Potential Marker Found for Stem Cell Population in the Inner Ear

Hearing loss is common as we get older. Presbycusis or age-related hearing loss results from the progressive death of sensory cells in the cochlea. Wouldn’t it be great if a stem cell population in the inner ear replaced these dying auditory sensory cells?

As it turns out, a stem cell population might exist in the mammalian inner ear and researchers from the UC Davis Comprehensive Cancer Center have identified a polysialylated glycoprotein that regulates neurodevelopment and is on the surface of cells in the adult inner ear. This glycoprotein acts as a marker of early cells in the inner ear and allows researchers to identify immature cells in the inner ear. This discovery was published in the journal Biochemical and Biophysical Research Communications and potentially opens the door to developing stem cell replacement treatments in the inner ear to treat certain hearing disorders.

“Hearing loss is a complex process and is usually regarded as irreversible,” said Frederic A. Troy II, principal investigator of the study from the UC Davis Comprehensive Cancer Center. “Finding this molecule in the inner ear that is known to be associated with early development may change that view.”

The existence of a marker for immature stem cells could make it possible to isolate neural stem cells from the adult inner ear in those people who suffer from hearing loss, culture and expand these cells in the laboratory, and then re-introduce them back into the inner ear as functioning neurons. These implanted cells might recolonize and establish themselves and improve hearing.

During development, certain glycoproteins (carbohydrate-protein linked molecules) are expressed on cell surfaces and serve critical functions essential to the normal growth and organization of the nervous system. One member of the class of cell-surface glycoproteins is an unusual molecule called polysialic acid or polySia. The large size of polySia allows it to fill spaces between cells and its strong electric charge repels other molecules. Therefore, polySia prevents cells from adhering or attaching to one another and thereby promotes cell movement to other areas.

Neural cell adhesion molecules (NCAMs) are also expressed on neuronal cell surfaces. As their name suggests, NCAMs help cells stick together and stay put. However, when NCAMs become modified with polysialic acid (or becomes “polysialylated”), the cells no longer adhere to other cells and are induced to migrate to new areas. Re-expression of the “anti-adhesive” polySia glycan on the surface of many adult human cancer cells facilitates their detachment, which enhances their metastatic spread.

Neural stem cells with these polySia-NCAMs on their cell surfaces play very important roles during embryonic development because these cells are able to travel throughout the body and differentiate into specialized cells. During adulthood, neural stem cells with polySia-NCAMs may migrate to injured areas and promote healing.

“The landscape of the cell surface of developing cells is decorated with a bewildering array of informational-rich sugar-protein molecules of which polysialylated NCAMs are of chief importance,” explained Troy. “During the life of a cell, these surface molecules are critical to cellular proliferation, self-renewal, differentiation and survival—essential processes for normal embryonic development and tissue regeneration in adults.”

It was already well-known that polySia-NCAM-expressing cells exist in the central nervous system, but Troy’s study is the first to document that they are also in the peripheral nervous system, and specifically in the spiral ganglia, those cluster of nerve cells in the inner ear that are essential to hearing.

Working with adult cells isolated from the inner ear spiral ganglia of guinea pigs, Troy and his team showed that these spiral ganglia cells expressed both polySia and NCAM. The polySia component was abundantly present on neural stem cells but was markedly reduced on mature cells. This implies that the polySia-NCAM complex is present on immature cells and can serve as a biomarker to identify these immature cells.

“Finding polySia-NCAM—a functional biomarker that modulates neuronal differentiation—on adult inner ear neural stem cells after differentiation gives researchers a ‘handle’ to identify and isolate these cells from among the many cells taken from a patient,” said Jan Nolta, director of the UC Davis Stem Cell Program and the university’s Institute for Regenerative Cures. “This discovery will enhance research into spiral ganglion neurons and may bring treatments closer to patients with hearing deficits.”

Lead author Park Kyoung Ho, a professor at Catholic University College of Medicine in Seoul, Korea, initiated the research for this article while on sabbatical leave in Troy’s laboratory at UC Davis. With his colleague and co-author, Yeo Sang Won, he is now planning clinical trials, based on the findings, in Korea with individuals who suffer from hearing disorders.

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).

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.

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

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.

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.

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.

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

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

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

LY411575

“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.

Embryonic Stem Cell-Derived Nerve Cells Restore Hearing to Deaf Animals

In a remarkable study, a research team from the University of Sheffield in England has improved the hearing of deaf animals by using embryonic stem cells. This result should certainly give new hope to those who suffer from hearing disorders.

In this study, an uncommon for of deafness that affects perhaps less than 1% and no more than 15% of all hearing-impaired patients, was treated. Even though this treatment would not benefit all cases of hearing impairment, the strategy developed in this study could be expanded to apply to other cases of deafness. Since these results are strictly pre-clinical in nature, it will be years before human patients might benefit from them.

This work used gerbils are a model system and the results were reported in the international journal Nature. The research team was led by Dr. Marcelo Rivolta and the scientists in his laboratory.

To induce deafness in the gerbils, the scientists ablated (killed off) those nerve cells that transmit auditory information from the ear to the brain. The nerve cells are called “spiral ganglia neurons” or SGNs, and if a patient suffers damage to the SGNs, they will not be able to receive a cochlear implant to restore their hearing. Therefore, this experiment attempted to replace these SGN cells in order the restore hearing.

Rivolta’s group used human embryonic stem cell (hESC) lines H7, H14, and Shef1 and treated them with two growth factors, FGF3 and FGF10. The combination of these two growth factors induced the expression of a whole host of genes normally found in SGN cells (for example, Pax8 & Sox2). These treatments converted the hESCs into otic neural progenitors (ONPs).

In order to destroy the SGN cells in the ears of gerbils, Rivolta and others used a drug called ouabain. This drug, when injected into the inner ear, will destroy the SGN cells and make the animals completely deaf. In the next experiment, Rivolta et al. transplanted the immature nerve cells into the ears of 18 gerbils. One ear received the transplantation, while the other ear was kept as is as a control.

10 weeks later, they used electrophysiology tests to measure the response of the brain to sound. Of the 18 gerbils transplanted with the hESC-derived ONPs, the animals had recovered their hearing by an average of 46%. The recovery differed from animal to animal, but it ranged from modest recovery to almost complete in others.

All animal subjects were kept on anti-rejection medications to prevent rejecting the implanted human cells. In order to prevent tissue rejection in human patients, either induced pluripotent stem cells should be used, or hESCs that match the tissue types in the patient.

Rivolta’s team is also in the process of making immature versions of a second kind of inner-ear cell, that is, the “hair cell” that detects the auditory vibrations in the cochlea. The induction of ONPs from hESCs tends to produce two types cells: ONPs and otic epithelial progenitors (OEPs), which are the precursors of cochlear “hair cells.” Since damage to hair cells is far more common in cases of hearing loss, implantation of such cells should be able to treat far more cases of hearing loss. Unfortunately, this has not yet been tested in animals, according to Rivolta.

Yehoash Raphael of the University of Michigan, who didn’t participate in the work, said it’s possible the stem cell transplants worked by stimulating the gerbils’ own few remaining nerve cells, rather than creating new ones. But either way, “this is a big step forward in use of stem cells for treating deafness.”

Likewise, Lawrence Lustig of the University of California, San Francisco, said, “It’s a dynamite study (and) a significant leap forward.”

Some Induced Pluripotent Stem Cell Lines Cause Tumors When Transplanted into Mouse Cochleas

Japanese researchers have been carefully evaluating the safety of different stem cell lines to determine the tendency of these cells to form tumors when transplanted into mice. Such studies have made it abundantly clear that the tendency for cell lines to form tumors depends upon the cell line and where it is transplanted (see Blum & Benvenisty, The Tumorigenicity of Human Embryonic Stem Cells. Advances in Cancer Research, Volume 100, 2008, Pages 133–158). However, little is known about the cochlea and the tendency of stem cells to cause tumors when transplanted into the cochlea. Therefore, Takayuki Nakagawa of Kyoto University and his group examined the results of stem cell transplantation into mouse cochlea.

Nakagawa made it clear that his motivation for this work is to achieve successful stem cell transplantation into the cochlea to treat hearing loss. He said: “Hearing loss affect millions of people world-wide. Recent studies have indicated the potential of stem cell-based approaches for the regeneration of hair cells and associated auditory primary neurons. These structures are essential for hearing and defects result in profound hearing loss and deafness.”

In this study, Nakagawa’s group transplanted embryonic stem cells and three distinct clones of mouse induced pluripotent stem cells into the cochlea of adult mice. According to Nakagawa; “Our study examined using induced-pluripotent stem cells generated from the patient source to determine if they offer a promising alternative to ES (embryonic stem) cells. In addition, the potential for tumor risk from iPS cells needed clarification.”

Upon transplantation into the cochlea, each cell line showed a distinct ability to form neural structures and integrate into the adult cochlea four weeks after transplantation. Some cells showed poor survival in the cochlea and one induced pluripotent stem cell line formed tumors in the cochlea. “To our knowledge, this is the first documentation of teratoma formation in cochleae after cell transplantation,” said Nakagawa.

These data demonstrate the necessity of screen individual iPS cell lines before their use, since some lines have greater tumor-causing potential than others.  Furthermore, it essential for researchers to design and develop screens to eliminate tumorigenic iPS cell lines.

John Sladek from the University of Colorado School of Medicine said: “While this study do not look at the ability of the transplanted cells to repair hearing loss, it does provide insight into the survival and fate of transplanted cells.  It highlights the importance of factors such as knowing the original source of the cells and their degree of differentiation to enable the cells to be ranked in order of their likelihood of forming tumors.”

Stem Cell Treatments for Hearing Loss?

In mammals, loss of hearing is irreversible because neurons in the cochlea and so-called “hair cells” do not regenerate. More than half of the population over the age of 60 suffers from severe hearing loss. Replacing cells in the inner is an important goal for regenerative medicine.

The ear consists of three main compartments. The outer ear consists of the cup-like structure on the sides of our heads called “pinnae,” and the opening to the middle called “external auditory meatus” (EAM). The EAM terminates at the eardrum. Behind the ear lies the middle ear, inside which is housed three small bones called the “auditory ossicles.” The auditory ossicles are attached to the eardrum and when the eardrum vibrates as a result of air pressure disturbances caused by sound waves traveling through the air, the ossicles vibrate with the eardrum and set up a series of vibrations on the other side of the middle ear where the ossicles are attached to the so-called oval window.

The vibrations that occur at the oval window are passed into the cochlea, which is derived from the Latin word for snail-shell.

This coiled structure contains two main compartments, one of which extends throughout the cochlea, and the other of which surrounds this central compartment.  The central compartment is called the scala media, and the surrounding compartments are the scala vestibuli (above) and the scala timpani (below).  The scala media contains an organ called the Organ of Corti, and this structure is responsible for producing the signals that are interpreted by the brain as hearing.

The organ of Corti consists of a series of cells with hair-like extensions that called “hair cells.” When the vibrations from the oval window are transmitted to the cochlea, the fluid in the scala vestibuli and scala timpani vibrates and these vibrations are transmitted into the scala media.  The vibrations of the scala media causes a membrane above the hair cells, called the “tectorial membrane” to vibrate and this vibrating tectorial membrane, into which the tips of the hair cells are embedded, moves the hair cell extensions back and forth.  The louder the sound, the greater the degree to which the tectorial membrane vibrates.  Also, the frequency of the sound varies the type of vibrations in the scala media and each hair cell releases neurotransmitters to the neurons that connect with them only when vibrations of the proper frequency activate them.  These activated hair cells release neurotransmitters to the connecting neurons and these neurons take those signals to the brain where they are interpreted and turned into sound sensations.

Sensoineural deafness results from the death of neurons that innervate the hair cells, or the hair cells themselves.  Replacing the hair cells or the connecting neurons is one of the main goals of regenerative medicine.  To that end, several labs have injected neural stem cells, embryonic stem cells or neural stem cells made from embryonic stem cells into the cochleas of deaf animals.  These experiments have shown that injected embryonic stem cells can survive when injected into the cochlea (see Hildebrand MS, et al., J Assoc Res Otolaryngol.2005 Dec;6(4):341-54), and injected cells can even differentiate into cell types that are specific for the cochlea (see Coleman B, et al., Cell Transplant.2006;15(5):369-80).  Inject neural stem cells made from embryonic stem cells can even extend axons that move into the cochlea and make contact with hair cells (Corrales CE,, et al. J Neurobiol. 2006 Nov;66(13):1489-500).  The difficulty with these cells is that they are not originally from the ear and might not differentiate fully into the tissue they are trying to repair.

What is a better cell type for repairing the inner ear?  Fetal ears contain a stem cell population that was identified in 2007 by Wei Chen in Marcelo Rivolta’s lab from the University of Sheffield (Chen et al., Hear Res. 2007 Nov;233(1-2):23-9).  Rivolta’s lab has also designed protocols for isolating and expanding these cells in vitro.  These fetal auditory stem cells form structures that resembled those found in organ of Corti.  The electrophysiological profiles of these cells also greatly resembled those observed in organ of Corti cells.  Also, the auditory stem cells expressed a great many of the genes found in developing inner ear cells when induced with various growth factors and small molecules (Chen W, et al., Stem Cells. 2009 May;27(5):1196-204).

These cell lines be used to cure deafness?  That is a different question, but they can certainly be used as a model system for drug screening, toxicity, and testing therapies to cure hearing loss.  Treating defects of the inner ear have many challenges, and while such cells are a first start, they represent the beginning of what might be a viable source of treatment for hearing loss.

There is also much to say about the fetal source of these cells.  Fetal cells were used to treat Parkinson’s disease and Huntington’s chorea.  The use of brain tissue from aborted fetuses is gruesome to say the least, and while these experiments did not cause the death of the pre-born baby, they represent an acceptance of the killing of unborn children that is execrable.  While the knowledge that has been gained from these experiments is certainly useful, it was gained over the bodies of innocent victims of children who were killed because they were an inconvenience.  This should disturb and sicken us.  The fact that it often doesn’t is a testimony of our moral deafness as a nation.