WO2021072595A1

WO2021072595A1 - Medical use of mesenchymal stem cells in treatment of hearing impairment

Info

Publication number: WO2021072595A1
Application number: PCT/CN2019/111034
Authority: WO
Inventors: 许益超; 黄美月; 陈彦聪; 蔡宏逸
Original assignee: 玛旺干细胞医学生物科技股份有限公司; 许益超
Priority date: 2019-10-14
Filing date: 2019-10-14
Publication date: 2021-04-22

Abstract

Provided in the present invention is the medical use of mesenchymal stem cells, in particular the medical use of mesenchymal stem cells of human skin in the treatment of a hearing impairment. The hearing impairment includes, but is not limited to, a sensorineural hearing impairment, mixed hearing impairment and central hearing impairment.

Description

Medical use of mesenchymal stem cells in the treatment of hearing disorders

Technical field

This application relates to the medical use of mesenchymal stem cells, especially the medical use of human skin mesenchymal stem cells in the treatment of hearing disorders.

Background technique

According to statistics, the hearing impaired population in Taiwan has exceeded 120,000 at the end of 2017. Another research report pointed out that there are nearly 28 million people with hearing impairment in China, and in 2013, about one billion people worldwide suffered from different degrees of hearing impairment. Children’s hearing impairment may affect language learning and intellectual development. For adults, it may also cause inconvenience in life and work difficulties. For the elderly, hearing impairment is more likely to reduce their social interaction with others. Causes a sense of loneliness and is more likely to develop dementia earlier.

The process of auditory perception is basically to first make the sound pass through the auricle of the outer ear, the outer auditory canal to the eardrum, and then to the three ossicles located in the middle ear cavity, and then enter the inner ear through the oval window, and from the inner ear located on the cochlear basement membrane. Inner hair cells convert sound signals into nerve signals, which pass through the auditory nerve and brainstem, and finally reach the auditory area of the brain to produce hearing. A problem at any point in this path may cause hearing impairment. Hearing disorders can be caused by many different causes, including genetic defects, aging, noise damage, pathogenic microbial infections, childbirth complications, ear trauma, drug or poison damage, etc. According to the location of occurrence, hearing impairment can be divided into five types: (1) Conductive hearing impairment: occurs in the outer or middle ear. The causes include cerumen embolism, foreign body, inflammation, tympanic membrane perforation, middle ear effusion, and ossicles. Rupture or dislocation, etc., usually belong to mild to moderate hearing impairment, which can be improved by drugs or surgery; (2) Sensorineural hearing impairment: it occurs in the inner ear or auditory nerve, usually because the auditory hair cells in the cochlea are damaged Or it is caused by lack of it. The etiology includes viral infection, ototoxic drug poisoning, aging, noise damage, etc. Patients suffering from this type of hearing impairment are usually less sensitive to high-frequency sounds; (3) Mixed hearing impairment, patients are combined Suffering from conductive hearing impairment and sensorineural hearing impairment; (4) Central hearing impairment, which occurs in the central auditory nervous system, and causes include aging, brain injury, neuropathy, etc.; and (5) Functional hearing impairment, patients No organic disease on the auditory canal occurred, but due to psychological or emotional factors, the sensitivity to sound decreased.

For example, platinum-containing chemotherapeutic drugs are widely used to treat sarcomas, malignant epithelial tumors, lymphoma and germ cell tumors, such as head and neck cancer, brain tumors, ovarian cancer, bladder cancer, and non-small cell lung cancer. Among them, cisplatin is used (cisplatin; cis-dichlorodiammine platinum (II)) is the most representative. However, a serious side effect of this type of chemotherapeutic drugs is ototoxicity, which significantly reduces the quality of life of cancer patients. The ototoxicity of cisplatin mainly occurs in the cochlea and is generally believed to be related to the production of reactive oxygen species. Cisplatin is absorbed by stria vascularis, cochlear fluid, and hair cells to enter the cochlea tissue, which activates the third isomer of NADPH oxidase and increases the content of reactive oxygen species in the cochlea. On the one hand, this leads to the exhaustion of endogenous antioxidants (such as glutathione), allowing reactive oxygen species to attack cells, on the other hand, it activates the transcription factor NFκB to produce a large amount of pro-inflammatory cytokines, resulting in external hair cells, Spiral ganglion cells and striatal blood vessel border cells undergo apoptosis, which leads to hearing impairment (see Paken et al., J Toxicol, 2016, Vol. 2016, Article ID 1809394).

Since the hair cells cannot regenerate after death, hearing impairment involving hair cells is often irreversible and cannot be restored or improved by surgery or medication. Patients can only seek improvement by wearing hearing aids. Therefore, the industry has a strong demand for medical methods and pharmaceutical compositions that can effectively treat hearing impairment and are safer and have fewer side effects.

Summary of the invention

Mesenchymal stem cells (MSCs) are multipotent stem cells, which have the ability of cell proliferation and multidirectional differentiation, and can differentiate into chondrocytes, adipocytes, sclerocytes, etc. In recent years, because mesenchymal stem cells have the function of repairing and replacing damaged neurons, they have been actively used in the development of treatments for brain trauma, stroke and neurodegenerative diseases (Hosseini et al., Int J Stem Cells 8:191 -9; and Yoo SW et al., Exp Mol Med 40:387-97). In addition, mesenchymal stem cells also have the properties of regulating human immune function and reducing cell oxidative stress. Through the action of exocrine microvesicles, it can regulate the imbalance between immune function and oxidative stress in the body.

By establishing an animal model of hearing impairment, the inventors of the present application found that mesenchymal stem cells, especially human mesenchymal stem cells, such as human skin mesenchymal stem cells, can effectively improve this animal model of hearing impairment. The disclosure of this application shows that mesenchymal stem cells can bring beneficial effects on hearing impairment, and can be used as an alternative or supplementary medicine for the treatment of hearing impairment.

Therefore, according to the first aspect of the present invention, it provides a use of a mesenchymal stem cell composition in the preparation of a medicament for the treatment of hearing disorders in an individual, wherein the mesenchymal stem cell composition comprises mesenchymal stem cells And a pharmaceutically acceptable carrier.

According to a second aspect of the present invention, it provides a method for treating hearing impairment in an individual, the method comprising administering to the individual an effective amount of a mesenchymal stem cell composition; wherein the mesenchymal stem cell composition comprises Mesenchymal stem cells and pharmaceutically acceptable carriers.

In a preferred embodiment, the mesenchymal stem cells are skin mesenchymal stem cells. In another preferred embodiment, the mesenchymal stem cells are derived from humans. In a more preferred embodiment, the mesenchymal stem cells are human skin mesenchymal stem cells.

In a preferred embodiment, the hearing impairment is selected from sensorineural hearing impairment, mixed hearing impairment and central hearing impairment. In a more preferred embodiment, the hearing impairment is selected from sensorineural hearing impairment.

In a preferred embodiment, the sensorineural hearing impairment is caused by ototoxic drugs. In a more preferred embodiment, the ototoxic drug is selected from platinum-containing chemotherapeutic drugs. In a most preferred embodiment, the platinum-containing chemotherapeutic drug is selected from cisplatin.

In a preferred embodiment, the individual is a human.

In a preferred embodiment, the mesenchymal stem cell composition is prepared as an injection form of a sterile liquid solution or suspension.

Description of the drawings

Figure 1 is the process of establishing a mouse model of hearing impairment, showing that cisplatin was injected through the abdominal cavity every day from day 0 to day 5, and human skin mesenchymal stem cells were injected through the tail vein on day 8;

Figure 2 is a bar graph showing the performance of each group of mouse models in the auditory brainstem response (ABR) test; and

Fig. 3 is a photograph of tissue analysis after immunofluorescence staining, showing the condition of hair cells in the cochlea tissue of each group of mouse models.

Detailed ways

Unless otherwise stated, the following terms used in this specification and appended claims have the following definitions. It should be noted that the indefinite article "a" or "an" used in this specification and claims is intended to mean one or more than one, such as "at least one", "at least two" or "at least three ", and does not just refer to a single one. In addition, the terms "including," "including," and "having" used in the claims are open-ended terms and do not exclude elements that are not mentioned. Unless otherwise stated, the term "or" generally encompasses "and/or." The term "about" used throughout the specification and appended claims is used to describe and represent minor changes that do not materially affect the nature of the present invention.

In the specification of this application, the term "mesenchymal stem cells" or "MSCs" for short refers to multipotent stem cells (multipotent stem cells) derived from adult stromal tissues. These adult stromal tissues include but are not limited to bone marrow, umbilical cord, amniotic membrane, Amniotic fluid, adipose tissue, pulp cavity, skeletal muscle and skin. MSCs have the ability to self-renew and have the ability to differentiate into cells of the mesenchymal cell lineage. The MSC used in this application can be collected from humans, rats, mice, sheep, cows, pigs, dogs, cats, horses, and non-human primates (such as monkeys, gorillas, and chimpanzees). In a preferred embodiment, the MSCs are derived from humans. In a preferred embodiment, the MSCs used in this application are isolated from the skin, namely skin mesenchymal stem cells (SMSCs). Compared with other MSCs, SMSCs have the advantages of abundant sources, convenient materials, high number and purity of isolated cells, and the advantages of maintaining the characteristics of stem cells after multiple subcultures. In a more preferred embodiment, the skin mesenchymal stem cells are human skin mesenchymal stem cells.

Since MSCs gradually lose their viability as the number of generations increases, MSCs from the 1st to 10th generations are usually used, and MSCs from the 2nd to 6th generations are preferably used.

MSCs can be collected from various sources by methods known in the art, usually stem cells are isolated from tissues. Subsequently, the MSCs were incubated in an appropriate medium for a period of time, and then the supernatant was collected. For example, in the specific example of collecting SMSCs, the skin tissue is first obtained from the skin of the provider through a surgical operation, then the tissue is digested with collagenase, the cell clusters precipitated after centrifugation are washed with phosphate buffer, and then The cells were cultured in a culture medium and other cells were removed to collect SMSCs. In a preferred embodiment, the collection of MSCs can also include isolating MSCs from cell culture by using differences in surface antigen markers. Non-limiting examples of isolation methods include magnetic cell sorting (MACS), fluorescence activated cell sorting (FACS), and flow cytometry sorting (FCS). If more than 95% of the collected cells have the three cell surface markers CD90, CD73 and CD105, it can be confirmed that they are human skin mesenchymal stem cells. Subsequently, the collected MSCs are cultured in an appropriate medium for at least 3 hours, preferably 3 to 120 hours, more preferably 24 to 96 hours, for example 72 to 96 hours.

The above-mentioned "medium" refers to any liquid medium containing in vitro culture for supporting human or animal cells. In a preferred embodiment, the medium is a basic medium containing basic nutrients such as inorganic salts, amino acids and vitamins. Examples of basal media suitable for the present invention include but are not limited to Eagle's Basic Medium (Basal Medium Eagles; BME), Minimum Essential Medium (MEM), Dubbock's Modified Eagle's Medium ( Dulbecco's Modified Eagle's Medium; DMEM), nutrient mixed medium F-10 (HAM's F-10), nutrient mixed medium F-12 (HAM's F-12), or a combination thereof. These media can be easily prepared or purchased from the market. In a more preferred embodiment, the medium is DMEM.

If necessary, blood components such as serum, plasma, and platelet-rich plasma may be added to the culture medium to support the growth of the cultured cells. In a preferred embodiment, the medium is supplemented with serum, such as DMEM supplemented with serum. The "serum" referred to here refers to a liquid preparation derived from human or animal blood, in which blood cells, fibrinogen and fibrin are removed to provide nutrients for cell growth, especially Refers to serum preparations derived from humans or animals living in non-epidemic areas, including but not limited to human serum, fetal bovine serum (FBS), calf serum, adult bovine serum or serum from other animals, such as from Serum preparation for horses and camels. The amount of serum in the culture medium is in the range of about 0.5 to 20% by volume based on the total volume of the culture medium. If necessary, the cell culture medium can be further supplemented with other components, such as vitamins, proteins and sugars, growth factors (such as FGF and EGF), antibiotics (such as penicillin, streptomycin, and tetracycline), fungicides, hormones, and antioxidants Wait. For details on cell culture media and culture methods, please refer to Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal Cell Culture Alan R. Liss, New York (1984) and Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons Ltd., Chichester, England, United Kingdom, 1996.

The term "culture" refers to the maintenance of MSCs under in vitro conditions that are conducive to the growth and survival of MSCs. The method of culturing MSCs belongs to the conventional and routine methods in the technical field involved in this application. This application uses standard methods to cultivate MSCs using aseptic processing and manipulation. Generally, the optimum temperature for culture is about 35 to 37°C. In a preferred embodiment, the cells are cultured in a carbon dioxide incubator. The carbon dioxide incubator is usually set at a constant temperature (for example, 37°C), a stable CO ₂ level (for example, 5%), a constant pH (for example, pH 7.2-7.4), and a relatively high relative saturation humidity (for example, 95%), To simulate the growth environment of cells in organisms.

After the culture is completed, the MSCs culture can be processed by conventional means such as centrifugation or filtration to remove the aqueous part, and then trypsin is used to detach the MSCs from the attached surface, thereby harvesting the cells.

As disclosed in this specification, MSCs, especially SMSCs, such as SMSCs derived from human skin tissue, have the effect of treating hearing disorders. The term "hearing impaired" as used in this application generally refers to an individual's decreased sensitivity to sound perception. For human individuals, "hearing impairment" mainly refers to their low sensitivity to the frequency of daily speech. According to the standards promulgated by the World Health Organization (WHO) in 1997, the severity of hearing impairment can be divided into 4 levels according to the average hearing threshold at four frequencies of 500Hz, 1000Hz, 2000Hz and 4000Hz: 26-40 decibels (dB) It belongs to mild hearing impairment, 41 to 60 decibels are moderate, 61 to 80 decibels are severe, and greater than 80 decibels are depth. Clinically, hearing impairment can be diagnosed through hearing tests. These hearing tests include (1) behavioral or subjective hearing tests, such as pure tone audiometry, tuning fork tests, voice hearing tests, etc.; and (2) physiological or objective tests Hearing tests, such as auditory brainstem response (ABR) test, impedance audiometry, oto-acoustic emissions, etc. Additional tympanic hearing tests, computed tomography and magnetic resonance imaging can also be performed to assist specialists in diagnosis. In a preferred embodiment, the hearing impairment includes, but is not limited to, conductive hearing impairment, sensorineural hearing impairment, mixed hearing impairment, and central hearing impairment. In a more preferred embodiment, the hearing impairment is selected from the group consisting of sensorineural hearing impairment, mixed hearing impairment and central hearing impairment. In a more preferred embodiment, the hearing disorder is selected from sensorineural hearing disorders, in particular sensorineural hearing disorders involving degeneration, injury or death of hair cells in the cochlea. The damage of the hair cells can be evaluated by the histochemical analysis of Example 3 below and/or the audiograms obtained from the above-mentioned hearing examination showing the loss of sensitivity to mid- and high-frequency sounds.

In a preferred embodiment, the hearing impairment is caused by congenital genetic defects of hair cells (such as mitochondrial DNA mutations, OTOF gene mutations), acquired chemical or physical damage (such as ototoxic drug poisoning, noise Injuries), infectious pathogen infections (such as meningitis, mumps, measles, scarlet fever, influenza) or degeneration of senile sexual function (such as insufficient blood supply to hair cells due to vascular sclerosis). In a more preferred embodiment, the hearing impairment is caused by an ototoxic drug. The term "ototoxic drug" used in this application refers to a drug that may cause damage to the outer hair cells and/or inner hair cells in the cochlea after administration, which includes but is not limited to (1) aminoglycoside antibiotics , Such as gentamicin, kanamycin, polymyxin, dihydrostreptomycin, neomycin, etc.; (2) anti-cancer agents, including platinum-containing chemotherapeutic drugs (such as cisplatin, carboplatin (carboplatin) ), nedaplatin, oxaliplatin, lobaplatin and miriplatin), vinblastine anticancer agents, methotrexate, etc.; (3) Antimalarial drugs, such as quinine; (4) diuretics, such as ethacrynic acid, furosemide; (5) steroids, such as corticotropin, prednisone ); (6) Non-steroidal analgesics, such as salicylic acid, diclofenac, and ibuprofen; (7) β receptor blockers, such as practolol, metoprolol ); (8) Antiepileptic agents, such as phenytoin and ethosuximide; (9) Mood modifiers, such as prozac, alprazolam; and other drugs, such as Thalidomide (thalidomide). More preferably, the ototoxic drug is selected from the above platinum-containing chemotherapeutic drugs, with cisplatin being the most preferred.

The inventor of the present application established an animal model of hearing impairment by administering cisplatin to mice through the ototoxicity of cisplatin. The present application evaluated the medical effect of MSCs on hearing impairment by administering MSCs to this animal model. The auditory brainstem response test described in Example 2 and the histochemical analysis described in Example 3 below consistently show that the typical symptoms of hearing impairment caused by cisplatin, such as hearing loss and ear hair Cell damage can be improved by administering MSCs. It is worth noting that the administration of ototoxic drugs is selected as a representative hearing impairment trigger mode, because the typical hearing impairment symptoms caused by them also commonly appear in hearing impairments caused by other factors.

Although not wishing to be bound by any theory, we speculate that when MSCs enter the body of patients with hearing impairment, they can secrete a variety of growth factors, cytokines and chemokines, which can enhance cell survival, repair cells, and reduce inflammation. MSCs may also reduce the oxidative pressure of cells by releasing exocrine microvesicles and prevent them from going to apoptosis.

Therefore, this application covers the medical use of MSC for the treatment of hearing impairment in an individual, and a method of treating hearing impairment in an individual, the method comprising administering an effective amount of MSC to the individual.

The term "individual" as used herein is intended to encompass human or non-human vertebrates, such as non-human mammals. Non-human mammals include domestic animals, companion animals, laboratory animals, and non-human primates. Non-human individuals also include, but are not limited to, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, minks, and rabbits. It should be understood that the preferred individuals are humans, especially human patients suffering from or at risk of hearing impairment.

The term "treatment" in this specification means to reverse, alleviate, delay the onset or inhibit the progress of the mood disorders described in this specification or one or more of their symptoms. In some specific cases, treatment can be performed after one or more symptoms develop. Treatment can also be continued after the symptoms are resolved to delay its recurrence.

MSCs can be formulated together with a pharmaceutically acceptable carrier to prepare a mesenchymal stem cell composition suitable for administration to an individual. "Pharmaceutically acceptable carrier" as used in this application means an inert substance used as a carrier for MSCs, which has no toxicity, irritation, pyrogenicity, antigenicity, or hemolysis to the site of application, and no The substantial pharmacological activity will not hinder the exertion of the beneficial effects of the MSCs. These pharmaceutically or cosmetically acceptable carriers are familiar to those skilled in the art to which this application belongs (see Remington: The Science and Practice of Pharmacy, Niceteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John .,Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton,Pennsylvania 1975; Liberman,HAand Lachman,L.,Eds.,PharmaceuticalDosage Forms,MarcelDecker,NewYork,NY,1980;DosageDrugSystemDelivery , Seventh Ed. (Lippincott Williams & Wilkins 1999)). Generally, the amount of the pharmaceutically acceptable carrier is from about 1% to about 99.9%, preferably from about 50% to about 99%, based on the total weight of the composition. The suitable type of pharmaceutically acceptable carrier depends on the form of the composition.

MSCs can be administered to an individual by any suitable route. In a preferred embodiment, the mesenchymal stem cell composition is prepared as an injection, which is in the form of a sterile liquid solution or suspension, to introduce MSCs into an individual's body via veins, arteries, or spinal fluid. In this specific example, it is preferable that the pharmaceutically acceptable carrier is isotonic with the blood of the individual. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, physiological saline, balanced salt solutions (e.g., phosphate buffered physiological saline (PBS), Hank's balanced salt solution (HBSS)), vegetable oils, dextrose, glycerin , Ethanol, wetting agent, emulsifier and pH buffering agent. In some specific cases, it may be necessary to formulate the composition with a preservative (such as thimerosal or sodium azide) to facilitate long-term storage. The carrier may also contain other pharmaceutically acceptable excipients for changing or maintaining the pH, osmolarity, viscosity, transparency, color, sterility, stability, dissolution rate, or odor of the composition.

According to the present invention, the term "administration" includes allocating, delivering, or administering MSCs in a suitable composition to an individual by any suitable route, so as to administer the mesenchymal stem cell composition or its metabolome. It is delivered to the desired location in the individual, so that the composition or its metabolite group is brought into contact with the target cell or tissue. In a specific example, the mesenchymal stem cell composition is administered to the individual before, during, and/or after the onset of the emotional disorder. In a specific example, one or more therapeutic agents can be administered to the individual together with the mesenchymal stem cell composition. The mesenchymal stem cell composition may be administered before the one or more therapeutic agents (e.g. 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days). , 3 days, 4 days, 5 days, 6 days, 7 days or longer), at the same time or after (e.g. 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or longer) administration. The mesenchymal stem cell composition and the therapeutic agent can be administered by different schedules (for example, different plans), different administration routes, or different dosages.

The mesenchymal stem cell composition is administered to an individual in a therapeutically effective amount to induce the biological or drug response in cells, tissues, systems, animals or humans sought by researchers, veterinarians, doctors or other clinicians, preferably stable , Improve or alleviate one or more symptoms of the disease condition in the individual, such as tinnitus, hair cell death, hearing loss or loss, ear stuffiness, intermittent sound blur, poor or distorted speech recognition, hearing hallucinations , Avoid social interaction, dizziness, and headaches. Therefore, the term "effective amount" refers to the amount of the mesenchymal stem cell composition that produces the medicinal effect of observing any of the above-mentioned symptoms when an effective amount of the composition is administered to an individual. Although the effective amount is usually determined by comparing them with the effect observed in the absence of the mesenchymal stem cell composition disclosed herein (ie, the control group), the actual dose is calculated according to the specific route of administration selected . The actual dose can be calculated according to the particular route of administration selected. Those skilled in the relevant art will routinely further refine the calculations required to determine the appropriate dosage. Therefore, when administered to a human individual, it is preferably 1×10 ⁴ cells/kg body weight/day to 1×10 ⁷ cells/kg body weight/day per day, weekly or twice a week, for example, 5× The mesenchymal stem cell composition is administered in an amount of ^{10 5} cells/kg body weight/day to 5×10 ^{6 cells/kg body weight/day.}

In a preferred embodiment, the mesenchymal stem cell composition is used for autologous cell therapy, that is, mesenchymal stem cells are obtained from an individual in advance, cultured in vitro, and then transplanted back to the same individual. As known to those skilled in the art to which this application belongs, autoimmune therapy has relatively few side effects, high safety, and is not prone to allergies and rejection.

In another specific example, the composition mainly consists of MSCs and the aforementioned pharmaceutically acceptable carrier. The "mainly composed of" as used herein means that the described combination of components does not exclude the inclusion of other undescribed components that do not substantially affect the properties and functions of the aforementioned composition. In another specific example, the composition of the present application only consists of MSCs and the aforementioned pharmaceutically acceptable carrier.

The following examples are only used to illustrate the present invention, but not to limit the scope of the present invention. The data contained in the examples are presented as mean±standard deviation. Stuart’s t-test was used for two-group comparisons, and single-way analysis of variance was used to compare the mean differences between multiple groups. When the p value is less than 0.05, it is considered as a significant difference.

Preparation Example 1: Preparation of skin mesenchymal stem cells

The human skin sample was cut into a size of about 3 mm ^{2 and} transferred to an enzyme reaction solution containing neutral protease (dispase) and collagenase, and reacted at 37°C for 16 hours. The skin sample fragments and the enzyme reaction solution were transferred to a centrifuge tube, and an equal volume of Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical Co., St. Louis, Missouri, USA) supplemented with an appropriate amount of serum was added for enzyme neutralization. Centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and add DMEM medium containing serum and antibiotics to suspend the cells and move them to a new petri dish, and place them in a carbon dioxide incubator at 37°C for culture. Change the fresh medium every 3-4 days, and perform subcultures when the cell density is about 80% full. Flow cytometry was used to detect the CD markers of skin mesenchymal stem cells, that is, the expression of cell surface antigens CD73, CD90 and CD105, to confirm that the cultured cells are skin mesenchymal stem cells.

The aqueous part of the skin mesenchymal stem cell culture is removed, and the cells are washed, and then the cells are trypsinized to remove the cells from the culture container, and then the cells are suspended in an aqueous carrier for later use.

Example 1: Mouse Model of Hearing Impairment

8-week-old C57BL/6 female mice were purchased from Lesco Biotechnology Co., Ltd. (Taipei City). Animals are kept in a sterile environment with automatic 24-hour temperature and humidity control. The room temperature is maintained at 20-22°C, humidity is 50% ± 5%, and the lighting cycle is 12/12 hours day and night. The animals are fed standard rodent feed. As shown in Figure 1, from day 0 to day 5, cisplatin (purchased from Sigma–Aldrich Chemical Co., St. Louis, Missouri, USA) was injected intraperitoneally every day from day 0 to day 5 at a dose of 4 mg/kg mouse body weight to establish Animal model of hearing loss. The control group was intraperitoneally injected with an equal volume of 0.25% dimethyl sulfoxide (DMSO).

The animals were randomly divided into five groups, including (I) DMSO-vehicle control group: from day 0 to day 5, 100 μL of an aqueous solution containing 0.9% sodium chloride and 0.25% dimethyl sulfoxide was injected intraperitoneally every day, On the 8th day, 100 μL of MSC-free vehicle was injected through the tail vein (n=6; that is, there are 6 mice in this group); (II) DMSO-MSC control group: from day 0 to day 5, through every day Intraperitoneal injection of 100 μL of an aqueous solution containing 0.9% sodium chloride and 0.25% dimethyl sulfoxide, 100 μL of the MSC preparation prepared in Preparation Example 1 (3×10 ⁶ cells) (n=6) was injected into the tail vein on the 8th day (III) CIS-vehicle group: daily intraperitoneal injection of 100 μL of cisplatin (dissolved in 0.9% NaCl) from day 0 to day 5, and intravenous injection of 100 μL of MSC-free carrier on day 8 (n= 9); (IV) CIS-MSC group: daily intraperitoneal injection of 100 μL of cisplatin from day 0 to day 5, and intravenous injection of 100 μL of MSC preparation prepared in Preparation Example 1 (1×10 ⁶ ) on day 8 Cells) (n=9); and (V) CIS-MSC group: daily intraperitoneal injection of 100 μL of cisplatin from day 0 to day 5, and intravenous injection of 100 μL of MSC preparation prepared in Preparation Example 1 on day 8 (3×10 ⁶ cells) (n=9).

Example 2: Auditory brainstem response (ABR) test

As shown in Figure 1, for the animal model established in Example 1, the auditory brainstem response (ABR) test was used to evaluate the hearing threshold of each group of animals before the administration of cisplatin on day 0 and on the 15th day, and records were recorded The difference is the threshold offset (in decibels (dB)). The larger the threshold shift value, the more severe the hearing loss caused by cisplatin. In the ABR test, ketamine (40 mg/kg) and xylazine (xylazine; 10 mg/kg) were used to anesthetize the animals, and the animals were kept warm during the entire test on a hot pad. Insert the probe electrode under the skin of the animal's head, and insert the reference electrode under the skin under the animal's ear shells, and then use the ABR workstation (BIOPAC Systems) to give a series of low-frequency 4kHz, medium-frequency 8kHz or high-frequency 12kHz tone bursts. The ABR waveform is averaged in the interval of 10 milliseconds. The sound intensity changes every 10 decibels around the hearing threshold. Based on ABR records, blind analysis was performed by two independent observers.

Figure 2 is a histogram drawn based on the average values obtained from three independent ABR tests. It shows that the mice in group (III) injected with cisplatin, whether given a series of low-frequency, intermediate-frequency, or high-frequency sound bursts, its The threshold deviation of the ABR test was higher than that of the mice injected with DMSO in group (I), and the statistical analysis reached the significance of p<0.05. This indicates that the mice injected with cisplatin showed significant hearing loss compared to the control group, and this application successfully established an animal model of hearing loss. After the injection of MSC preparations (groups (IV) and (V)), the threshold shift caused by cisplatin was significantly improved under high-frequency audible bursts, compared with group (III) Up to a statistically significant difference, but no significant difference was observed under low-frequency and mid-frequency tone bursts. This is because mice have keen hearing for high-frequency sounds above 12kHz, but are relatively insensitive to low-frequency and mid-frequency sounds. The results of this example indicate that injection of MSC preparation has the effect of improving hearing loss.

Example 3: Histochemical analysis

On the 22nd day, the mice in each group of Example 1 were anesthetized with isoflurane, and were first perfused with normal saline and then with paraformaldehyde phosphate buffer. The cochlear tissue located in the inner ear of the animal was collected and fixed with freshly prepared 4% paraformaldehyde at 4°C for 30 minutes. The cochlear tissue was embedded in a frozen tissue embedding agent, and then sliced into a thickness of 10 microns. At 37°C, all the sections were incubated with 4',6-diamidino-2-phenylindole, multiple MYO7A antibodies, and phalloidin to display the nucleus of ear hair cells by fluorescent staining. Myosin and cytoskeleton. After washing three times with phosphate buffer, the condition of ear hair cells was observed under a laser conjugate focus microscope (model TCS-SP2; Leica Microsystems Heidelberg GmbH, Heidelberg, Germany).

Figure 3 shows that the ear hair cells of the mice in the group (III) injected with cisplatin showed obvious defects (as indicated by the arrow) than the ear hair cells of the mice in the group (I) injected with DMSO. This once again points out that this application has successfully established an animal model of hearing loss. Compared with the group (III), the mice (groups (IV) and (V)) that were given MSC preparations through the tail veins had significantly improved ear hair cell defects.

The above embodiments are only for illustrating the present invention, but not for limiting the present invention. Various alterations or changes made by those skilled in the relevant fields without departing from the technical scope of the present invention should also belong to the protection category of the present invention.

Claims

A use of a mesenchymal stem cell composition in the preparation of a medicament for treating hearing disorders in an individual, wherein the mesenchymal stem cell composition comprises mesenchymal stem cells and a pharmaceutically acceptable carrier.
The use according to claim 1, wherein the mesenchymal stem cells are skin mesenchymal stem cells.
The use according to claim 2, wherein the skin mesenchymal stem cells are human skin mesenchymal stem cells.
The use according to claim 1, wherein the hearing impairment is selected from the group consisting of sensorineural hearing impairment, mixed hearing impairment and central hearing impairment.
The use according to claim 4, wherein the hearing impairment is selected from sensorineural hearing impairment.
The use according to claim 5, wherein the sensorineural hearing impairment is caused by ototoxic drugs.
The use according to claim 6, wherein the ototoxic drug is selected from platinum-containing chemotherapeutic drugs.
The use according to claim 7, wherein the platinum-containing chemotherapeutic drug is selected from cisplatin.
The use according to claim 1, wherein the individual is a human.
The use according to claim 1, wherein the mesenchymal stem cell composition is prepared as an injection form of a sterile liquid solution or suspension.