Stem Cell Differentiation Stage Factors (SCDSFs) Taken from Zebrafish Embryo During Organogenesis and Their role as Epigenetics Regulators able to reverse Neurosensory Hearing Loss

Hearing dysfunctions can be classified by type, degree, configuration, time of onset, aetiology, and finally, consequences on speech development. They can be divided into conductive, mixed, central types and sensorineural. Conductive hearing loss (CHL) results from interference with the mechanical transmission of sound through the external and middle ear; it can be congenital, as a consequence of anatomic abnormalities, but it can commonly be acquired following middle ear inflammatory pathologies. Sensorineural hearing loss (SNHL) results from failure to transduce vibrations to neural impulses in the cochlean and is a consequence of an irreversible damage to the differentiated cells which make up the organ of hearing and the acoustic paths at various levels. Mixed hearing loss involves a combination of these two types in the same ear. Studies in neuroscience field have shown that the prevention of cell degeneration is only possible if all the factors taken at the different stages of stem cells’ multiplication and differentiation are administered together. We have demonstrated this in a recent study on the ability of SCDSFs to prevent neurodegeneration in hippocampal cells of the CA1 zone in mice. This study confirms previous findings demonstrating that early developmental zebrafish embryo extracts could act as a modulator of senescence in human mesenchymal stem cells (hMSC) isolated from many adult tissues. These findings have open a promising way for the approaches promoting the rejuvenation and regeneration of different tissues, by-passing stem cell transplantation. In the present clinical trial we have used SCDSFs to study the possible reversion of neurosensory hearing loss, until now considered an irreversible condition.


INTRODUCTION
Hearing loss, the most common form of human sensory deficit, is the partial or total inability to hear sound in one or both ears. It may be a sudden or a progressive impairment that gradually gets worse over time. Depending on the cause, it can be mild or severe, temporary or permanent. It may be a bilateral loss occurring in both ears or unilateral. Hearing loss may be fluctuating, that is, varying over time-improving at times and getting worse at other times. In other cases, hearing loss is stable, not changing at all with time. Hearing loss is caused by many factors, including genetics, age, exposure to noise, illness, chemicals, and physical trauma. Hearing loss may affect all ages, delaying speech and learning in children, and causing social and vocational problems for adults. [1] Hearing dysfunctions can be classified by type, degree, configuration, time of onset, etiology, and finally, consequences on speech development. They can be divided into conductive, mixed, central types and neuro sensory hearing loss [2]. Conductive hearing loss results from interference with the mechanical transmission of sound through the external and middle ear; it can be congenital, as a consequence of anatomic abnormalities, but it can commonly be acquired following middle ear inflammatory pathologies. Neuro sensory hearing loss results from failure to transduce vibrations to neural impulses in the cochlea and usually is a consequence of an irreversible damage to the differentiated cells which make up the organ of hearing and the acoustic paths at various levels. [3] Mixed hearing loss involves a combination of these two types in the same ear [1] Clearly in the etiology of the neuro sensory hearing loss are relevant many chemical toxic factors, like many pharmacological substances or physic agents like noise which induce degenerative or apoptotic damages, which however manifest themselves in a monotonous way with the loss not only of the cytological structure but also of the dedicated function: in this case the acoustic ability.
In the binomial structure-function, as it is reasonable to expect a neuro sensory hearing loss in the case of lesion, it is equally reasonable to expect that an auditory recovery demonstrated with a tonal audiometric examination is supported by cellular regeneration. Such correspondence is considered These researches demonstrated that SCDSFs are significant in activating important genes, which counteract human cells senescence. Indeed, these factors represent very effective tools to increase stem cell expression of multipotency, reducing the expression of the beta-galactosidase marker and enhancing the stemness genes Oct-4, Sox-2 and c-Myc. [5] Furthermore, it was possible to activate the gene expression of TERT, the catalytic subunit of telomerase, and the transcription of Bmi-1, [6][7] which plays a role in counteracting senescence, as a key repressor of telomerase-independent aging. [8][9].
Based on researches on stem cell rejuvenation and differentiation, we have also conducted studies on the prevention of cell degeneration and tissue regeneration without stem cell transplantation. [10] Studies in this field have shown that the prevention of cell degeneration is only possible if all the factors taken at the different stages of stem cells' multiplication and differentiation are administered together. We have demonstrated this in a recent study on the ability of SCDSFs to prevent neurodegeneration in hippocampal cells of the CA1 zone in mice [11]. This experiment demonstrates that the degeneration of the cells of a tissue can be avoided only by the administration of all the differentiating factors able to regenerate and differentiate the stem cells of that tissue, that is to say when the information is complete and redundant. This study confirms previous findings demonstrating that early developmental zebrafish embryo extracts could act as a modulator of senescence in human mesenchymal stem cells (hMSC) isolated from many adult tissues [7-12-13, 14]. These findings have open a promising way for the approaches promoting the rejuvenation and regeneration of different tissues, by-passing stem cell transplantation.
In the present clinical trial we have used SCDSFs to study the possible reversion of neurosensory hearing loss, until now considered an irreversible condition.

RESULTS
The audiograms at times T0 and T1 were evaluated. The comparison The comparison of the audiometric test between the single frequencies examine by setting the cut off to a gain of 10 decibels or higher; or to a recovery of one sensitivity previously totally absent in certain frequencies of the traces on the single frequencies examine by setting the cut off to a gain of 10 decibels or higher; or to a recovery of one sensitivity previously totally absent in certain frequencies. holistic approach leaves the theorical contexts and actually enters the context of medical praxis as information medicine. The present observational work carried out, while presenting some limits, as its essentiality does not allow to evaluate the action of the epigenetic factors at the single levels of the auditory function from the organ of the Corti to the cerebral cortex, is fully inserted as a therapy worthy of attention in the context of fragility associated with secretory phenotype (SAPS). In the future observational researches, it will be our concern to submit each patient affected by neurosensory hearing loss on examination with evoked potentials and speech audiometry. It is reasonable to hope that the simple approach, which leverages functional aspects of diagnostics, can become a model of investigation in individual specialist branches to validate the use of cell regeneration factors in degenerative pathologies, still lacking in therapy.

MATERIALS AND METHODS
The tonal audiometric test, in its simplicity, is able to provide data on the efficiency of the auditory function in its complexity. That is, he can select the so-called transmissive hearing loss from those of the type neuro-sensory. In the context of the latter, however, it is unable to carry out a topodiagnosis; such circumstance does not fall within the scope of the present work, as all the cells that make up the cochlear and nervous pathway are to be considered perennial. In this way the tonal audiometric examination carried out at time T0 and after 2 months of administration of regenerating factors at time T1, will allow us to extend a useful action for the implementation of hearing ability.
The audiometer used in this clinical trial is of the type: LEDISO AD629 BY INTERACOUSTICS.
The examinations were carried out in a silent cabin and in acoustic rest and from the same operator, a factor often requested by neurosurgeons to check with patient treated with radiotherapy against acoustic neurinoma. The comparison of the graphs obtained at time T0 and T1 (after 2 months of therapy) was carried out evaluating each single frequency recorded on the abscissa (250,500,1000,2000,4000,8000 Hz) crossing the intensity plotted in ordinate in dB scale. Gain values equal to or greater than 10 dB were considered significant. The timing of administration at 2 months, for the audiological follow-up, was obtained following pilot cases suffering from chronic atrophic rhinitis, through nasal cytology. Precisely, serial nasal cytologies performed at 10 days of interval had shown the first sign of regeneration of the ciliary apparatus, of which they are provided cells the nasal epithelium, at 2 months. Therefore the administration of 2 months was chosen as a useful period to evaluate a tissue regeneration. Regarding the evaluation of the effectiveness of the perceptive ability or at least the possibility of support a correlation between changes in tonal audiometric thresholds and epigenetic factors, some pilot cases with neurosensory hearing loss had been preliminarily investigated also with vocal audiometry and evoked potentials examination. This methodological premise, which turned out to be encouraging, allowed us to believe that the tonal audiometry alone could be sufficient for the present research. Two products containing the SCDSFs where prepared: one product, named Cell Integrity Brain, contains tablets to dissolve in the mouth which in addition to the SCDSFs are consisting of some anti-oxidant substances, like L-Glutathion, some vitamins like vitamin A, B2, B6, C, D, E, substances like zinc, extracts of Curcuma longa and of Bacopa Monnieri. This product was conceived to protect the cognitive function (Bacopa Monnieri), to preserve the cells from oxidative stress (Vit. C, E, curcuma longa) and to preserve the immune system (Vit A, B6, C, D. Zinc). Another product, named Cell Integrity Age contains tablets to dissolve in the mouth which in addition to SCDSFs are consisting of many other anti-oxidants substances like Resveratrol, Coenzyme Q10, some vitamins like vitamin A, B1, B6, B12, C, D, E, folic acid, and some different substances like Rhodiola Rosea, N-Acetylcarnitine Hydrochloride, Nacetylcysteine, Creatine, and some extracts like Curcuma longa and Blakcurrent. This last integrative product was conceived first of all with the scope to prevent aging, to support the body energy and to reduce the fatigue. It should be emphasize that in any case all the substances added to the 2 products have never shown the ability to regenerate tissues: at experimental level, only SCDSFs have demonstrated the ability to regenerate different tissues in several experimental studies as already reported. The two nutraceutical products were used in this way: three daily