KR20110066443A - Neural differentiation of human adipose tissue-derived stem cells - Google Patents

Abstract

PURPOSE: A method for differentiating human adipose-derived stem cells to nerve cells is provided to effectively treat neurologic disorder or hearing loss with minimized side effects. CONSTITUTION: A method for differentiating to nerve cells for treating neurologic disorder or hearing loss comprises: a step of obtaining human adipose-mesenchymal stem cells; a step of inducing the differentiation of the mesenchymal stem cells in a medium containing bFGF; and a step of inducing secondary differentiation.

Description

Neural differentiation of human adipose tissue-derived stem cells

The present invention relates to a method for differentiating neurons from human adipose derived stem cells, and more particularly, to a method for producing a large amount of homogenous stem cells from a human adipose cells in a simple and efficient manner and from the stem cells It relates to a method of differentiation of optimized neurons.

Despite the remarkable achievements of medical development, there are still intractable diseases that cannot be fixed by modern medicine.

Parkinson's disease, also known as intractable disease, chronic diseases such as Alzheimer's disease, and various cancers (cancer) damaged by tissues and organs to treat the treatment is usually a method of treatment with medication or surgery. However, this treatment has been pointed out as a problem by damaging many other normal cells. Recently, cell replacement therapy, which supplies cells destroyed or damaged by diseases from the outside, has been suggested as an effective treatment. In such cell therapies, stem cells capable of proliferating and differentiating into tissues in need of regeneration are in the spotlight.

The stem cells are cells prior to differentiation into each cell constituting the tissue, and are capable of infinite proliferation in an undifferentiated state and have a potential of being differentiated into cells of various tissues by specific differentiation stimulation.

Among them, mesenchymal stem cells are attracting attention as core cells because they can differentiate into bone, cartilage, muscle, tendon, ligament, and nerve tissue. Currently, the tissues of origin of these mesenchymal stem cells include bone marrow, cord blood, periosteum, synoviium, and the like. However, although there are many mesenchymal stem cells in bone marrow, there are many limitations in the use due to the pain and burden on patients, and the periosteum and synovial membrane have many difficulties in harvesting and the number of cells obtained is limited.

Recently fat contains stem cells (WO00 / 53795; WO03 / 022988; WO01 / 62901; Zuk, PA, et al., Tissue Engineering, Vol. 7, 211-228; Zuk, PA, et al , Molecular Biology of the cell, Vol. 13, 4279-4295, 2002) .A higher amount of stem cells can be obtained from fat than in other tissues such as bone marrow and has been reported to have a higher density of stem cells. It is troublesome to treat adipose tissue, which is a source of protein, with enzymes such as collagenase. Also, retinoic acid is responsible for the differentiation of differentiated cells, the time required for differentiation of neurons, and the differentiation efficiency into neurons. There is a limitation in that it is not possible to simply generate a large amount of stem cells. However, in spite of the above limitations, in order to apply stem cells and precursor cells to regenerative medicine, there is a need for a method for generating homogeneous stem cells and precursor cells in large quantities.

Accordingly, the present inventors have found a method for generating a large amount of homogenous stem cells from human adipocytes in a simple and efficient manner, and a method for differentiating efficiently optimized neurons from the stem cells, and completed the present invention.

An object of the present invention is to provide a method for producing a large amount of homogeneous mesenchymal stem cells from human fat cells at a simple and low cost.

Another object of the present invention to provide a differentiation-inducing medium for the differentiation of neurons for the treatment of hearing loss diseases or neurological diseases from the mesenchymal stem cells generated and a culture method using the same.

Another object of the present invention is to provide a neuron for the treatment of hearing loss diseases or neurological diseases cultured using the optimal differentiation maintenance medium.

Another object of the present invention to provide a cell composition for the treatment of deafness or cerebral nervous system disease using neurons as an active ingredient.

In order to achieve the above object, the present invention provides a method for producing a large amount of homogeneous mesenchymal stem cells from human adipocytes at low cost and a nerve for treating deafness or brain nervous system disease from the generated mesenchymal stem cells. It provides a differentiation induction medium for differentiation of cells and a culture method using the same.

The present invention provides a neuronal cell for the treatment of hearing loss disease or cerebral nervous system disease cultured using the optimal differentiation maintenance medium and a cell composition for the treatment of hearing loss disease or cerebral nervous system disease comprising the neuron as an active ingredient.

Hereinafter, the present invention will be described in detail.

Adipose-derived mesenchymal stem cells of the present invention are characterized by obtaining adipose derived mesenchymal stem cells by treating trypsin at a concentration of 0.02 to 0.07% containing EDTA from human adipose cells.

The method of differentiation of neurons for the treatment of deafness or cerebral nervous system diseases of the present invention is characterized in that the primary neuronal differentiation and secondary neuronal differentiation induction proceeds sequentially, wherein the primary neuronal differentiation is a growth factor. Cultured for 5 to 10 days in primary neuronal differentiation induction medium containing phosphorus basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), the mesenchymal stem cells induced primary neuronal differentiation are forskolin Induced secondary neuronal differentiation for 3 to 5 days in the secondary neuronal differentiation induction medium containing (forskolin), characterized in that the differentiation into neurons.

Primary neuronal differentiation induction medium according to the present invention is characterized in that it contains 10 to 30 ㎍ / ㎖ bFGF and 80 to 120 ㎍ / ㎖ EGF, and contains a serum of 0.5 to 2% concentration, 2 Primary neuronal differentiation induction medium is characterized by containing 5 to 15 concentrations of forskolin, and 3 to 10% concentration of serum.

The present invention

a) obtaining adipose derived mesenchymal stem cells from human fat cells;

b) inducing primary differentiation of the obtained mesenchymal stem cells in primary neuronal differentiation induction medium containing basic growth factors (bFGF) and epidermal growth factor (EGF) as growth factors; And

c) inducing secondary differentiation of the primary differentiation-induced mesenchymal stem cells in a secondary neuronal differentiation induction medium containing forskolin, and then differentiating into neurons;

It provides a method for differentiation of neurons for the treatment of deafness or cerebral nervous system disease comprising a.

Adipose-derived mesenchymal stem cells according to the present invention have a very important meaning for the differentiation of a large amount of neurons in the last stage.

In the production method of the present invention, the adipose-derived mesenchymal stem cells of step a) is obtained by treating trypsin at a concentration of 0.02 to 0.07% containing EDTA human fat cells, more specifically human fat Adipocytes isolated from the adipose tissues were made using tweezers, etc., and then treated with 0.05% trypsin containing EDTA within 5 minutes, and cultured for 1 to 2 weeks. Obtained.

In order to confirm the differentiation ability of the obtained adipose derived mesenchymal stem cells into mesodermal cells, oil red O staining, alkaline phosphatase staining and Alcian blue staining result, the mesenchymal stem cells are fat cells, bone It was confirmed that all of the differentiation capacity into mesodermal cells such as cells and chondrocytes. See FIG. 2.

In the production method of the present invention, the primary differentiation of step b) comprises the mesenchymal stem cells obtained in step a) containing the basic growth factor (bFGF) and EGF (epidermal growth factor) as growth factors. It is induced by incubation for 5 to 10 days in the primary neuronal differentiation induction medium, wherein the primary neuronal differentiation induction medium is 10 to 30 µg / ml bFGF and 80 to 120 µg / ml EGF and 0.5 to 2 It is characterized in that it contains a serum concentration of%, the bFGF and bEGF is made into a high concentration stock (stock), frozen and stored at the required time can be added to the primary neuronal differentiation induction medium, inducing primary neuronal differentiation Change the medium every two days, preferably one week.

In the production method of the present invention, the second differentiation induction of step c) is the primary differentiation-induced mesenchymal stem cells in step b) in the secondary neuronal differentiation induction medium containing forskolin (3) Induced by incubation for 5 to 5 days, the secondary neuronal differentiation induction medium is characterized in that it contains 5 to 15 concentrations of forskolin and 3 to 10% of the serum, the forskolin ) Can be stored in high concentration stock (stock), refrigerated and melted when necessary and added to the secondary neuronal differentiation induction medium, and secondary neuronal differentiation induction is preferably maintained for 4 days.

The primary and secondary neuronal differentiation inducing media can differentiate mesenchymal stem cells into neurons when used sequentially for a suitable period. If the order of use of the medium is changed or if one or more of the medium is not used, differentiation into desired neurons may be hindered, so that the composition, sequential processing and culture period of the differentiation-inducing medium according to the present invention are important. Has

More specifically, 20 μg / ml of bFGF and 100 μg / ml of EGF were added to DMEM medium containing 1% serum for neuronal differentiation of the mesenchymal stem cells obtained. After primary neuronal differentiation was induced, 10 neuronal cell differentiation was induced for 4 days by adding 10 forskolin to DMEM medium containing 5% serum.

In addition, in order to confirm the differentiation efficiency of neurons differentiated from human adipocytes according to the present invention, as a result of confirming the protein expressed in the cells by using cell immunofluorescence staining, neuronal markers (Tuj1, NF-L) , And MAP2) can be confirmed that a large amount of differentiation efficiency into neurons, and the morphology of the cells can be more surely observed to differentiate into neuron-like cells. See FIG. 3.

The differentiation method of neurons for the treatment of deafness or cerebral nervous system disease according to the present invention can obtain a large amount of homogeneous mesenchymal stem cells in a short time at a low cost, the differentiation induction medium according to the present invention and a culture method using the same The primary neuronal differentiation and secondary neuronal differentiation induction due to the sequential progression has the advantage of producing a large amount of stable neurons.

The present invention provides a neuron differentiated from human adipocytes by a method of differentiating neurons for the treatment of deafness or cerebral nervous system disease of the present invention.

In order to evaluate the functional maturation of neurons differentiated by the differentiation method of the present invention, electrophysiological analysis of neurons was performed by a patch clamp technique. As a result, it was confirmed that the internal current of the voltage-dependent sodium channel was measured to functionally mature as a neuron. See FIG. 4.

The present invention provides a cell composition for the treatment of hearing loss diseases or neurological disorders, which comprises neurons differentiated from fat cells of humans as an active ingredient in the differentiation method of neurons for the treatment of hearing loss diseases or cerebral nervous system diseases.

The deafness or cerebral nervous system disease is characterized in that selected from the group consisting of deafness, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, stroke, ischemia and neurological diseases caused by spinal cord injury.

More specifically, the neuron suspension (5 × 10 ⁵ cells / kg) differentiated by the differentiation method of the present invention was injected into the inner ear of the guinea pig of the hearing loss model using a microinjector at a rate of 2 μl / min. The auditory brainstem response was measured at 2 week intervals for 8 weeks. As a result, it was confirmed that the hearing of guinea pigs, which had a threshold response of 80 to 90 decibels, recovered to about 45 decibels. As a result of confirming the survival of the hair cells in the corti organs, the cell survival rate of more than 90% was confirmed. See FIG. 5.

The cell composition for the treatment of deafness or cerebral nervous system disease of the present invention can be formulated by a method known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid or injection of a suspension, if necessary. For example, it is formulated by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, suspending agent, stabilizer, binder, and the like into a unit dosage form required for generally accepted pharmaceutical practice. I think of doing. The active ingredient amount in the formulation is such that a suitable dose in the range indicated can be obtained. In addition, a sterile composition for injection may be prescribed according to a conventional formulation practice using an excipient such as distilled water for injection.

As the aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose or other auxiliary agents, for example, D-sorbitol, D-mannose, D-mannitol can be used in combination with a suitable dissolution aid.

Administration to the patient is preferably implanted directly into the inner ear by otolaryngological surgery following local anesthesia.

The therapeutically effective amount of the neurons is not particularly limited thereto, but is preferably 1 × 10 ⁴ cells / kg to 1 × 10 ⁸ cells / kg, and preferably 1 × 10 ⁵ cells / kg to 1 × 10 ⁷ cells / kg. More preferably, it is most preferably 5 × 10 ⁵ cells / kg to 5 × 10 ⁶ cells / kg.

The method of administration is not particularly limited, but direct administration to the inner ear is most preferred.

The neuron according to the present invention, in the treatment of hearing loss, by using the differentiation of neurons from mesenchymal stem cells present in adipocytes by the method of the present invention to solve the problem of the supply of nerve cells and therapeutic problems including immune rejection response There is an advantage that can be solved.

Differentiation method of neurons for the treatment of deafness or brain nervous system diseases of the present invention according to the present invention is due to the advantage of producing a large amount of homogenous mesenchymal stem cells in a simple and efficient manner in a short time using inexpensive trypsin It is effective to produce efficiently optimized neurons.

In addition, according to the present invention, the method for differentiation of neurons for the treatment of deafness or cerebral nervous system disease according to the present invention is stable from mesenchymal stem cells by culturing sequentially for a suitable period of time using primary and secondary neuronal differentiation induction medium. There is an advantage that can generate a large amount.

Neuronal cells for the treatment of deafness or cerebral nervous system diseases according to the present invention are effective in the treatment of deafness or cerebral nervous system diseases through direct or indirect transplantation into not only deafness, but also in the neurological disorders of the brain. There is an advantage that can be minimized.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art to which the present invention belongs, the gist of the present invention in the following description and the accompanying drawings Descriptions of well-known functions and configurations that may be unnecessarily blurred are omitted.

[ Example  1] derived from fat cells of human Mesenchymal stem cells  Isolation and Cultivation

After receiving the patient's permission for the part of the adipose tissue to be removed after surgery during the operation at Chonnam National University Hospital, a portion of the adipose tissue was cut out and stored in physiological saline to be immediately used for the experiment. The cut adipose tissue was finely chopped using tweezers and placed in 0.05% trypsin (Gibco, USA) containing EDTA and reacted for 5 minutes in a 37 ° C. incubator, followed by 10% FBS (Hyclone, USA) and 100 U / ml. It was cultured in a 37 ° C., 5% CO ₂ incubator with DMEM (Hyclone, USA) medium containing penicillin-streptomycin (Hyclone, USA).

The culture was incubated while changing the medium every 1 to 2 weeks in the incubator, after culturing to remove the medium and washed with phosphate-buffered saline (PBS) to remove the remaining excess medium. Human adipose tissue-derived stem cells (hADSCs) were obtained using 0.05% trypsin containing EDTA, diluted 1: 2 with fresh medium, and used in the following examples.

[ Example  2] derived from fat cells Mesenchymal stem cells  Into mesodermal cells Eruption  Confirm

To determine whether the adipocyte-derived mesenchymal stem cells obtained in Example 1 can differentiate into mesodermal cells such as adipocytes, bone cells and chondrocytes, the adipocyte-derived mesenchymal stem cells are tissue-specific. Differentiation was induced by culturing in each differentiation medium that induces differentiation.

Adipose-derived mesenchymal stem cells were cultured in adipose cell differentiation medium (Gibco, USA) every two days with medium exchange for one week, followed by oil red O staining to confirm their differentiation into adipocytes.

As a result, as can be seen in Fig. 2A, it was confirmed that the mesenchymal stem cells were differentiated into adipocytes because the fat droplets stained red by oil red O were generated in the cells.

In addition, adipose-derived mesenchymal stem cells were cultured in a bone cell differentiation medium (Gibco, USA) every two weeks for two weeks with medium exchange, followed by alkaline phosphatase staining to confirm their differentiation into bone cells.

As a result, as can be seen in Figure 2 B, it was confirmed that the mesenchymal stem cells were differentiated into bone cells from the increase of intracellular alkaline phosphatase activity.

Finally, the adipose-derived mesenchymal stem cells were cultured and cultured every two days for three weeks in chondrocyte differentiation medium (Gibco, USA) and subjected to alcian blue staining to confirm their differentiation into chondrocytes.

As a result, as can be seen in Figure 2 C, it was confirmed that the mesenchymal stem cells were differentiated into chondrocytes from the fact that the cartilage matrix components of the chondrocytes were blue stained in the Alcian blue stain.

[ Example  3] Neuronal Differentiation of Adipose-derived Mesenchymal Stem Cells

The adipocyte-derived mesenchymal stem cells obtained in Example 1 were cultured in a general medium containing 10% FBS and penicillin-streptomycin in DMEM medium during subculture. For differentiation into neurons, 20 µg / ml bFGF and 100 µg / ml EGF were added to DMEM medium containing 1% FBS to induce primary neuronal differentiation for 1 week while changing medium every two days. , Secondary neuronal differentiation was induced for 4 days by adding 10 forskolin to DMEM medium containing 5% FBS. Thereafter, the cells were cultured in a basal medium containing no compound for induction of neurons for 1 week to confirm that the differentiation conditions were maintained, and an untreated group kept in the basal medium was used as a control.

[ Example  4] Confirmation of Differentiation from Adipocytes into Differentiated Neurons

(One) Cell immunofluorescence ( immunofluorescence cytochemistry method )

To confirm the differentiation efficiency of neurons differentiated from adipocytes, cell-immunofluorescence staining was used to identify proteins expressed in cells.

The cover slip (Fisher Scientific, USA) was coated with 2% gelatin, then coated with glutaraldehyde and glycerin, washed 10 times with distilled water. The adipocyte-derived mesenchymal stem cells obtained in Example 1 were detached from the container with 0.05% trypsin containing EDTA, and then incubated in DMEM medium containing 10% FBS for one day by planting in a coating cover slip prepared by the above method. It was.

Neuronal cells differentiated by the method of Example 3 were treated with methanol previously placed in the refrigerator for 30 minutes at room temperature, fixed, and washed three times with PBS. 0.5% Triton X-100 was then permeated for 15 minutes at room temperature and washed three times with PBS. Thereafter, blocking buffer containing 5% goat plasma was treated for 40 minutes to prevent nonspecific binding. Primary antibodies Tuj1 (Chemicon, USA), NF-L (Chemicon, USA) and MAP2 (Chemicon, USA) diluted with blocking buffer were reacted overnight at 4 ° C. to bind cells of experimental groups pretreated with the above method. After washing three times with PBS, and reacted with Alexa546 (Molecular probe, USA) secondary antibody diluted 1: 500 in blocking buffer for 1 hour at dark room temperature.

The reaction results were analyzed histologically by LSM710 confocal microscope (Confocal, Carl Zeiss, Germany).

As a result, as shown by the fluorescence picture of FIG. 3, the neuronal marker was not expressed in the untreated control group, but the neuronal marker was treated in the group treated with the neuronal inducer compound. It was confirmed that the differentiation efficiency was high, and the morphology of the cells could be more surely observed to differentiate into neuron-like cells.

(2) electrophysiological analysis electrophysiological recording )

In order to evaluate the functional maturation of neurons differentiated by the method of Example 3, electrophysiological analysis of the neurons was performed. The adipocyte-derived mesenchymal stem cells obtained in Example 1 were planted in a gelatin coated cover slip by the method presented in the immunological analysis and the sodium current was recorded using the whole-cell patch clamp method. The recording electrode was manufactured using a fine glass electrode maker (Narishige, Japan) and microforge (Narishige, Japan) to have a resistance of 3 to 5 MPa. Membrane voltage fixation and current measurements were made with a computer using an Axopatch 200B patch clamp amplifier (Axon, USA) via the Digidata 1200B (Axon, USA) interface. The recording and data analysis of the sodium current obtained from the membrane voltage control and the experimental results were carried out using pClamp 7 software (Axon, USA), and the oscilloscope was used to observe the current change simultaneously. The current is filtered at 10 kHz, and the series resistance is compensated using the series resistance compensation circuit of the Axopatch 200B patch clamp amplifier, and the pClamp 7 is not applied to the leakage current. Calibration was done using the software.

As a result, as shown in FIG. 4, inward currents were measured in cells differentiated into neurons, and these internal currents were selectively inhibited by voltage-dependent sodium channels, tetrodotoxin (TTX). Inhibited by 100 nM, the internal current was partially recovered when tetrodotoxin was removed and the current-voltage related function was bell-shaped, typical voltage-dependent sodium channel. This is a result of confirming that the fat-derived mesenchymal stem cells differentiated into neurons has induced the functional maturation by having the electrophysiological characteristics of neurons.

[ Example  5] Transplantation in deafness model from adipocytes to differentiated neurons

(1) Preparation of transplanted neuron

As cells to be transplanted in vivo, the adipocyte-derived mesenchymal stem cells obtained in Example 1 were used. In the primary and secondary neuronal differentiation induction medium, the neurons induced differentiation into neurons were washed twice with PBS, treated with 0.05% trypsin containing EDTA, detached from the culture vessel, and suspended in saline. It was. The suspended neuron number was prepared to be 5 × 10 ⁵ cells / kg and put in ice to prepare a transplant.

(2) transplantation into the inner ear of deafness animal model

One week after 5% neomycin (neomycin) was injected into the guinea pig's inner ear, an animal with a threshold response of about 80 to 90 decibels was audited using an auditory brainstem response (ABR). One week after the deafness model was prepared, the suspension of the prepared neural cell suspension was injected into the inner ear of the guinea pig at a rate of 2 μl / min using a microinjector. Auditory brainstem induced reactions were measured at 2 weeks interval for 8 weeks after transplantation.

As a result, as shown in the results of FIG. 5, it was confirmed that the hearing of the guinea pig was restored to about 45 decibels, and then, tissue fragments were prepared to confirm the survival of hair cells in corti organs, and immunostaining using hematoxylin and eosin. As a result, it was confirmed that the living cells were stained. As a result, the control group injected with physiological saline only showed less than 30% of living cells, whereas the group of transplanted stem cells differentiated with neurons showed a cell survival rate of 90% or more.

This is not only the result of confirming that the neuron cell according to the present invention can be treated by a patient with deafness or cerebral nervous system disease to treat deafness or neurological disease, but also presenting a method of treatment.

Figure 1 shows the morphology of the adipocyte-derived mesenchymal stem cell culture 7 days after the optical microscope,

Figure 2 confirms the differentiation ability of the adipocyte-derived mesenchymal stem cells of the present invention into adipocytes, bone cells and chondrocytes,

(A: oil red O staining, B: alkaline phosphatase staining, C: alkian blue staining)

Figure 3 shows the results of immunochemical analysis of neurons differentiated from human adipocytes by cell immunohistochemical staining method according to the present invention,

(A: Tuj1 antibody, B: NF-L antibody, C: MAP2 antibody)

Figure 4 shows the results of electrophysiological analysis of the neuronal cells differentiated from adipocytes of the present invention by the patch clamp method,

Figure 5 shows the results of confirming the survival rate of hemotoxin and eosin staining of the inner hair cells, external hair cells and neuroblastoma cells of Corti organs after transplanting neurons differentiated from adipocytes of the present invention into the inner ear of a model of hearing loss. .

(A: control group, B: experimental group)

Claims (11)

a) obtaining adipose derived mesenchymal stem cells from human fat cells; b) inducing primary differentiation of the obtained mesenchymal stem cells in primary neuronal differentiation induction medium containing basic growth factors (bFGF) and epidermal growth factor (EGF) as growth factors; And c) inducing secondary differentiation of the primary differentiation-induced mesenchymal stem cells in a secondary neuronal differentiation induction medium containing forskolin, and then differentiating into neurons; Differentiation method of neurons for the treatment of deafness or cerebral nervous system disease comprising a. The method of claim 1, The adipose-derived mesenchymal stem cells of step a) is differentiation method of neuronal cells for the treatment of deafness or cerebral nervous system disease, characterized in that the human fat cells obtained by treating trypsin at a concentration of 0.02 to 0.07% containing EDTA. . The method of claim 1, The primary differentiation induction of step b) is in the primary neuron differentiation induction medium containing the mesenchymal stem cells obtained in step a), the growth factors bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor) Differentiation method of neurons for the treatment of deafness or cerebral nervous system disease, which is induced by culturing for 5 to 10 days. The method of claim 3, The primary neuronal differentiation induction medium comprises 10 to 30 μg / ml bFGF and 80 to 120 μg / ml EGF. The method of claim 4, wherein The primary neuronal differentiation inducing medium further comprises 0.5 to 2% of serum concentration of the method for differentiating neurons for the treatment of hearing loss disease or cerebral nervous system disease. The method of claim 1, Induction of the secondary differentiation of step c) is induced by incubating the primary differentiation-induced mesenchymal stem cells in step b) for 2 to 5 days in a secondary neuronal differentiation induction medium containing forskolin Differentiation method of nerve cells for the treatment of deafness or cerebral nervous system disease, characterized in that. The method of claim 6, The secondary neuronal differentiation inducing medium is 5 to 15 concentrations of forskolin (forskolin) characterized in that the differentiation of neurons for the treatment of hearing loss disease or cerebral nervous system disease, characterized in that. The method of claim 7, wherein The secondary neuronal differentiation induction medium further comprises a serum of 3 to 10% concentration of differentiation of neurons for the treatment of deafness or cerebral nervous system disease. Neural cells differentiated from human fat cells by the differentiation method according to any one of claims 1 to 8. A cell composition for the treatment of deafness or cerebral nervous system disease comprising the nerve cell according to claim 9 as an active ingredient. The method of claim 10, The deafness or cerebral nervous system disease is deafness, Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, stroke, ischemia and spinal cord injury, characterized in that selected from the group consisting of deafness or neurological disease Therapeutic cell composition.

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