Research Of Using Stem Cell Approaches In Management For Hearing Loss

Hearing loss is a condition that greatly affects the quality of life of millions of people around the world. In 2012, WHO reported that around 360 people in the world suffer from hearing loss, which is equivalent to 5. 3% of the world's population. The prevalence of regions with the highest rates of hearing loss in children is South Asia, Asia Pacific which includes Indonesia and sub-Saharan Africa.

The incidence of hereditary deafness is quite high, namely one child in a thousand births, born with deafness. Besides that one in one thousand normal hearing births will also be deaf before adulthood. Based on the age onset of the patient, deafness can affect oral language skills, cognitive development and psychosocial development. The prevalence of hearing loss is increasing, due to the increasing world population and higher noise pollution.

One of the biggest challenges in the treatment of cochlear disorders is finding management for hearing loss caused by loss of cochlear hair cells or spiral ganglion neurons. In mammalian cochlea, there are two types of sensory cells namely hair cells and auditory neurons, which are only produced during the fetus. The capacity to replace damaged sensory cells is lost immediately after birth. This makes damage to subsequent sensory cells can cause permanent deafness. Although knowledge of the mechanisms of hair and neuronal cells has increased exponentially over the past two decades, therapy for deaf patients remains elusive. Clinically, the function of lost hair cells can be repaired even if not entirely by electrical stimulation of the auditory nerve by heating implantation of electronic devices. Cochlear implants continue to be the only therapeutic option for severe deafness, but the success of the application depends on the function of sensory neurons. The sensation of hearing through the implant in the cochlea is not the same as normal hearing. Thus learning to interpret the sound signals produced by postoperative cochlear implants is very important. The cochlear implant serves to deliver sound signals to spiral ganglion neurons in lieu of damaged hair cells. Therefore the number of good spiral ganglion neurons plays a major role in the efficacy of cochlear implants. Thus cochlear implants are quite invasive and can also cause complications such as facial parese and infection.

Several cases have been found of rejection reactions to cochlear implants used. Besides that the patient will depend on the equipment that will be used for life. The equipment used in koklear implants is still classified as quite expensive. Currently other therapies are being sought to find a broader biological treatment. The latest development in stem cell technology is a new hope for deaf people. Besides that there is still an opportunity to take cochlear implants in unsuccessful cases using stem cells. In recent years a lot of research has been done on stem cells as a new approach to mammalian hair cell regeneration therapy. This is evidenced by the discovery that ear hair cells can be produced in vivo from embryonic stem cells, adult cochlear stem cells, mesenchymal stem cells and nerve stem cells. These stem cells are pluripotent and can theoretically regenerate into various cell types in the cochlea.

Induction of stem cells to differentiate into desired cells with the help of certain media induction has long been carried out by researchers. This is done to see the ability of stem cell differentiation itself. This knowledge is expected to be useful in finding treatment for cells that are severely damaged or cells that do not have regeneration abilities such as hair cells and cochlear nerve cells. It is expected that the induction media tested will be able to directly stimulate the cell regeneration process, or cells that have been successfully induced in the culture media can be transplanted to the desired organ. Some media used to induce cochlear cells include Epidermal Growth Factor (EGF) combined with Insulin-like Growth Factor 1 (IGF-1). Both of these growth factors are used by Qin et al. (2011) to induce nerve progenitor cells obtained from induction of mesenchymal stem cells (SPM), into cochlear hair cells with satisfactory results. Another study conducted by Jeon et al. (2007) who used a combination of IGF-1 growth factor, EGF, and basic fibroblast growth factor (bFGF) only succeeded in converting SPM into nerve progenitor cells, even after adding Neurotrophin-3 (NT-3) and Brain-derived Neurotrophic Factor (BDNF). The success of inducing these cells into cochlear hair cells in this study was obtained after the administration of plasmid transfection by carrying the Atonal Mouse 1 gene.

Several studies have shown that SPM can be induced into cochlear hair cells. SPM is known to have the ability to differentiate into nerve progenitor cells which is one of the characteristics of cochlear hair cells. Process of Cochlear Hair Cell DifferentiationOne of the most decisive stages in the development of cochlea is the process of specifying neurosensory progenitors and diversification of various cells in the cochlea. In chickens and mice, the neurosensory pathway produces sensory nerve cells, sensory hair cells and supporting cells. There are 2 main pathways that occur during the cochlear formation process. First, neurosensory progenitors produce nerves (neuroblasts) or sensory precursors. Second, after the nerve has been delimited, the progenitor still in the epithelium develops into cochlear hair cells or supporting cells. Differentiation of cochlear nerve cells and hair cells is influenced by Neurog1, Atoh1. Notch signal pathways also play an important role in the two pathways above as a precursor determinant in passing alternative cell pathways with a lateral inhibition mechanism. Another signaling pathway that plays a role in the development of cochlea is FGF. The FGF pathway plays a role in the regulation of the formation of several cochlear sensory organs which induce oticus placode, oticus vesicle formation and regulation of hair cell morphogenesis. FGF also induces the expression of the Sex determining region Y (SRY) -box 2 (Sox2) on the otic placode which later determines the direction of cell differentiation into cochlear nerve cells or hair cells.

Another protein that plays an important role in the specification of cochlear hair cells and nerve cells in the neurosensory pathway of the oticus is Sox2. Sox2 is a subfamily of Sox genes which are transcription factors. Sox 2 can activate Neurogenin (Neurog1) and Atonal basic helix loop helix (BHLH) transcriptor factor (Atoh1) and experience downregulation of differentiated nerve cells and cochlear hair. Sox2 expression appears to remain high in supporting cells. This shows that the supporting cells in the cochlea may retain their progenitor property. Neurog1 will regulate the process of nerve cell differentiation while Atoh1 can induce cochlear hair cell formation and rapidly downregulate after the cell becomes mature. This downregulation process is very important because Sox2 and Atoh1 can block mature hair cell markers, namely Myosin VIIa. The hair cell bundle is formed by stereocilia sequences whose storied height resembles steps and a single kinocilium behind the longest stereocilia.

Hair cells

Stereosilia are in the cicular plate, a rigid platform formed from the actin filaments in the apical cytoplasm of hair cells in the region that connects between the hair cells and supporting cells. Actin filaments decrease from stereosilia to the cuticular plate as rootlets which are crosslinked into actin tissue. In addition to actin, the cuticular plate contains spectrin which are interconnected proteins between actin filaments and have elastic deformation retaining properties. There is also tropomyosin, a protein that binds the actin circumference and strains it. Various myosin families of motor proteins, types Ic, VI, VIIa and 15-all unconventional (non-muscle) isotypes, are also localized in the area of the cuticular and stereocilia plates. Mice with mutations in the myosins VI, VIIa or 15 genes all show deafness and balance abnormalities and abnormalities in the stereosiliary bundle. In mutant mice, myosin 6 is damaged (Snold's waltzer), stereocilia look fused and longer.

In mice where mutations in myosin 15 (Shaker 2) occur, stereosilia appear to be high in height indicating that isotope myosin has a role in stereosilia growth, but the function is clearly unknown. Mutations of the Myosin VIIa gene are responsible for USH1B. Interestingly, similar anomalies are seen in Corti's elderly organs and may be related to hearing loss due to the influence of age. It has been estimated that myosin 6 can be involved in holding the apical plasma membrane of hair cells to the cuticular plate in the area between basal stereosilia so that individual stereosilia can be maintained and if there is a defect the membrane area can become fragmented. In mice in which myosin 15 mutates high stereocilia becomes reduced which indicates that this myosin isotype may have a role in maintaining stereosilia, but its proper function is still unclear.

Mutations in the Myosin

VIIa gene are responsible for USH 1B13. Mutant mouse strains that carry this mutation have shorter stereosilia. Myosin 1c is located near the insertion point on the tip-link tip (in the longer stereocillium stem) and is thought to be involved in adaptation motors that close the transduction channel when stereocillium is exposed to sustained stimulation.

In studies involving genetic manipulation the molecular structure of the myosin 1c found that this molecule was indeed involved in adaptation to vestibular hair cells. Myosin VIIa also plays a role in controlling the tension of tips. In the cochlea, defects in myosin cause a decrease in the sensitivity of the process of opening the transduction channel by stereosilia deflection. This causes functional absence of Myosin VIIa, channels are generally closed and tension in the tip-link is significantly reduced.

Auditory nerve

The formation of the auditory nerve begins during initial cochlear aids. The cochlea is a remarkable picture of the process of organogenesis. This is because all the cochlear hair cells and nerve cells that function as hearing and balance devices and all associated non-sensory tissues in them come from an embryonic source called the otic placode. The otic tract is an epithelial cell on the surface of the embryo adjacent to the developing neural tube. At the beginning of embryogenesis, otic placodes are invaded to form otic vesicles, which are adjacent to hindbrain and notochord. At the beginning of the 9th day of the embryo (E9) in mice, neuroblasts began delamination of the otic vesicles and joined to form the vestibular cochlear ganglion (CVG), long before the inner ear obtained a complex three-dimensional structure. As development progresses, CVG neurons separate themselves to form a spiral ganglion, which crosses along the cochlear channel, and the vestibular ganglion, which is on the surface of the inner ear and innervates the sensory epithelium of the vestibular structure. Recent studies have shown that spiral ganglion neurons are increasingly determined by transcription tissue that works in the otic vesicle region which contains precursors for neurons, hair cells and supporting cells. The development of nerve cells is affected by signaling pathways from certain proteins including FGF, Wnt, Sox2 etc.

Sensory Nerve Deafness

The most common features of sensory nerve deafness are damage or loss of cochlear sensory cells. Sensory nerve deafness is a hearing loss that results from damage to the inner ear (cochlea) or to the auditory nerve from the cochlea to the auditory center in the brain. Sensory nerve deafness often cannot be treated with medication or by surgery. This type of deafness is a type of deafness that most often causes permanent hearing loss. Sensory nerve deafness reduces the ability of the sufferer to hear sounds clearly. Even when the sound is released loud enough, the sufferer still cannot hear clearly. Some causes of sensory nerve deafness include:

1. Medications that can damage cochlear cells (ototoxic)
2. Genetic
3. The degeneration process / Presbycusis
4. Head trauma
5. Congenital cochlear malformation
6. Noise exposure.

Pathophysiology of cochlear hair cell death

Noise exposure and ototoxic drugs can cause the formation of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) in the cochlea, which eventually triggers apoptosis which is caspase-bound. ROS has been detected in the cochlea immediately after noisy exposure and persists for 7-10 days. ROS spreads from the basal end of the organ of Corti to the apex area. Exposure to sustained ROS can cause continuous hair cell disorders Mitochondria also produce Apoptosis-inducing Factors (AIF) and EndoG apoptotic nucleos into the cytosol of hair cells due to noise exposure. Translocation of the apoptotic factor to the nucleus triggers apoptosis. Activation from the c-Jun N-terminal kinase (JNK) / mitogen-activated protein kinase (MAPK) pathway also plays a role in the process of apoptosis of the cochlear outer hair cells in response to oxidative stress.

Management of Sensory Nerve Deafness

Cochlear implants

The cochlear implant is a small electronic device that can help provide sound stimulation through mechanico-electric transduction to the auditory nerve. The cochlear implant consists of two basic parts, namely an external sound processor that functions like a microphone and the implanted component that transmits the stimulus to the auditory nerve. Implants are usually used in severe deafness caused by loss of progressive hair cells in the organ of Corti. Loss of hair cells caused by aminoglycoside and cisplatin (chemotherapy) drugs can be restored by cochlear implantation. The procedure for implanting (implant) the internal component begins with a small postauricular incision followed by mastoidectomy to access the middle ear. Round window niches are identified and drilled to enter the tympanic scale of the cochlea (cochleostomy). Electrodes are inserted through the cochleostomy into the cochlea. The operation is carried out by an otologist and takes about 1. 5 hours to complete.

Growth factor

Previous studies have been conducted on the ability of growth factors to induce hair cell proliferation. Hair cells are known to lose their proliferation ability from the embryonic stage. Growth factors are expected to trigger hair cell proliferation during the postpartum stage because they have the ability to induce proliferation in cultured hair cells. The effects of various types of growth factors on utricular cell proliferation were investigated quantitatively by taking and culturing mouse utricle cells. Among the several growth factors tested, EGF, Transforming Growth Factor α (TGFα), bFGF, and IGF-1 were able to induce proliferation. In particular, the combination of FGF and IGF-1 or TGFα has a combined effect. Similar mitogenic effects have been confirmed in EGF and TGFα in vestibular organ cultures. In addition, EGF causes very large hair cell formation even in cochlear organ cultures in neonatal rats. Giving growth factors in cochlear disorders through blood vessels is considered quite difficult due to the presence of the cochlear barrier. One alternative to giving is through the tympanic scale where in this area there is perilymph which is directly related to axons and nerve synapses. The method of administering from neurotropic molecules to the perilymph fluid from the cochlea can be given through the tympanic cavity with a diffusion system or can be given directly through a round window. Although the cause of deafness is partly an acute process of damage to the hair cells and cochlear nerves, many other causes are gradual damage. In these circumstances, a long-term infusion of growth factors may be needed to maintain the spiral ganglion nerve population so that damage does not occur. Several studies have found a way of chronic administration of neutropin and other growth factors into the cochlea. One method that has been successful is by administering BDNF or NT-3 infusion to the tympanic scale of mice that have been previously transmitted using osmotic mini pumps.

Gene therapy

It is known that non-viral vectors, including plasmids, have advantages over other vectors. Non-viral vectors proved to be less toxic and resulted in less inflammation although on the one hand the transduction level was relatively lower. In the latest study, it was also known that the use of nanoparticles to transport DNA-polylysine particles into the cochlea of mice could be done. However, most studies focus on the use of viral subtypes, such as adenovirus, adeno-related viruses, herpes viruses, helper-dependent adenoviruses and lentiviruses. Of this virus subtype, adeno-related viruses have the greatest potential, because they do not cause ototoxicity. Various subtype of adeno-associated viruses have been successfully used to transport genes in the cochlea but have caused little damage to the organ of Corti. Kilpatrick et al. (2011) examined that adeno-associated viruses serotypes 1, 2, 5, 6 and 8 had good gene expression in hair cells and basal cells, as well as the cochlear nerve and spiral ganglion. One of the drawbacks is that these vectors can only carry fragments to a limit of 5 kb. This limits the function of the virus transduction.

Kesser and Lalwani (2009) reported that adenovirus can overcome the limitations of adeno-associated viruses. A study shows that the lentiviral vector does not spread beyond the cochlea, it is able to minimize toxicity to surrounding tissues. Regarding the method of administering viral vectors, most studies assume that the best method is cochleostomy or direct injection through a circular membrane using enzymatic processes with collagenase. Math1 in mice plays a role in transcription factors for sensory differentiation of cochlear basal cell hair cells. Izumikawa et al. (2005) reported that atonal 1 homologs can improve hearing hearing of deaf mice, get good results at both cellular and functional levels.

Oshima et al. (2010), shows how to differentiate stem cells from hair cells with their bundle structure and function. They use mouse embryonic stem cells and induced pluripotent stem cells, which are directed to become ectoderm that can respond to growth factors that induce otic cells. The resulting otic progenitor cells are directed towards various differentiation conditions, one of which directs the cell into the epithelial group which shows cells that resemble hair cells with stereosilia bundles. This bundle cell group responds to mechanical stimuli with movements that resemble the flow of adult hair cell transduction.

Stem Cell Therapy

Basic aspects of stem cells Stem cells or stem cells are cells that are not / not yet specialized and have the ability / potential to develop into various types of specific cells that form various body tissues. Stem cells have two distinctive characteristics, namely

Differentiate is the ability to differentiate into other cells. Stem cells can develop into a variety of specific (specific) cell types such as nerve cells, heart muscle cells, skeletal muscle cells, pancreatic cells and others.

Self regenerate / self-renew, namely the ability to renew or regenerate itself. Stem cells are able to make copies of cells that are exactly the same as themselves through cell division.

Induction of Stem Cells into Cochlea Hair Cells

Some researchers have tried inducing stem cells into cochlear hair cells by trying various induction media. This is expected to be useful in finding treatment for hair cells and cochlear nerve cells that do not have regeneration ability. It is expected that the induction media tested will be able to directly stimulate the cell regeneration process, or cells that have been successfully induced in the culture medium can be transplanted to the desired organ. Some of the media used to induce cochlear cells include EGF combined with IGF 1. Both growth factors used by Qin et al. (2011) to induce nerve cell progenitors obtained from mesenchymal stem cell (SPM) induction, into cochlear hair cells with satisfactory results. In another study conducted by Jeon et al. (2007) using a combination of IGF-1, EGF, and bFGF growth factors only succeeded in converting SPM into neural progenitor cells even after NT-3 and BDNF were added, but the success of inducing these cells into cochlear hair cells was obtained after plasmid transfection by carrying Math1 gene. But in a study conducted by Li, Liu & Heller (2003) it was found that adult utricular stem cells can be induced into a spherical form which is the origin of cochlear hair cells using a combination of EGF, IGF-1 and bFGF. The spherical cells are seen expressing Nestin mRNA. In addition the spherical cells also presented pax-2, bone morphogenetic protein (BMP) -4 and BMP 7 which are a combination of markers of cochlear cell growth. This change indicates that the media might be able to induce cells further towards cochlear hair cells. The culture process was then continued for 2 weeks with non-serum media. In the immunohistochemical examination, cells were seen presenting myosin VIIa and Brn3. The cochlear hair cell bundle is formed by actin filament (F-actin) and actin binding protein, espin and myosin VIIa, which can be a marker of the hair cell phenotype.

Conclusion

Stem cells are an alternative treatment to overcome this permanent hearing loss, either through direct transplantation of stem cells in the cochlea, or the administration of growth factors as a factor in the induction of stem cells in the cochlea. This development certainly still requires a long and continuous research. This study is expected to be the beginning of the development of hearing loss management through stem cell approaches, especially from dental pulp stem cells.