

“When we started these experiments, I actually was thinking that there would not be a response,” Wagers said. “We had been using similar kinds of approaches in other tissues, regenerative tissues, tissues that we know have the capacity to heal themselves after they’ve been injured. But the heart is not well known for doing that, and so I was quite convinced that there would be no response. When I saw the dramatic difference in heart size that was very apparent after this exposure of an old animal to young blood, it was very clear that we had to figure out what was going on,” she said.

“The blood is full of all kinds of things,” the biologist said, “and trying to narrow down what might be the responsible factor was going to be a big challenge. I think that’s where the collaboration was so wonderful; in that we could take advantage of the expertise in both of our laboratories to really home in on what might be the responsible substance.”

Lee explained, “We thought it was interesting right away, and we repeated it right away. But we had to show that this was not a blood pressure effect, that the young mice didn’t just cause the old mice to have lower blood pressure. We had to build a custom device to measure blood pressures off their tails. It took a year to do the analysis to show that it was not a blood pressure effect.

“After about 2½ years we were convinced, and said, ‘we really have to identify this factor.’ It took about six months to find something, and another year to be convinced that it was real,” Lee said. “We looked at lipids; we looked at metabolites. Then we set up a collaboration with a startup company in Colorado, called SomaLogic that had an interesting technology for analyzing factors in blood. And by working closely with SomaLogic, we found the likely factor.”

They discovered that at least one of the factors responsible for heart rejuvenation was GDF-11, “a member of a very important family of proteins called TGF-beta proteins, for transforming growth factor. There are around 35 members of the family,” Lee said. “Some have been very well studied, and this is one that is relatively obscure.”

The work was supported in part by HSCI, the National Institutes of Health, and the American Heart Association.

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.


“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.

Induced Pluripotent Stem Cell Transplant Claims Debunked

When this report first appeared, it seemed too good to be true and it turns out it probably was. Nobel Laureate Shinya Yamanaka at Kyoto University announced his remarkable discovery of induced pluripotent stem (iPS) cells in 2006. However, another Japanese researcher, Hisashi Moriguchi, made an even more earth-shaking claim earlier this year. Moriguchi, who was a visiting researcher at the University of Tokyo, claimed to have modified iPS technology to treat a person with terminal heart failure. The patient was allegedly surgically treated in February, 2012, according to a front-page article in the Japanese newspaper Yomiuri Shimbun. The article also said that the patient was healthy. If this was true, this would certainly be an earth-shaking result. An unidentified head of a Tokyo-based organization devoted to helping children with heart problems, told Yomiuri Shimbun, “I hope this therapy is realized in Japan as soon as possible.”

The Nippon News Network had posted a video of Moriguchi presenting his research at the New York Stem Cell Foundation, but they have since removed this video.

Unfortunately, once the journal Nature was altered to this report, they contacted Harvard Medical School and Massachusetts General Hospital (MGH), where Moriguchi claimed to have performed this work. Both institutions denied that Moriguchi had even done such a procedure. According David Cameron, a spokesperson for Harvard Medical School, “No clinical trials related to Dr Moriguchi’s work have been approved by institutional review boards at either Harvard University or MGH.” Likewise, the public affairs officer for MGH, Ryan Donovan, said “The work he is reporting was not done at MGH.”

There are other problems with Moriguchi’s work. Moriguchi reported that he had invented a method to reprogram cells using just two chemicals: a small molecule that inhibits a small RNA called “microRNA-145” and another molecule that binds the TGF-β receptor. However, a University of Tokyo stem-cell researcher, Hiromitsu Nakauchi, said that he has never “heard of success with that method.” Nakauchi even said that before this week he had never heard of Moriguchi.

Another bizarre claim made by Moriguchi was that he could differentiate iPS cells into heart muscle cells by utilizing a ‘supercooling’ method that he had invented. Nakauchi said that this was “another weird thing.”

Moriguchi never published his technique in a peer-reviewed journal, but in a book about advances in stem-cell research (see Moriguchi, H., Mihara, M., Sato, C. & Chung, R. T. in Embryonic Stem Cells — Recent Advances in Pluripotent Stem Cell-Based Regenerative Medicine (ed. Atwood, C.) 359–370 (InTech, 2011)). In this book, there are paragraphs copied almost verbatim from other papers. For example, a section under the heading “2.3 Western blotting” is identical to a passage from a 2007 paper by Yamanaka (see Takahashi, K. et al. Cell 131, 861–872 (2007)). Furthermore, section 2.1.1 describes human liver biopsies but the information in this section matches the number of patients and timing of specimen extractions described in an earlier article, but the name of the institution has been changed (see Thenappan, A. et al. Hepatology 51, 1373–1382 (2010)).

Nature contacted Moriguchi and he stood by his publication. He told Nature, “We are all doing similar things so it makes sense that we’d use similar words.” However, he did admit to using other papers “as reference.”

With respect to his reported supercooling technique, Moriguchi cited a paper of his own in Scientific Reports, which is published by the Nature Publishing Group. Nature, however, noted that this paper describes supercooling of human ovaries for preservation (Moriguchi, H., Zhang, Y., Mihara, M. & Sato, C. Sci. Rep. 2, 537 (2012)). The paper has nothing to do with the differentiation of iPS cells into cardiac cells. Moriguchi said that a journal referee had recommended that he leave the latter experiment out of the paper “because it’s basically the same technology”.

Moriguchi said that he did most of the contentious work himself, including safety research in pigs. However, the initial surgery and some of a further five similar procedures in other patients that took place from August onwards, and while, according to Moriguchi, other researchers were supposedly involved in some of these procedures, he would not provide any names.

Where did Moriguchi acquire the surgical expertise to perform these procedures? Moriguchi initially told Nature that earned a medical degree at the Tokyo Medical and Dental University, and that he learned surgery there. However, in his later conversation with Nature, Moriguchi said that he has a nursing degree from the institution and not a medical degree.

The University of Tokyo confirmed that Moriguchi held a position there from 2006 to 2009, during which he studied “medical economics” and “evaluation of clinical technologies.” Currently, he is a visiting researcher at the university, working in the laboratory of Makoto Mihara in the university hospital’s cosmetic-surgery section, where, according to a secretary, he “comes in once or twice a week.”

Moriguchi also claimed to have a laboratory at MGH and Harvard Medical School, but these institutions only confirmed that Moriguchi was a visiting fellow at MGH in 1999–2000, but he has not been associated with the hospital or the medical school since then.

Nature asked Moriguchi who had funded his iPS cell procedures and where they had been carried out, where his ethical review had taken place and which good manufacturing practice (GMP) facility had produced the necessary clinical-grade iPS cells, Moriguchi referred again to MGH and Harvard Medical School, but he could not name the head of the ethical review board or any contacts at the GMP facility.

Jerome Ritz, co-director of the Connell O’Reilly Cell Manipulation Core Facility at Harvard Medical School, told Nature, “We have not produced any iPS cells for any patients in our facility. I can’t imagine what other facility might have produced these cells.”

What do we have? We have a Japanese researcher who is a liar and who has as much of a problem telling the truth as Barak Obama. This clinical trial clearly never happened and Moriguchi should be banned from further stem cell work.

Unique Drug Responses of Stem Cells from Parkinson’s Patients

Induced pluripotent stem cells (iPSCs) are made from adult cells by means of genetic engineering techniques that introduce specific genes into the adult cell and force it to de-differentiate into an embryonic-like cell. This procedure might provide cells for therapeutic uses some day, but this technology must overcome the mutations introduced into these cells by this procedure and the tumors they can cause. Until then, iPSCs will remain off-limits as therapeutic tools.

That does not disqualify iPSCs as tools for research and even therapeutic investigation. This present paper that comes from a collaborative effort led by Ole Isacson, professor of neurology at McLean Hospital and Harvard Medical School in Boston, uses this very strategy to examine the response of patients with particular forms of Parkinson’s disease to various drugs.

Parkinson’s disease is a progressive, insidious disease that affects a portion of the brain called the midbrain. Within the midbrain is a black body called the substantia nigra, which is Latin for “black stuff.” The substantia nigra is rich in neurons that release a neurotransmitter called “dopamine.”

First of all, to review, neurotransmitters are chemicals that neurons (the cells that make and transmit nerve impulses to other neurons in the brain) use to talk to each other. Neurotransmitters bind to the surfaces of nearby neurons and initiate the production of a nerve impulse. If the neuron receives enough neurotransmitter, it will generate a nerve impulse. Neurons typically can only respond to particular neurotransmitters. The neurotransmitters to which they respond elicit particular responses from them.

Parkinson’s disease results from the death of dopamine-releasing neurons in the midbrain. These neurons connect to cells of the “striatum.” The striatum is responsible for balance, movement control, and walking. Dopamine, produced in the substantia nigra, passes messages between the striatum and the substantia nigra, and when the cells of the substantia nigra deteriorate, which is the case of Parkinson’s disease, there is a corresponding decrease in the amount of dopamine produced between these cells. The decreased levels of dopamine cause the neurons of the striatum to fire uncontrollably, and this prevents the patient from properly controlling their direct motor functions.

Most of the cases of Parkinson’s disease are spontaneous and have no apparent cause. However, there are several types of inherited forms of Parkinson’s and mutations in approximately 17 different genes are associated with inherited forms of Parkinson’s disease.  Of these, only nine have been studied in any detail.  Nevertheless, two genes in particular are important in this paper.

Isacson found two Parkinson’s patients with inherited forms of the disease.  One of then had a mutation in the LRRK2 (Leucine-rich repeat kinase-2) gene, which encodes the Dardarin protein and is intimately involved in the onset of Parkinson’s disease.  The other had a mutation in the PINK1 gene (PTEN induced putative kinase 1), which encodes a protein known to enter mitochondria (the powerhouses of the cell).  Isacson used cells from each patient to make iPSCs.  He also used additional patients, and he had a total of 3 patients with mutations in LRRK2 and two with mutations in PINK1.

Because mutations in LRRK2 and PINK1 are thought to interfere with the function of mitochondria in neurons, Isacson examined the mitochondria of these patient-specific iPSCs.  When compared to mitochondria from volunteers without Parkinson’s disease, Isacson found that the Parkinson’s patient-specific iPSCs were much more susceptible to damage after exposure to toxins.  Thus, the mitochondria of these patient-specific iPSCs were certainly much more fragile than normal mitochondria.

Could this mitochondrial fragility be ameliorated with medicines?  Isacson tested the ability of particular substance to mitigate this condition in the patient-specific iPSCs.  A supplement called Q10, which is known to aid mitochondrial function was administered the to Parkinson’s patient-specific iPSCs, was given to the cells, and all cells were prevented from experiencing mitochondrial damage after exposure to toxins.  However, when a different drug called rapamycin was administered, the results were very different.  Rapamycin diminishes the immune response of an organism, and therefore, it can spare weak cells from being cleared by the immune system.  Rapamycin prevented damage in the cells with mutations in LRRK2, but not those with mutations in PINK1.

This paper shows how iPSC-based research can lead to information that can fashion personal treatments for each patient.  Even though this work focused on Parkinson’s disease, there are many other diseases that could benefit from iPSC-based research.