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Otology

Findings Break Silence on Stem Cells in Inner Ear

Research Suggests Regenerative Therapy for Hearing Loss

he embryonic stem cell, a holy grail for cell and developmental biologists, has become the subject of a more controversial quest over the last few years as some policymakers have clamored to ban its use and alternative sources of stem cells have been uncovered. Scientists have found non-embryonic stem cells in a variety of tissues, including bone marrow and brain. But in the October issue of Nature Medicine (and online Aug. 31), Stefan Heller, HMS assistant professor of otology and laryngology at Massachusetts Eye and Ear Infirmary, reveals perhaps the most surprising source of stem cells to date, the inner ear of adult mice.

Stefan Heller (standing) and postdoctoral fellow Huawei Li were surprised at how easily pluripotent cells can be isolated from the mouse inner ear. These stem cells not only yield hair-like hair cells that may prove useful in restoring deafness, but when transplanted into chick embryos, can give rise to cells in all three germ layers.

Heller began researching the inner ear to elucidate how it converts sound energy into electrical signals. Though this process is poorly understood, it is known to depend on mechanoreceptor cells, tiny hair cells that are part of the organ of Corti, a region of the cochlea that is directly connected to the auditory nerve. In humans and other mammals, loss of these hair cells is a major cause of deafness, and is irreversible.

Regeneration of similar hair cells has been observed, however, in another part of the mammalian inner ear, a section of the vestibular organ called the utricle. This, plus the finding some 15 years ago that the avian equivalent of the organ of Corti can recover from total hair cell loss, suggests that promoting hair cell proliferation in the human inner ear may prove a viable treatment for hearing loss.

The key to this potential therapy is learning more about the factors that turn stem cells into hair cells. “Hair cells are terminally differentiated,” explained Heller, so they cannot undergo cell division. The only way for avian or human hair cells to regenerate is by proliferation and transformation of progenitor cells.

With this in mind, Heller set out to find the right mix of ingredients that would coax hair cells from stem cells, a feat that has never been achieved in any laboratory. “Others were skeptical,” Heller recalled, adding that it was particularly hard getting grant agencies to listen. Yet fueled by the enthusiasm of postdoctoral research fellow Huawei Li, the project took shape.

Cells from Inner Space

Li started by following procedures for isolating mouse forebrain stem cells. These are usually maintained in culture as floating colonies, or spheres of densely packed cells, which can divide ad infinitum. Deciding he had nothing to lose, he ran parallel experiments using utricles isolated from the mouse inner ear. “The surprise,” revealed Heller, “was that we were able to isolate sphere-forming cells much faster and easier from the utricle. It seems to have very robust sphere-forming cells that are relatively simple to isolate.”

But would these spheres form hair cells? As it turns out, only some of the sphere cells were true stem cells with the capacity for self-renewal. The remainder seemed to have already progressed to the level of progenitors because Li found they expressed proteins usually found only in particular cell types. Some sphere cells, for example, tested positive for nestin, a neuronal progenitor cell marker, while others expressed bone morphogenetic proteins and Pax-2, found in the developing inner ear. Significantly, Li discovered that after maintaining the spheres for around two weeks, a subset of cells began to express specialized hair cell proteins like

F-actin, espin, and myosin VIIA. All these proteins are found in the mechanosensitive bundle that protrudes from the apical surface of the hair cell. Sure enough, when Li examined the morphology of these progenitors, he found they had protruding hairlike fibers.

To see if the utricular stem cells could form hair cells in vivo, Li and Heller grafted some of the cells into newly formed chicken embryos. For this they used cells isolated from the utricle of the ROSA26-6 mouse. This animal has been genetically engineered to ubiquitously express the enzyme beta-galactosidase, which produces a blue color in the presence of its substrate X-Gal. When Li examined the chicken embryos seven days later, he found blue mouse cells interspersed among native chicken cells in the developing cochlea. Furthermore, both types of cell were expressing myosin VIIA, suggesting that they were true hair cells.

Pluripotency

The fact that subsets of the utricular sphere cells express different cellular markers suggested that the spheres may be capable of differentiating into cells of many different types. Li and Heller again used the chicken–mouse model as an in vivo test for the pluripotency of the utricular stem cells. They grafted sphere cells into early chicken embryos, then examined the distribution of the mouse cells several days later. Li found the blue cells in the heart, kidney, liver, and skin, suggesting that the spheres give rise to cells in all three germ layers.

“The next step,” commented Heller, “is to prove that these cells are functional. If we stimulate the hair bundle there should be a mechanosensitive channel that opens, and that would give rise to an influx of cations that is measurable.” He also plans to use cochlear explants to test ways of introducing new cells, before finally trying to restore lost function in whole mice.

—Tom Fagan

Green immunofluorescence labeling of the hair cell marker myosin VIIA, shows the similar morphology of hair cells from mouse cochlea (upper) and hair cell-like cells generated from mouse inner ear stem cells (lower). Nuclei of surrounding cells (upper) are highlighted by the blue dye DAPI. Hair like cells do not stain positive for pan-cytokeratin (red fluorescence, lower), a protein found in inner ear support cells.