Indian Journal of Biotechnology Vol 7, April 2008, pp 178-182

Stem cell based therapy to restore nearly normal hearing K Ananda Krishna1 and K R S Sambasiva Rao* Center for Biotechnology, Acharya Nagarjuna University, Guntur 522 510, India Received 13 November 2006; revised 22 August 2007; accepted 20 November 2007 Hair cells with stereocilia are the sensory receptors of the inner ear that are located in the organ of corti of the cochlea, involved in detecting sound, and are connected with the nerve fibers that stretch from the inner ear to the brain. These hair cells convert sound information to electric signals, which are then sent to the higher brain centers. In humans, hair cell damage results in permanent hearing impairment; whereas, birds have the capacity to rebuild a damaged inner ear and various studies have demonstrated hair cell regeneration. Recently, stem cell research has triggered many programmes around the world to examine the possibility for regenerating the hair cells from embryonic/adult stem cells. The important spin-off of the research would be to find signaling mechanisms that regulate hair cell regeneration to restore nearly normal hearing in humans. The current review focuses on stem cell-based therapy with particular emphasis to summarize the recent progress. Keywords: Stem cells, hair cell, cochlea

Introduction The cochlea1-3 is a snail-like structure with three fluid filled sections—two channels for the transmission of pressure and a sensitive organ of corti that detects pressure impulses and responds with electrical impulses, which travel along with the auditory nerve to the brain. Hair cells (mechanoreceptor cells)4,5 are the sensory receptors of the inner ear (with stereocilia), which are located in the organ of corti of the cochlea involved in detecting sound and are connected with the nerve fibers that stretch from the inner ear to the brain. These hair cells convert sound information to electric signals that are then sent to the higher brain centers6,7. The auditory nerve from the ear carries information to the brain that is directly sent to the processing areas. The hair cells (Fig. 1) are arranged in four long parallel columns on a delicate strip of tissue called basilar membrane. They detect movements of fluids in the channels caused by angular acceleration about an axis perpendicular to the plane of the channel. Thus, the tiny relative movement of the layers of the membrane sufficiently triggers the hair cells to send small voltage pulse (action potential) down the associated nerve fiber that travels to the auditory areas of the brain for processing. These tiny floating _______________ *Author for correspondence: Tel: 91-863-2293400; Fax: 91-863-2293378 E-mail: [email protected]

particles aid in the process of stimulating the hair cell as they move along with the fluid. Taking the electrical impulses from the cochlea and the semicircular channels, the auditory nerve makes connections with the brain enabling the process of hearing. As the time progresses, the hearing loss may occur as a result of death of hair cell population in cochlea that is induced because of hereditary causes or various mechanical/chemical/environmental insults (ototoxic drugs, acoustic trauma, aging, injury, and diseases). Hair cells regenerate in the mature avian cochlea; but in humans the loss of cochlear hair cells leads to permanent hearing loss because of the lack of regenerative response8. The application of stem cell therapy may repair cochlea to replace the damaged hair cells. In mammals, the very presence of vestibular stem cell population may imply the possibility of hair cell regeneration. A recent study has suggested that the postnatal cochlear tissue contained cells capable of producing hair cells in vitro9. Later, experimental methods were developed to isolate and culture pure hair cell progenitors from postnatal rat cochleae to facilitate studies on hair cell regeneration10. A side population of hair cells from postnatal mouse cochlea was able to differentiate into hair cell-like cells11. Further, the newborn mouse cochleae with sphere-forming cells were shown to differentiate into cells expressing hair cell markers, which implicate their application in hair cell

KRISHNA & RAO: STEM CELL BASED THERAPY TO RESTORE NORMAL HEARING

(a)

179

(b

Fig. 1—The schematic diagram (a) and EM (b) showing hair cells

regeneration12. Various types of stem cells (embryonic, neural and hematopoietic stem cells) were used to regenerate hair cells. However, there are limitations and difficulties in culturing hair cells. Scientists learn to regenerate hair cell and the clinical use of techniques to regenerate hair cells in humans may at least be a decade away. In the current review, authors focus on stem cell and their potential clinical application to restore nearly normal hearing with particular emphasis to summarize the recent progress. Regeneration Capacity of Hair Cells in Birds and Mammals The regeneration of hair cells in birds was first reported 19 years ago. Since then myriad studies were performed in quest to understand the mechanism behind regeneration process and to restore nearly normal hearing. Initially, it was assumed that regeneration capacity of hair cells was absent in the inner ears of birds and mammals. However, studies had shown that birds have the capacity to rebuild a damaged inner ear. Various studies have demonstrated hair cell regeneration. In chick, cochlea hair cells were recovered after gentamicin toxicity13. Hair cells in the avian inner ear were regenerated with functional recovery after administration with aminoglycoside14. Following gentamicin and kanamycin induced damages, the hair cells in the chick cochlea were also shown to regenerate and recover15. Exposures to an intense pure tone stimulus in coturnix quail demonstrated their potential to

regenerate hair cell throughout life16. Moreover, studies on gicerin (a novel member of the immunoglobulin superfamily) and neurite outgrowth factor in the chick suggested that gicerin might be associated with cell proliferation in the auditory epithelium, and played a role in neurite extension of the acoustic ganglion cells in conjunction with neurite outgrowth factor17. The cell proliferation in avian inner ear sensory epithelia was induced by insulin-like growth factor-I and insulin18. Studies on c-myc mRNA in the chick basilar papilla revealed that cmyc gene expression might be an important molecular event in the hair cell regeneration19. Even the immune system was implicated in hair cell regeneration20, because the involvement of macrophages and their secretory products (cytokines) were able to influence the proliferation of vestibular supporting cells and the survival of statoacoustic neurons21. Earlier, it was believed that mature mammals normally do not regenerate new hair cells. However, recent studies have shown that hair cell regeneration in mammals may be inducible and its loss may be restored22,23. In ototoxic-poisoned organ of corti explants of rats, retinoic acid (bioactive metabolite of vitamin A, retinol) was shown to stimulate the regeneration of mammalian auditory hair cells24. In mammals, transforming growth factor-α significantly increased cell proliferation, indicating that inner ear hair cell damage is reversible25,26. Furthermore, studies in chick inner ear following acoustic trauma suggested that basic fibroblast growth factor was

180

INDIAN J BIOTECHNOL, APRIL 2008

related to the proliferation of the supporting cells and the extension of the nerve fibers27. Recent studies on the influence of growth factors on the proliferation of pure rat utricular epithelial cells suggested that fibroblast growth factor-2 and insulin-like growth factor-1 may regulate the proliferation step during hair cell development and regeneration28. Moreover, studies suggested that glial growth factor enhances supporting cell proliferation in rodent vestibular epithelia29. The studies using cytosine arabinoside (Ara-C) suggests that these supporting cells can convert into hair cells in the acoustically damaged chick inner ear30. Studies also implicated that heregulin (a member of the neuregulin family) may play vital role in hair cell regeneration, following ototoxic damage, and enhance proliferation of supporting cells in postnatal rat utricular epithelial sheet cultures after gentamicin treatment31. In another study, bone morphogenetic protein signaling was shown to regulate hair cell generation32. Oghalai et al33 developed a technique to harvest living human hair cells, which laid foundation to study and regenerate hair cell. More recent findings showed that fetal otocyst cells transplanted into gentamicin-treated explants of vestibular sensory epithelia survived, indicating that they have potential to migrate into damaged area and differentiate into hair cell34. A chain of events occur when hair cells of the inner ear are subjected to noise and ototoxic damage, resulting in permanent hearing loss because of hair cell destruction35. In birds, in the event of hair cell death, the supporting cells are to transdifferentiate into hair cells. The injured hair cells ejected from sensory epithelium are replaced by supporting cells by forming new hair cells (without a mitotic event) and with the progress in time these cells make connections with tectorial membrane plus afferent and efferent cochlear nerves. The auditory nerve reconnects hair cells to the central nervous system. However, this process of transdifferentiation of supporting cells into hair cells does not occur spontaneously in mammals. It is not clear whether the mammalian hair cell and cochlear neurons fail to regenerate as a result of lack of appropriate signal or due to the inability of supporting cells to divide. In-depth understanding of both cellular and molecular events that are responsible for hair cell regeneration plus identifying specific hair cell markers will facilitate studies on the mechanisms of hair cell regeneration and help restore the normal functions of hearing.

Stem Cell Based Therapy Embryonic and adult stem cells also appear to be the precursors of hair cells. The bone marrow derived mesenchymal stem cells/cord blood stem cells may also differentiate into hair cells when grown in suitable medium condition to restore the nearly normal hearing. Studies have shown that bone marrow mesenchymal stem cells were progenitors (in vitro) for inner ear hair cells36. Adult olfactory precursor cells were shown to differentiate into hair cell-like cells in culture37. Recently, protocols were developed to generate inner ear progenitor cells from murine embryonic stem cells. Further, these protocols were modified to culture human embryonic stem cells that expressed similar set of inner ear markers38. Furthermore, investigation of genes (Serpinh1, Slc2a10, Sparc) involved in inner ear function showed upregulation after addition of epidermal growth factor39. The murine embryonic stem cells in the presence of epidermal growth factor were committed to hair cell lineage. Thus, the focus of the future research should have been: (i) to transdifferentiate embryonic/adult stem cells to hair cells using a suitable culture medium, (ii) to identify the cellular and molecular events that produce hair cell regeneration, and (iii) to inject stem cells/precursor cells into the inner ear/graft them successfully to restore the nearly normal hearing in humans. However, there are limitations and difficulties in culturing hair cells. New techniques need to be developed to identify and isolate stem cells, culture them in artificial environment, provide essential growth factors, and stimulate them to differentiate into progenator cells. These cells when grafted into the inner ear must be capable of giving rise to new functional hair cells. In addition, hair cell regeneration is a complex process. The supporting cells must receive signals to undergo mitosis and transdifferentiate into new functional hair cells. Recently, studies have shown that Math1 [a transcription factor encoded by the gene Atoh1 (Math1)], a positive regulator of inner hair cell differentiation, was essential for inner ear hair cell genesis40,41; whereas, Hes1, a negative regulator of inner ear hair cell differentiation, antagonized Math142. Thus, a balance between Math1 and Hes1 expression is important for the production of inner ear hair cells. Further, it was shown that Hes1 and Hes5 participate together and control inner ear hair cell production43 and the role of Hes6 remains to be

KRISHNA & RAO: STEM CELL BASED THERAPY TO RESTORE NORMAL HEARING

addressed44. And Atoh1 gene therapy to regenerate hair cells using adenovectors is a novel therapeutic approach to improve hearing in mammals45. The pluripotent stem cells from the adult mouse inner ear were able to differentiate into hair cell-like cells46. In vitro, murine embryonic stem cells were used to generate hair cells, which expressed their characteristic markers when integrated into the developing inner ear at sites of epithelial injury47. In mice, neural stem cells restored hearing loss, which demonstrates their potential to differentiate into inner ear hair cells48. In the adult mouse, degenerated spiral ganglion neurons were replaced with adult neural stem cell, which illustrated their ability to repair the injured inner ear49. Further, neural stem cells injected into the inner ear after ischemic insult showed their therapeutic application in preventing damage to hair cells50. Further, studies have shown that mouse embryonic stem cell-derived neural progenitors have potential to restore neural network in the peripheral vestibular system51. Recently, the EYFPexpressing embryonic stem cell-derived mouse neural progenitor cells were injected into the cochlear nerve trunk in immunosuppressed animals after destroying the cochlear nerve cells (leaving hair cells intact), which resulted in regeneration of neurons52. Further, mouse neonate cochlea harboured stem cells were capable to differentiate into hair cells53. Thus, the application of stem cell therapy to produce hair cells for improving the disease conditions may offer a window of possibility for an effective treatment and cure. Conclusion Recent researches are more inclined toward regenerative therapeutic strategy to restore the normal functions of hearing with the application of stem cell therapy. In birds, hair cells in the inner ear can regenerate naturally after damage. The nerve cells appear to re-connect with the regenerated hair cells without any difficulty due to the growth factors that attract nerve fibers to the hair cells. Studies have revealed that small numbers of supporting cells can multiply in the inner ear tissue (guinea pigs, mice, rats) reinforcing the possibility that human hair cells might be induced to regenerate using growth factors. The most important challenge for the future is to induce the stem cells to transdifferentiate into mature hair in humans to restore nearly normal hearing. It can be envisaged that stem cell therapy may one day restore normal hearing and balance function in humans. It is possible if the hair cell regeneration can be induced or grafted successfully in humans, which could improve

181

hearing of millions of people who are suffering from deafness. Further, studies to understand the capability of precursors to transform into fully functional hair cell have demonstrated the functional capability of newly formed hair cells and revealed the molecular events involved in hair cell regeneration, which would play a vital role in the treatment strategies. References 1 Robles L & Ruggero M A, Mechanics of the mammalian cochlea, Physiol Rev, 81 (2001) 1305-1352. 2 Wever E G, Electrical potentials of the cochlea, Physiol Rev, 46 (2005) 102-127. 3 Wever E G, The physiology of hearing: The nature of response in the cochlea, Physiol Rev, 13 (1933) 400-425. 4 Kelley M W, Hair cell development: Commitment through differentiation, Brain Res, 1091 (2006) 172-185. 5 Cheng A G, Cunningham L L & Rubel E W, Mechanisms of hair cell death and protection, Curr Opin Otolaryngol Head Neck Surg, 13 (2005) 343-348. 6 Fettiplace R, Active hair bundle movements in auditory hair cells, J Physiol, 576 (2006) 29-36. 7 LeMasurier M & Gillespie P G, Hair-cell mechanotransduction and cochlear amplification, Neuron, 48 (2005) 403-415. 8 Malgrange B, Differentiation, protection and regeneration of hair cells and auditory neurons in mammals, Bull Mem Acad R Med Belg, 160 (2005) 276-286. 9 Doetzlhofer A, White P M, Johnson J E, Segil N & Groves A K, In vitro growth and differentiation of mammalian sensory hair cell progenitors: A requirement for EGF and periotic mesenchyme, Dev Biol, 272 (2004) 432-447. 10 Zhai S, Shi L, Wang B E, Zheng G, Song W et al, Isolation and culture of hair cell progenitors from postnatal rat cochleae, J Neurobiol, 65 (2005) 282-293. 11 Savary E, Hugnot J P, Chassigneux Y, Travo C, Duperray C et al, Distinct population of hair cell progenitors can be isolated from the postnatal mouse cochlea using side population analysis, Stem Cells, 25 (2007) 332-339. 12 Wang Z, Jiang H, Yan Y, Wang Y, Shen Y et al, Characterization of proliferating cells from newborn mouse cochleae, Neuroreport, 17 (2006) 767-771. 13 Cruz R M, Lambert P R & Rubel E W, Light microscopic evidence of hair cell regeneration after gentamicin toxicity in chick cochlea. Arch Otolaryngol Head Neck Surg, 113 (1987) 1058-1062. 14 Tucci D L & Rubel E W, Physiologic status of regenerated hair cells in the avian inner ear following aminoglycoside ototoxicity, Otolaryngol Head Neck Surg, 103 (1990) 443-450. 15 Li S, Zhu H & Zhang Q, Hair cell regeneration and recovery following gentamicin induced damage in the chick cochlea. Zhonghua Er Bi Yan Hou Ke Za Zhi, 29 (1994) 89-91. 16 Ryals B M & Westbrook E W, Hair cell regeneration in senescent quail, Hear Res, 50 (1990) 87-96. 17 Kajikawa H, Umemoto M, Taira E, Miki N, Mishiro Y et al, Expression of neurite outgrowth factor and gicerin during inner ear development and hair cell regeneration in the chick, J Neurocytol, 26 (1997) 501-509. 18 Oesterle E C, Tsue T T & Rubel E W, Induction of cell proliferation in avian inner ear sensory epithelia by insulin-

182

19

20 21 22 23

24

25 26

27

28

29

30 31

32 33 34

35 [[

INDIAN J BIOTECHNOL, APRIL 2008 like growth factor-I and insulin, J Comp Neurol, 380 (1997) 262-274. Li H & Wang J, Study on c-myc mRNA and its product expression during hair cell regeneration in the chick basilar papilla, Lin Chuang Er Bi Yan Hou Ke Za Zhi, 13 (1999) 173-175. O’Halloran E K & Oesterle E C, Characterization of leukocyte subtypes in chicken inner ear sensory epithelia, J Comp Neurol, 475 (2004) 340-360. Warchol M E, Immune cytokines and dexamethasone influence sensory regeneration in the avian vestibular periphery, J Neurocytol, 28 (1999) 889-900. Forge A, Li L, Corwin J T & Nevill G, Ultrastructural evidence for hair cell regeneration in the mammalian inner ear, Science, 259 (1993) 1616-1619. Warchol M E, Lambert P R, Goldstein B J, Forge A & Corwin J T, Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans, Science, 259 (1993) 1619-1622. Lefebvre P P, Malgrange B, Staecker H, Moonen G & van de Water T R, Retinoic acid stimulates regeneration of mammalian auditory hair cells, Science, 260 (1993) 692-695. Lambert P R, Inner ear hair cell regeneration in a mammal: Identification of a triggering factor, Laryngoscope, 104 (1994) 701-718. Yamashita H & Oesterle E C, Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor alpha and epidermal growth factor, Proc Natl Acad Sci USA, 92 (1995) 3152-3155. Umemoto M, Sakagami M, Fukazawa K, Ashida K, Kubo T et al, Hair cell regeneration in the chick inner ear following acoustic trauma: Ultrastructural and immunohistochemical studies, Cell Tissue Res, 281 (1995) 435-443. Zheng J L, Helbig C & Gao W Q, Induction of cell proliferation by fibroblast and insulin-like growth factors in pure rat inner ear epithelial cell cultures, J Neurosci 17 (1997) 216-226. Gu R, Marchionni M & Corwin J T, Glial growth factor enhances supporting cell proliferation in rodent vestibular epithelia cultured in isolation, Soc Neurosci Abstr, 22 (1996) 520. Adler H J & Raphael Y, New hair cells arise from supporting cell conversion in the acoustically damaged chick inner ear, Neurosci Lett, 205 (1996) 17-20. Zheng J L, Frantz G, Lewis A K, Sliwkowski M & Gao W Q, Heregulin enhances regenerative proliferation in postnatal rat utricular sensory epithelium after ototoxic damage, J Neurocytol, 28 (1999) 901-912. Pujades C, Kamaid A, Alsina B & Giraldez F, BMPsignaling regulates the generation of hair-cells, Dev Biol, 292 (2006) 55-67. Oghalai J S, Holt J R, Nakagawa T, Jung T M, Coker N J et al, Harvesting human hair cells, Ann Otol Rhinol Laryngol, 109 (2000) 9-16. Kim T S, Kojima K, Nishida A T, Tashiro K, Lee J E et al, Expression of calretinin by fetal otocyst cells after transplantation into damaged rat utricle explants, Acta Otolaryngol Suppl, 551 (2004) 34-38. Forge A & Li L, Apoptotic death of hair cells in mammalian vestibular sensory epithelia, Hear Res, 139 (2000) 97-115.

36 Jeon S J, Oshima K, Heller S & Edge A S, Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells, Mol Cell Neurosci, 34 (2007) 59-68. 37 Doyle K L, Kazda A, Hort Y, McKay S M & Oleskevich S, Differentiation of adult mouse olfactory precursor cells into hair cells in vitro, Stem Cells, 25 (2007) 621-627. 38 Rivolta M N, Li H & Heller S, Generation of inner ear cell types from embryonic stem cells, Methods Mol Biol, 330 (2006) 71-92. 39 De Silva M G, Hildebrand M S, Christopoulos H, Newman M R, Bell K et al, Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway, Acta Otolaryngol, 126 (2006) 1148-1157. 40 Zheng, J L & Gao W Q, Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci, 3 (2000) 580-586. 41 Bermingham N A, Hassan B A, Price S D, Vollrath M A, BenArie N et al, Math1: An essential gene for the generation of inner ear hair cells, Science, 284 (1999) 1837-1841. 42 Zheng J L, Shou J, Guillemot F, Kageyama R & Gao W Q, Hes1 is a negative regulator of inner ear hair cell differentiation, Development, 127 (2000) 4551-4560. 43 Zine A, Aubert A, Qiu J, Therianos S, Guillemot F, et al, Hes1 and Hes5 activities are required for the normal development of the hair cells in the mammalian inner ear, J Neurosci, 21 (2001) 4712-4720. 44 Qian D, Radde-Gallwitz K, Kelly M, et al, Basic helix-loophelix gene Hes6 delineates the sensory hair cell lineage in the inner ear, Dev Dyn, 235 (2006) 1689-1700. 45 Izumikawa M, Minoda R, Kawamoto K, Abrashkin K A, Swiderski D L, et al, Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals, Nat Med, 11 (2005) 271-276. 46 Li H, Liu H & Heller S, Pluripotent stem cells from the adult mouse inner ear, Nat Med, 9 (2003) 1293-1299. 47 Li H, Roblin G, Liu H & Heller S, Generation of hair cells by stepwise differentiation of embryonic stem cells, Proc Natl Acad Sci USA, 100 (2003) 13495-13500. 48 Tateya I, Nakagawa T, Iguchi F, Kim T S, Endo T et al, Fate of neural stem cells grafted into injured inner ears of mice, Neuroreport, 14 (2003) 1677-1681. 49 Hu Z, Wei D, Johansson C B, Holmstrom N, Duan M et al, Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear, Exp Cell Res, 302 (2005) 40-47. 50 Hakuba N, Hata R, Morizane I, Feng G, Shimizu Y et al, Neural stem cells suppress the hearing threshold shift caused by cochlear ischemia, Neuroreport, 16 (2005) 1545-1549. 51 Kim T S, Nakagawa T, Kita T, Higashi T, Takebayashi S et al, Neural connections between embryonic stem cellderived neurons and vestibular hair cells in vitro, Brain Res, 1057 (2005) 127-133. 52 Corrales C E, Pan L, Li H, Liberman M C, Heller S et al, Engraftment and differentiation of embryonic stem cellderived neural progenitor cells in the cochlear nerve trunk: Growth of processes into the organ of corti, J Neurobiol, 66 (2006) 1489-1500. 53 Yerukhimovich M V, Bai L, Chen D H, Miller R H & Alagramam K N, Identification and characterization of mouse cochlear stem cells, Dev Neurosci, 29 (2007) 251-260.