CN113481160B

CN113481160B - Retinal pigment epithelial cell induction culture medium and application thereof

Info

Publication number: CN113481160B
Application number: CN202110962908.0A
Authority: CN
Inventors: 张晗
Original assignee: Beijing Taidong Biotechnology Co ltd
Current assignee: Beijing Taidong Biotechnology Co ltd
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2022-04-29
Anticipated expiration: 2041-08-20
Also published as: CN113481160A

Abstract

The invention relates to a retinal pigment epithelial cell induction culture medium and application thereof. The induction medium is Neurobasal medium containing the following components: b27 replenisher, 1 mM-5 mM of L-glutamine, 0.5 mM-3 mM of sodium pyruvate, 0.05 mM-0.15 mM of beta-mercaptoethanol, 30 mM-120 mM M, cAMP 0.5.5 mM-3 mM of L-ascorbic acid, 30 nM-120 nM of BMP inhibitor, 5 MuM-20 Mu M, ALK of GSK-3 beta inhibitor, 5 MuM-25 Mu M of SHH and 15 ng/ml-60 ng/ml of SHH. The culture medium can be used for directional differentiation culture of human retinal pigment epithelial cells as raw materials to obtain dopaminergic neuron-like cells capable of producing dopamine.

Description

Retinal pigment epithelial cell induction culture medium and application thereof

Technical Field

The invention relates to the technical field of cell culture, in particular to a retinal pigment epithelial cell induction culture medium and application thereof.

Background

Parkinson's disease is one of the most common neurodegenerative diseases in the elderly population. Parkinson patients often suffer from resting tremor, stiffness, hypokinesia, gait, balance and autonomic dysfunction, depression and dementia. From an etiological point of view, parkinson's disease is a nigral-striatal neuronal projection caused by the loss of dopamine-producing neurons in the substantia nigra pars compacta, which is responsible for motor symptoms during the disease. Starting from changes in mesencephalic dopamine neurons, this disease ultimately affects forebrain neurons, such as cortical neurons. Over the past decade, several susceptibility factors have been identified. This study suggests that protein misfolding, abnormally increased oxidative stress, mitochondrial dysfunction, and damage to the ubiquitin-proteasome and autophagy-lysosomal systems all may contribute to the development of parkinson's disease.

Parkinson patients currently receive a variety of medications including levodopa, dopamine agonists and monoamine oxidase B inhibitors. Among them, levodopa is the most effective drug, but undesirable side effects are often observed, including fluctuations in PD symptoms and dyskinesias. Deep Brain Stimulation (DBS) of the bilateral subthalamic nucleus or the Globus Pallidus Internuclear (GPi) improves the condition of patients, reduces their daily dose of levodopa, and improves levodopa-related motor complications and dyskinesias. Nevertheless, DBS has been reported to cause severe depression. Despite improvements in symptom management, none of these therapies can prevent disease progression. It is desirable to arrest the progression of the disease by replacing diseased neurons, and cell therapy has been used in clinical trials to treat animals with parkinson's disease or parkinson's disease patients. In this regard, fetal-derived Neural Stem Cells (NSCs) are the first cell type studied, followed by mesenchymal stem cells, dopaminergic neurons differentiated from embryonic stem cells (ES), and induced pluripotent stem cells (iPS). However, safety issues, ethical issues, and the ability to over-transplant dopamine production in cells limit clinical use in parkinson's disease patients. In order to find an alternative cell source for parkinson's disease treatment, an open-label pilot study using human fetal retinal pigment epithelial cells reported that was observed within 12 months after transplantation, was able to improve the patient's condition. However, a randomized double-blind study using the same retinal pigment epithelial cells did not demonstrate the same therapeutic effect. Furthermore, ethical issues remain in the clinical use of fetal tissues.

The inventors of the present invention have previously conducted studies on inducing differentiation of retinal pigment epithelial cells into dopaminergic neuron-like cells, but the induced culture method adopted still has a problem of low dopamine production.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention relates to a retinal pigment epithelial cell induction culture medium, which is a Neurobasal culture medium containing the following components:

b27 replenisher, 1 mM-5 mM of L-glutamine, 0.5 mM-3 mM of sodium pyruvate, 0.05 mM-0.15 mM of beta-mercaptoethanol, 30 mM-120 mM M, cAMP 0.5.5 mM-3 mM of L-ascorbic acid, 30 nM-120 nM of BMP inhibitor, 5 MuM-20 Mu M, ALK of GSK-3 beta inhibitor, 5 MuM-25 Mu M of SHH and 15 ng/ml-60 ng/ml of SHH.

Alternatively, the retinal pigment epithelial cell induction medium as described above is Neurobasal medium comprising:

b27 replenisher, L-glutamine 2 mM-4 mM, sodium pyruvate 1 mM-2 mM, beta-mercaptoethanol 0.05 mM-0.15 mM, L-ascorbic acid 50 mM-100 mu M, cAMP 1 mM-2 mM, BMP inhibitor 50 nM-100 nM, GSK-3 beta inhibitor 10 mu M-15 mu M, ALK inhibitor 10 mu M-20 mu M and SHH20 ng/ml-50 ng/ml.

Optionally, the retinal pigment epithelial cell induction medium is as described above, and the B27 supplement is added in an amount of 1/60-1/40 of the volume of the Neurobasal medium.

Alternatively, the retinal pigment epithelial cell induction medium as described above, and the BMP inhibitor is LDN 193189.

Alternatively, the retinal pigment epithelial cell induction medium as described above, wherein the GSK-3 β inhibitor is CHIR 99021.

Alternatively, the ALK inhibitor is SB431542 in a retinal pigment epithelial cell induction medium as described above.

The second aspect of the present invention relates to a retinal pigment epithelium cell culture method, which comprises inoculating the retinal pigment epithelium in the induction medium as described above for culture.

Optionally, in the method for culturing retinal pigment epithelial cells, the culture time is more than or equal to 7 days.

Optionally, in the retinal pigment epithelial cell culture method, the cells are subcultured once after 2-4 days of culture.

A third aspect of the invention relates to the use of an induction medium as described above, or a method as described above, for the preparation of dopaminergic neuron-like cells.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a culture solution and a differentiation method for efficiently and directionally inducing and trans-differentiating human retinal pigment epithelial cells into dopaminergic neuron-like cells in vitro, and can obtain abundant dopaminergic neurons in vitro. Compared with the existing mode for producing the dopaminergic neurons, the method has the advantages of convenience in operation, high safety, short differentiation and culture time, high differentiation efficiency, high yield, high differentiation purity and the like, and provides a new idea for the application of clinical transplantation of the dopaminergic neurons in treating the Parkinson's disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the results of immunofluorescence assays in accordance with an embodiment of the present invention; A) and B) results of experiments with Tyrosine Hydroxylase (TH) labeled with Alexa Fluor594, A) RPE cells, B) induced dopaminergic neuron-like cells; C) and D) Alexa Fluor 594-labeled dopamine Transporter (DAT) experimental results; C) RPE cells, D) dopaminergic neuron-like cells after induction;

FIG. 2 is a graph showing the dopamine content of the cell homogenate supernatant in one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed embodiments belong; the following definitions serve to better understand the teachings of the present invention by way of further guidance. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the embodiments will be apparent from the following detailed description and claims.

As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range and the recited endpoints.

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The symbol "%" as used herein, when it is solid with respect to the total material, generally means weight/weight percent, unless otherwise specified; when the total material is a liquid, it is generally referred to as a weight/volume percentage. Of course, for the case where the total material is liquid and the solute is liquid, the percentages that characterize the liquid solute generally refer to volume/volume percentages.

In the present invention, the "retinal pigment epithelial cells" are cells isolated from animals, preferably mammals, more preferably primates, and most preferably humans.

The culture medium can be used for directional differentiation culture of human retinal pigment epithelial cells serving as raw materials to obtain dopaminergic neuron-like cells capable of generating dopamine, can be used for direct transplantation or terminal differentiation into dopaminergic neurons, and can be used for cell transplantation treatment of neurodegenerative diseases, in-vitro disease model research, drug screening and other biomedical applications. The method has the characteristics of convenience and rapidness in operation, short culture time, high differentiation efficiency, high dopamine yield and the like.

SHH, Sonic Hedgehog Protein, is involved in the localization of precursor cells early in embryonic development and determines neuronal phenotype at specific sites, such as ventral midbrain dopamine neuronal phenotype, but SHH has been rarely reported to induce retinal pigment epithelial cells. The applicant unexpectedly finds that the dopamine yield of the induced cells can be effectively improved by matching SHH with L-ascorbic acid and cAMP with proper concentrations, so that the induced cells have better application scenes.

In some embodiments, the retinal pigment epithelial cell induction medium is Neurobasal medium comprising:

b27 supplement, L-glutamine 2mM, sodium pyruvate 1mM, beta-mercaptoethanol 0.1mM, L-ascorbic acid 50. mu. M, cAMP 1.5.5 mM, BMP inhibitor 100nM, GSK-3. beta. inhibitor 10. mu. M, ALK inhibitor 15. mu.M and SHH35 ng/ml.

In the present invention, the induction medium may be packaged in the form of a solution, or may be packaged in the form of a dry powder composition (e.g., lyophilized powder or spray-dried powder) and dissolved at the time of use. When the solution is prepared, the ratio of each component in the composition or the solution meets the set concentration when the working concentration is prepared. Usually in the form of working strength or mother liquor. In the present invention, "working concentration" is used to define the ratio of the main active components in the amplification medium when culturing retinal pigment epithelial cells, and the concentration of the main active components in the amplification medium may be the working concentration or a mother liquor that can be diluted to this concentration (for example, a mother liquor that is 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold concentrated).

It will be appreciated that the components such as L-glutamine, L-ascorbic acid, etc. may be substituted with L-glutamine sodium salt, L-ascorbic acid sodium salt or other salts, particularly soluble salts, as well as derivatives known in the art.

This application does not exclude the use with other media or the addition of some conventional nutritional supplements such as salts, antibiotics, vitamins, and amino acids to the media of the present application, and the amino acid supplement may include any amino acid including glycine, alanine, valine, leucine, isoleucine, arginine, lysine, aspartic acid, cysteine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, asparagine, glutamine, histidine, and serine. Antibiotics such as gentamicin, penicillin, streptomycin, ampicillin, kanamycin, and the like.

The medium of the present application may also contain a buffer, and the type thereof is not particularly limited and may be of a type well known to those skilled in the art. A "buffer" is a solution that resists pH changes by the action of its acid-base conjugate components. Various Buffers that can be employed depending on, for example, the desired pH of the buffer (as well as the microbial growth and metabolic characteristics, the microbial culture system, pH control and the medium used) are described in Buffers. In one embodiment, the buffer has a pH in the range of about 2 to about 9, alternatively about 3 to about 8, alternatively about 4 to about 7, alternatively about 5 to about 7. Non-limiting examples of buffers that will control pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations of these. In some embodiments, the buffer is selected from the group consisting of 3- (N-morpholino) propanesulfonic acid (MOPS) free acid, 3- (N-morpholino) propanesulfonic acid (MOPS) Na, hydroxyethylpiperazine ethanesulfonic acid (HEPES), and sodium bicarbonate.

In the present invention, the culture environment of the cells is, but not particularly limited to, about 5% CO₂And about 37 ℃.

In some embodiments, formulation aids, such as antifoam agents, may also be added to the medium. In some embodiments, the antifoaming agent of the medium formulation comprises an ionic or nonionic surfactant.

b27 supplement, L-glutamine 3mM, sodium pyruvate 1.5mM, beta-mercaptoethanol 0.1mM, L-ascorbic acid 75. mu. M, cAMP 1.5.5 mM, BMP inhibitor 75nM, GSK-3. beta. inhibitor 12.5. mu. M, ALK inhibitor 15. mu.M, and SHH35 ng/ml.

In some embodiments, the B27 supplement is added in an amount of 1/60-1/40, for example 1/50, of the volume of Neurobasal media.

In some embodiments, the BMP inhibitor is LDN 193189.

In some embodiments, the GSK-3 β inhibitor is CHIR 99021.

In some embodiments, the ALK inhibitor is SB 431542.

The invention also relates to a retinal pigment epithelium cell culture method, which comprises inoculating the retinal pigment epithelium in the induction medium for culture.

In some embodiments, the culturing period is greater than or equal to 7 days, such as 8, 9, 10, 11, 12, 13, 14 days.

In some embodiments, the cells are passaged once 2-4 days after culture.

The invention also relates to the use of the method as described above for the preparation of dopaminergic neuron-like cells.

The invention also relates to a method for treating the neurodegenerative disease caused by dopamine deficiency, which comprises the steps of culturing the retinal pigment epithelial cells by using the retinal pigment epithelial cell culture method, and transplanting safe and effective dopaminergic neuron-like cells obtained by induced culture into a subject.

In some embodiments, the neurodegenerative disease is parkinson's disease.

The phrase "safe and effective amount". As used herein, means that the amount of a compound or composition within the scope of reasonable medical adjustment is large enough to significantly effectively alleviate the symptoms or conditions being treated, but small enough to avoid serious side effects (at a reasonable benefit/risk ratio). The safe and effective amounts of the active ingredients in the pharmaceutical compositions used in the methods of the present invention will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the disease, the time of treatment, the concurrent condition, the particular active ingredient employed, the particular pharmaceutically acceptable excipient employed and such factors including the knowledge and skill of the attending physician.

The term "disease or condition" as used herein refers to a physical condition of the subject that is associated with a disease or condition described herein.

The term "subject" as used herein may refer to a patient or other animal receiving an agent or medicament of the invention for the treatment, prevention, alleviation and/or alleviation of a disease, disorder or condition described herein, including a warm-blooded animal, such as a mammal, like a primate, and preferably a human. Non-human primates are also subjects. The term subject includes domesticated animals such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.).

The cultured cells can be administered by transplantation in the form of a pharmaceutical composition. The pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent or carrier.

The term "pharmaceutically acceptable excipient, diluent or carrier" refers to an excipient, diluent or carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art, including but not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

Isolation and culture of human RPE cells

Human eyes from a 57 year old donor from the Beijing college of Homelan (members of the International eye Bank Association). The posterior ocular tissue remaining after penetrating corneal transplantation surgery was used to isolate the RPE cell layer. The tissue was incised at the serrated edge, the anterior and vitreous discarded, and the RPE layer exposed. RPE layers were dissected into small pieces and seeded into 6-well plates. RPE cells were cultured in MEM-a modified medium (Sigma-Aldrich, St. Louis, MO, USA), 2mM L-glutamine, penicillin/streptomycin (1:100), 1% sodium pyruvate, and 10% FBS (fetal bovine serum). Cells were cultured in an incubator humidified with 5% CO2 at 37 ℃ and the medium was changed every 3 days.

When the culture reached approximately 80% confluence, the medium was removed from the wells and washed with PBS. (cells are not allowed to exceed 90% confluence, as this may lead to cell detachment and increased levels of cell death). Adding 200. mu. LACCUTASE^TMAnd incubated at 37 ℃ for 2-3 min (to check if cells become rounded and detached). Cells were transferred to 15mL tubes, washed once with PBS, and then transferred to the same tube. Cells were diluted to 5mL with PBS and washed with 30Centrifuge at 0 Xg for 5 min. Followed by the step of induction culture.

Example 1

An induced culture method of retinal pigment epithelial cells.

Retinal pigment epithelial cells were plated at 2X 10 per well⁴The cells were plated at a density in 6-well plates in Neurobasal media containing B27 supplement (1: 50), L-glutamine 2mM, sodium pyruvate 1mM, beta mercaptoethanol 0.1mM, L-ascorbic acid 50. mu. M, cAMP 1.5.5 mM, LDN 193189100 nM, CHIR 902110. mu. M, ALK inhibitor SB 43154215. mu M, SHH35ng/ml Neurobasal media. Thereafter, the cells were passaged after 3 days. Cells were cultured in neuronal induction medium to day 7.

Example 2

An induced culture method of retinal pigment epithelial cells.

Retinal pigment epithelial cells were plated at 2X 10 per well⁴The cells were plated at a density in 6-well plates in Neurobasal media containing B27 supplement (1: 60), L-glutamine 5mM, sodium pyruvate 0.5mM, beta mercaptoethanol 0.15mM, L-ascorbic acid 30. mu. M, cAMP 3mM, LDN 193189120 nM, CHIR 90215. mu. M, ALK inhibitor SB 4315425. mu M, SHH 60ng/ml Neurobasal media. Thereafter, the cells were passaged after 3 days. Cells were cultured in neuronal induction medium to day 7.

Example 3

An induced culture method of retinal pigment epithelial cells.

Retinal pigment epithelial cells were plated at 2X 10 per well⁴The cells were plated in 6-well plates at a density of cells in Neurobasal media containing B27 supplement (1: 40), L-glutamine 1mM, sodium pyruvate 3mM, beta-mercaptoethanol 0.05mM, L-ascorbic acid 120. mu. M, cAMP 0.5.5 mM, LDN 19318930 nM, CHIR 902120. mu.m, ALK inhibitor SB 43154225. mu. M, SHH 15 ng/ml. Thereafter, the cells were passaged after 3 days. Cells were cultured in neuronal induction medium to day 7.

Immunofluorescence:

immunofluorescent staining was performed on example 2. Cells on coverslips were fixed for 15min in 4% PFA pre-chilled on ice. After three washes in phosphate buffer, the cells were blocked with 5% bovine serum albumin in phosphate buffered saline for 30min and then incubated with the appropriate primary antibody at 4 ℃ for 12 h.

The primary antibody was used at a dilution of 1:200 in solution A. After three washes, cells were incubated with AlexaFluor 594-labeled secondary antibody in blocking buffer for 1h at 37 ℃ followed by 5min incubation with DAPI. The coverslip was cleaned and mounted on a slide. The imaging of the cells was performed using an LSM 780 confocal microscope (carl zeiss).

The staining results are shown in fig. 1, and the tyrosine hydroxylase and the dopamine transporter are respectively stained through immunofluorescence detection; tyrosine hydroxylase is an enzyme that converts L-tyrosine to L-3, 4-dihydroxyphenylalanine (L-DOPA), and is also a precursor to dopamine. Dopamine transporter is a transmembrane transporter that controls the reuptake of dopamine from the outside of the cell to presynaptic neurons. The result shows that the induced retinal pigment epithelial cells can express the specific marker of the dopaminergic neuron.

Comparative example 1

An induced culture method of retinal pigment epithelial cells.

Retinal pigment epithelial cells were plated at 2X 10 per well⁴The cells were plated at a density in 6-well plates in Neurobasal media containing B27 supplement (1: 50), L-glutamine 2mM, sodium pyruvate 1mM, beta-mercaptoethanol 0.1mM, L-ascorbic acid 50. mu. M, cAMP 1.5.5 mM, LDN 193189100 nM, CHIR 902110. mu. M, ALK inhibitor SB 43154215. mu.M Neurobasal media. Thereafter, the cells were passaged after 3 days. Cells were cultured in neuronal induction medium to day 7.

Comparative example 2

An induced culture method of retinal pigment epithelial cells.

Retinal pigment epithelial cells were plated at 2X 10 per well⁴The cells were plated in 6-well plates at a density of B27 supplement (1: 50), L-glutamine 2mM, sodium pyruvate 1mM, beta mercaptoethanol 0.1mM, LDN 193189100 nM, CHIR 902110 μ M, ALK inhibitor SB 43154215 μ M, SHH35ng/ml Neurobasal nerve medium. Thereafter, the cells were passaged after 3 days. Cells were cultured in neuronal induction medium to day 7.

The cultured cells were detected by the following method.

Immunofluorescence:

cells on coverslips were fixed for 15min in 4% PFA pre-chilled on ice. After three washes in phosphate buffer, the cells were blocked with 5% bovine serum albumin in phosphate buffered saline for 30min and then incubated with the appropriate primary antibody at 4 ℃ for 12 h.

The primary antibody was used at a dilution of 1:200 in solution A. After three washes, cells were incubated with AlexaFluor 594-labeled secondary antibody in blocking buffer at 37 ℃ for 1h, followed by addition of DAPI for 5 min. The coverslip was cleaned and mounted on a slide. The imaging of the cells was performed using an LSM 780 confocal microscope (carl zeiss).

High performance liquid chromatography:

for HPLC sample preparation, 1X 10⁶Individual retinal pigment epithelial cells were homogenized in 200 μ L of 0.4M perchloric acid. The homogenate was centrifuged at 12,000 rpm for 20min at 4 ℃. High performance liquid chromatography analysis used a high performance liquid chromatography system with electrochemical detection and a Uniget C-18 reverse phase microcolumn as the stationary phase. 1L of mobile phase consisted of 8.84g of citric acid monohydrate, 10g of sodium acetate anhydride, 220mg of sodium octane sulfonate, 5mg of disodium ethylenediaminetetraacetate and 200mL of methanol. The flow rate was 0.4mg/mL, and 10. mu.L of the sample supernatant was directly injected into high performance liquid chromatography for analysis. The dopamine standard DOPAC (sigma-aldrich) was used to quantify and identify peaks on the chromatogram.

The cells cultured in example 1, comparative example 2, and the human RPE cells initially cultured were assayed for dopamine content in the cell homogenate supernatant by a high performance liquid chromatography method. As shown in FIG. 2, the dopamine contents of examples 1-3 were significantly increased (p <0.05) compared to those of comparative examples 1-2, and the dopamine content of example 2 was the highest.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The retinal pigment epithelial cell induction medium is characterized by comprising the following components:

b27 replenisher, 2 mM-4 mM L-glutamine, 1 mM-2 mM sodium pyruvate, 0.05 mM-0.15 mM beta-mercaptoethanol, 50 mu M-100 mu M, cAMP 1 mM-2 mM L-ascorbic acid, 50 nM-100 nM BMP inhibitor, 10 mu M-15 mu M, ALK inhibitor of GSK-3 beta inhibitor, 10 mu M-20 mu M SHH20 ng/ml-50 ng/ml;

wherein the B27 supplement is added in an amount of 1/60-1/40 of the volume of the Neurobasal medium; the BMP inhibitor is LDN 193189; the GSK-3 beta inhibitor is CHIR 99021; the ALK inhibitor is SB 431542.

2. A method for culturing retinal pigment epithelium, comprising inoculating retinal pigment epithelium in the induction medium according to claim 1 and culturing the same.

3. The method for culturing retinal pigment epithelial cells according to claim 2, wherein the culture time is 7 days or longer.

4. The method for culturing retinal pigment epithelial cells according to claim 2, wherein the cells are passaged once after 2 to 4 days of culture.

5. Use of the induction medium of claim 1, or the method of any one of claims 2 to 4, for the preparation of dopaminergic neuron-like cells.