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.

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.

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.