Method for inducing stem cells to differentiate into retinal pigment epithelial cells

Abstract

This application relates to a method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE), comprising the following steps: culturing the pluripotent stem cells in a first culture medium before the pluripotent stem cells exhibit cell state decline after cell culture, wherein the first culture medium comprises DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA), and L-glutamine. The method described in this application can significantly improve the efficiency of differentiation into RPE.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for inducing stem cells to differentiate into retinal pigment epithelial cells. Background Technology

[0002] Retinal degenerative diseases are the leading cause of blindness worldwide. In these diseases, damage and dysfunction of the retina pigmented epithelium (RPE) cells lead to degeneration of photoreceptor cells, ultimately affecting the patient's vision and causing blindness. Due to the non-regenerative nature of the RPE and photoreceptor cells, there are currently no effective treatments for these diseases in clinical practice.

[0003] Because stem cells possess the ability to proliferate indefinitely and differentiate into various cell types, somatic cells obtained from mutant patients can be reprogrammed into iPS cells and then induced into RPE cells. These cells can serve as disease models for drug screening, and after their function is restored, the gene-edited RPE cells can be used as an ideal source of transplantation cells. Reports have already confirmed the feasibility and safety of using stem cell-derived RPE cells in clinical research on human retinal degenerative diseases, although postoperative vision in patients still falls far short of the requirements for normal life.

[0004] Over the past decade or so, various methods have been reported to control the differentiation of stem cells into RPE cells, but these methods still have some drawbacks, such as being time-consuming, cumbersome, having large batch-to-batch variations, and having low cell yields.

[0005] Therefore, a method is needed to efficiently induce stem cells to differentiate into RPE cells. Summary of the Invention

[0006] This application provides a method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE). Mature RPE cells can be obtained using the method described in this application. Furthermore, the method described in this application has at least the following beneficial effects: high differentiation efficiency, short differentiation cycle, strong reproducibility, no need for exogenous components, and clinical applicability.

[0007] On one hand, this application provides a method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE), which includes the following steps: before the stem cells show cell state decline after cell culture, the stem cells are placed in a first culture medium for culture, wherein the first culture medium contains DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA) and L-glutamine.

[0008] In some embodiments, the first culture medium comprises DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX. TM .

[0009] In some implementations, the DMEM medium contains KnockOut TM DMEM and / or DMEM / F-12.

[0010] In some embodiments, the serum comprises a clinical-grade serum substitute.

[0011] In some embodiments, the serum contains KnockOut TM Serum substitutes.

[0012] In some embodiments, the NEAA comprises L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and / or glycine.

[0013] In some embodiments, the concentration of the serum in the first culture medium is about 10% (v / v) to about 30% (v / v).

[0014] In some embodiments, the concentration of β-mercaptoethanol in the first culture medium is about 0.1% (v / v) to about 1% (v / v).

[0015] In some embodiments, the concentration of NEAA in the first culture medium is about 0.5% (v / v) to about 2% (v / v).

[0016] In some embodiments, the concentration of L-glutamine in the first culture medium is about 0.5% (v / v) to about 2% (v / v).

[0017] In some embodiments, in the first culture medium, the GlutaMAX TM The concentration is approximately 0.5% (v / v) to approximately 2% (v / v).

[0018] In some embodiments, the first culture medium further comprises activin A.

[0019] In some embodiments, the concentration of Activin A is about 50 ng / mL to about 200 ng / mL.

[0020] In some embodiments, the first culture medium further comprises SU5402.

[0021] In some embodiments, the concentration of SU5402 is about 5 μM to about 20 μM.

[0022] In some embodiments, the stem cells are cultured in the first culture medium for about 5 to about 15 days.

[0023] In some embodiments, the cell state decay includes at least about 20% of the cells exhibiting a state selected from the group consisting of cell shrinkage, cell shedding, and cell apoptosis.

[0024] In some embodiments, the cell state decline occurs approximately 6 to approximately 15 days after the stem cells have been cultured.

[0025] In some embodiments, the stem cells achieve a cell fusion rate of approximately 70% prior to cell culture.

[0026] In some embodiments, the stem cells are digested prior to cell culture.

[0027] In some embodiments, after digestion, the stem cells are seeded at a ratio of about 1:2 to about 1:4.

[0028] In some embodiments, the seeding is placed in Fibronectin or Cell superior.

[0029] In some embodiments, the cell culture is performed in a second culture medium containing DMEM medium, B27 cell culture additive, N2 cell culture additive, and non-essential amino acids (NEAA).

[0030] In some embodiments, the cell culture is performed in a second culture medium comprising DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), and nicotinamide (NIC); or, the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), and 3-aminobenzamide (3ABA).

[0031] In some embodiments, the cell culture is carried out in the second culture medium for about 2 days.

[0032] In some embodiments, the cell culture is performed in a second culture medium comprising DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and nicotinamide (NIC); or, the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and 3-aminobenzamide (3ABA).

[0033] In some embodiments, the cell culture continues in the second culture medium for approximately 2 days.

[0034] In some embodiments, the cell culture is performed in a second culture medium containing DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), DKK1 protein, insulin-like growth factor-1 (IGF-1), and activin A.

[0035] In some embodiments, the cell culture continues in the second culture medium for approximately 2 days.

[0036] In some embodiments, the cell culture is carried out in a second culture medium, wherein the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), activin A, and SU5402.

[0037] In some embodiments, the cell culture continues in the second culture medium for about 1 day.

[0038] In some embodiments, the method further includes the step of digesting cells cultured with the RPE morphology.

[0039] In some embodiments, the method further includes the step of enriching cells with the RPE morphology cultured from the cells.

[0040] In some embodiments, the enrichment is performed using a method selected from the group consisting of flow cytometry (FACS) and immunomagnetic bead cell sorting (MACS).

[0041] In some embodiments, the stem cells include embryonic stem cells and / or induced basal stem cells.

[0042] In some embodiments, the stem cells include human cells.

[0043] In some embodiments, the stem cells are human embryonic stem cells.

[0044] In some implementations, the stem cells are obtained without destroying a human embryo.

[0045] On the other hand, this application provides a method for generating RPE cells, which includes the following steps:

[0046] a) Obtaining stem cells;

[0047] b) According to the method described in this application, the stem cells of step a) are induced to differentiate into RPE cells.

[0048] On the other hand, this application provides the use of the method described in this application in the preparation of a medicament for treating retinal diseases.

[0049] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0050] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0051] Figure 1-9 The images shown depict RPE cells differentiated using the method described in this application at different time points during cell culture.

[0052] Figures 10A-10C The image shows the expression of iPS marker genes and RPE marker genes in cells obtained using the method described in this application.

[0053] Figure 11A-11B The results shown are immunofluorescence identification assays of differentiated RPE cells at different time points in cell culture using the method described in this application.

[0054] Figure 12A-12B The results shown are phagocytosis assays of differentiated RPE cells at different time points in cell culture using the method described in this application. Detailed Implementation

[0055] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0056] Terminology Definition

[0057] In this application, the term "stem cell" generally refers to an undifferentiated cell that possesses the ability to self-renew while retaining different potentials to form differentiated cells and tissues. The stem cells can proliferate and produce a large number of differentiated or differentiateable daughter cells. In this application, the stem cells may include totipotent stem cells and / or pluripotent stem cells. Totipotent stem cells generally refer to stem cells capable of differentiating into embryonic and extraembryonic cell types. For example, totipotent stem cells may include embryonic stem cells (ES). Embryonic stem cells may include preimplantation embryos, artificially generated embryos (i.e., those produced through in vitro fertilization), etc., which are capable of dividing for a long time in culture without differentiation and / or have the ability to develop into cells and / or tissues of the three proteroderms—ectoderm, mesoderm, and endoderm. In this application, the stem cells can be obtained by methods that do not involve the destruction of human embryos. In this application, the stem cells may be vertebrate stem cells, such as mammalian stem cells, such as stem cells derived from humans, primates, or rodents. For example, pluripotent cells may be human stem cells.

[0058] In this application, the term "pluripotent stem cell" generally refers to a cell capable of self-replication and generating all cell types in a subject. The pluripotent stem cells can differentiate under appropriate conditions into cell types with three germ layers (e.g., cell types capable of differentiating into ectoderm, mesoderm, and endoderm). In this application, the pluripotent cells can also be maintained in an undifferentiated state for an extended period through in vitro culture. For example, the pluripotent stem cells may include hED cells, hiPS cells, hEG cells, and / or hEC cells.

[0059] In this application, the term "retinal pigment epithelial cell (RPE)" generally refers to a layer of pigment cells closely attached to the outer edge of the retinal sensory nerves. The retinal pigment epithelium consists of a single layer of hexagonal cells containing densely packed pigment granules. The RPE is closely connected to the underlying choroid and the retinal nerve cells above it. Its main functions include: controlling the fluid and nutrients in the subretinal space, acting as a blood-retinal barrier; synthesizing growth factors to adjust local structure; absorbing light and regulating electrical balance; regenerating and synthesizing visual pigments; phagocytizing and digesting the outer segments of photoreceptors; maintaining retinal attachment; and regenerating and repairing after injury. The RPE is generally considered an important tissue for maintaining photoreceptor function, but it is also affected by many diseases of the choroid and retina.

[0060] In this application, the term "non-essential amino acid (NEAA)" generally refers to amino acids that can be synthesized in the body and do not require external supplementation as a nutrient source. For example, most cells can synthesize said NEAA through glutamine breakdown, glycolysis, or the TCA cycle. In this application, said NEAA may include L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and / or glycine. For example, said NEAA may include L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine.

[0061] In this application, the term "GlutaMAX" TM "This usually refers to L-alanyl-L-glutamine. It can be a dipeptide alternative to L-glutamine. GlutaMAX" TM It can eliminate problems related to the spontaneous degradation of L-glutamine during cell incubation and culture. GlutaMAX TM It is soluble in water. GlutaMAX TM It has thermal stability.

[0062] In this application, the term "DMEM medium" may include DMEM / F12 medium. The DMEM / F12 medium can be a medium obtained by adding some components of F12 medium to DMEM medium. The DMEM / F12 medium can be used for serum-free culture. In this application, the DMEM medium may include KnockOut... TM DMEM.

[0063] In this application, the term "KnockOut" TM "DMEM" typically refers to a basal culture medium used for the growth of undifferentiated embryonic stem cells and induced basal stem cells. (KnockOut) TMDMEM may not contain L-glutamine. KnockOut TM DMEM can meet cGMP requirements. KnockOut TM DMEM may not contain serum.

[0064] In this application, the term "clinical-grade serum substitute" generally refers to a substitute that avoids the potential drawbacks of serum (e.g., cell differentiation, and / or heat inactivation) while still containing certain desired components of serum (e.g., cytokines). Because such clinical-grade serum substitutes avoid the risk of viral contamination, they can be directly used in clinical-level trials.

[0065] In this application, the term "KnockOut" TM "Serum substitute" typically refers to a serum-free additive suitable for culturing human and mouse pluripotent stem cells. (KnockOut) TM Serum substitutes can be used for stem cell culture and / or differentiation, for example, for the differentiation of iPS cells.

[0066] In this application, the term "Activin A" generally refers to a secreted protein belonging to the transforming growth factor (TGF) β family. Activin A can be a protein with a dimer structure. Activin A can be produced in bone marrow cells and immune cells within the bone marrow, and can regulate follicle-stimulating hormone (FSH), trigger hemoglobin production, signal dendritic cells to initiate an immune response, and play a role in angiogenesis. In this application, Activin A can be human Activin A.

[0067] In this application, the term "SU5402" generally refers to a multi-target receptor kinase inhibitor. For example, SU5402 can inhibit VEGFR2, FGFR1, and PDGF-Rβ. For example, SU5402 has an IC50 of [missing information - likely referring to an IC50 value] for VEGFR2, FGFR1, and PDGF-Rβ. 50 The values ​​can be 20nM, 30nM, and 510nM, respectively.

[0068] In this application, the term "cell state decline" generally refers to the process in which the cell's ability to proliferate, differentiate, and its physiological functions gradually decline during the cell cycle.

[0069] In this application, the term "B27 cell culture additive" generally refers to a serum-free additive that can be used for the growth and maintenance of short-term or long-term activity of hippocampal neurons and other central nervous system (CNS) neurons. The B27 cell culture additive may be developed by Gibco. The B27 cell culture additive may not contain vitamin A. For example, the B27 cell culture additive may include NeuroCult. TM SM1 Without Vitamin A (e.g., developed by STEMCELL).

[0070] In this application, the term "N2 cell culture additive" generally refers to an additive based on Bottenstein's N1 formulation, which may not contain serum. The N2 cell culture additive can be used for the growth and expression of late-stage mitotic neurons in primary cell cultures of neuroblastoma cells and peripheral nervous system (PNS) and central nervous system (CNS). The N2 cell culture additive can replace Bottenstein's N1 formulation.

[0071] In this application, the term "noggin" generally refers to NOG, which can be a homodimer composed of two non-glycosylated polypeptide chains. In this application, the gene number of recombinant noggin in NCBI can be 9241, and its protein accession number can be Q13253.

[0072] In this application, the term "DKK1 protein" generally refers to a member of the dipkopf-associated protein family. The DDK1 protein can inhibit the WNT signaling pathway involved in embryonic development, bind to LRP6 with high affinity, and prevent the formation of the Wnt-responsive Frizzled-Wnt-LRP6 complex. The DDK1 protein can influence eye development from a defined developmental time point and is crucial for the separation of the lens from the surface ectoderm via β-catenin-mediated Pdgfrα and E-cadherin expression. The human DKK1 protein accession number in GenBank is 22943.

[0073] In this application, the term "insulin-like growth factor-1 (IGF-1)" generally refers to a class of broad-spectrum growth factors with a chemical structure similar to insulin. IGF-1 plays an important role in promoting cell differentiation, proliferation, and individual growth and development. The accession number for human IGF-1 in GenBank is 3479.

[0074] In this application, the term "nicotinamide (NIC)" generally refers to an amide compound of nicotinic acid. It may have the following chemical formula:

[0075] In this application, the term "3-aminobenzamide (3ABA)" generally refers to a PARP inhibitor. It may have the following chemical formula:

[0076] In this application, the term "basic fibroblast growth factor (bFGF)" generally refers to basic fibroblast growth factor, or heparin-binding growth factor, which belongs to the FGF superfamily. The bFGF plays an important role in embryogenesis, tissue regeneration, wound healing, central nervous system development, angiogenesis, and tumor development. The bFGF can promote the division of mesodermal and neuroectodermal cells.

[0077] In this application, the term "retinal disease" generally refers to retinal pigment epithelium (RPE) atrophy or crystalline retinal degeneration. RPE atrophy typically refers to degenerative changes in the retinal pigment epithelium (RPE) characterized by cell death or dysfunction. Age-related macular degeneration or retinitis pigmentosa (RP) is usually accompanied by retinal pigment epithelium atrophy. Retinitis pigmentosa (RP), also known as retinal pigment disease, is generally a group of hereditary ophthalmic diseases. Its inheritance patterns include autosomal recessive, dominant, and X-linked inheritance, as well as bigenic and mitochondrial inheritance. Common early symptoms include night blindness and narrowed visual field, where the patient can see objects directly in front but cannot see objects slightly to the left or right; vision will gradually disappear afterward. Retinitis pigmentosa (RP) can include unilateral primary retinitis pigmentosa, quadrant primary retinitis pigmentosa, central or paracentral primary retinitis pigmentosa, achromic retinitis pigmentosa with white dot retinal degeneration, crystalline retinitis pigmentosa, venous lipophilic retinitis pigmentosa, arteriole-preserving retinitis pigmentosa, Leber congenital amaurosis, and retinitis pigmentosa in other syndromes. The main symptoms of crystalline retinitis can include a lens (a transparent covering) in the cornea, small, yellow or white crystalline deposits in the light-sensitive tissues of the retina, and progressive atrophy of the retina, choroidal capillaries, and choroid. Crystalline retinitis can also be caused by mutations in the CYP4V2 gene.

[0078] In this application, the terms “comprising” or “including” generally mean including the expressly specified features, but do not exclude other elements.

[0079] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Invention Details

[0081] On one hand, this application provides a method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE). This application significantly improves the efficiency of obtaining RPE from stem cell differentiation by using a suitable cell culture medium at an appropriate stem cell culture stage. The method described in this application has good reproducibility, is simple to operate, and the obtained RPE can be directly used for clinical treatment of patients, and / or the number and / or cell activity of obtained RPE are significantly improved.

[0082] On one hand, this application provides a method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE), which includes the following steps: before the stem cells show cell state decline after cell culture, the stem cells are placed in a first culture medium for culture, wherein the first culture medium contains DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA) and L-glutamine.

[0083] In this application, the first culture medium may contain DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX. TM .

[0084] All components involved in the first culture medium are components that meet clinical requirements (e.g., meet GMP standards). The first culture medium described in this application and / or the method described in this application can both meet clinical requirements. RPE cells obtained using the first culture medium described in this application and / or the method described in this application can also meet clinical requirements.

[0085] In this application, the DMEM culture medium may not contain serum. For example, the DMEM culture medium may contain KnockOut. TM DMEM and / or DMEM / F-12.

[0086] In this application, the serum may contain clinical-grade serum substitutes. For example, the serum may contain KnockOut. TM Serum substitutes.

[0087] In this application, the NEAA may comprise alanine, glutamic acid, asparagine, aspartic acid, proline, serine, and / or glycine. The NEAA may comprise L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and / or glycine. The NEAA may comprise at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acids.

[0088] In this application, the concentration of the serum in the first culture medium can be from about 10% (v / v) to about 30% (v / v). For example, the concentration of the serum can be from about 10% (v / v) to about 30% (v / v), from about 11% (v / v) to about 30% (v / v), from about 12% (v / v) to about 30% (v / v), from about 13% (v / v) to about 30% (v / v), from about 14% (v / v) to about 30% (v / v), from about 15% (v / v) to about 30% (v / v), from about 16% (v / v) to about 30% (v / v), from about 17% (v / v) to about 30% (v / v), from about 18% (v / v) to about 30% (v / v). % (v / v) - about 30% (v / v), about 19% (v / v) - about 30% (v / v), about 20% (v / v) - about 30% (v / v), about 10% (v / v) - about 28% (v / v), about 10% (v / v) - about 26% (v / v), about 10% (v / v) - about 24% (v / v), about 10% (v / v) - about 22% (v / v), about 10% (v / v) - about 20% (v / v), or about 15% (v / v) - about 25% (v / v). For example, the concentration of the serum may be at least about 10% (v / v), at least about 11% (v / v), at least about 12% (v / v), at least about 13% (v / v), at least about 14% (v / v), at least about 15% (v / v), at least about 16% (v / v), at least about 17% (v / v), at least about 18% (v / v), at least about 19% (v / v), at least about 20% (v / v), at least about 21% (v / v), at least about 22% (v / v), at least about 23% (v / v), at least about 24% (v / v), at least about 25% (v / v), at least about 26% (v / v), at least about 27% (v / v), at least about 28% (v / v), at least about 29% (v / v), or at least about 30% (v / v).

[0089] In this application, the concentration of β-mercaptoethanol in the first culture medium can be from about 0.1% (v / v) to about 1% (v / v). For example, the concentration of the β-mercaptoethanol may be about 0.1% (v / v) to about 1% (v / v), about 0.2% (v / v) to about 1% (v / v), about 0.4% (v / v) to about 1% (v / v), about 0.5% (v / v) to about 1% (v / v), about 0.6% (v / v) to about 1% (v / v), about 0.7% (v / v) to about 1% (v / v), about 0.8% (v / v) to about 1% (v / v), about 0.1% (v / v) to about 0.9% (v / v), about 0.2% (v / v) to about 0.9% (v / v), about 0.4% (v / v) to about 0.9% (v / v), or about 0.5% (v / v) to about 0.9% (v / v). For example, the concentration of the β-mercaptoethanol may be at least about 0.1% (v / v), at least about 0.2% (v / v), at least about 0.3% (v / v), at least about 0.4% (v / v), at least about 0.5% (v / v), at least about 0.6% (v / v), at least about 0.7% (v / v), at least about 0.8% (v / v), at least about 0.9% (v / v), or at least about 1.0% (v / v).

[0090] In this application, the concentration of NEAA in the first culture medium can be from about 0.5% (v / v) to about 2% (v / v). For example, the concentration of NEAA can be from about 0.5% (v / v) to about 2% (v / v), from about 0.6% (v / v) to about 2% (v / v), from about 0.7% (v / v) to about 2% (v / v), from about 0.8% (v / v) to about 2% (v / v), from about 0.9% (v / v) to about 2% (v / v), from about 1.0% (v / v) to about 2% (v / v), from about 0.5% (v / v) to about 1.9% (v / v), or from about 0.5% (v / v) to about 1.8% (v / v). (v), about 0.5% (v / v) to about 1.7% (v / v), about 0.5% (v / v) to about 1.6% (v / v), about 0.5% (v / v) to about 1.5% (v / v), about 0.5% (v / v) to about 1.4% (v / v), about 0.5% (v / v) to about 1.3% (v / v), about 0.5% (v / v) to about 1.2% (v / v), about 0.5% (v / v) to about 1.1% (v / v) or about 0.5% (v / v) to about 1% (v / v). For example, the concentration of the NEAA may be at least about 0.5% (v / v), at least about 0.6% (v / v), at least about 0.7% (v / v), at least about 0.8% (v / v), at least about 0.9% (v / v), at least about 1.0% (v / v), at least about 1.1% (v / v), at least about 1.2% (v / v), at least about 1.3% (v / v), at least about 1.4% (v / v), at least about 1.5% (v / v), at least about 1.6% (v / v), at least about 1.7% (v / v), at least about 1.8% (v / v), at least about 1.9% (v / v), or at least about 2.0% (v / v).

[0091] In this application, the concentration of L-glutamine in the first culture medium can be from about 0.5% (v / v) to about 2% (v / v). For example, the concentration of L-glutamine can be from about 0.5% (v / v) to about 2% (v / v), from about 0.6% (v / v) to about 2% (v / v), from about 0.7% (v / v) to about 2% (v / v), from about 0.8% (v / v) to about 2% (v / v), from about 0.9% (v / v) to about 2% (v / v), from about 1.0% (v / v) to about 2% (v / v), from about 0.5% (v / v) to about 1.9% (v / v), or from about 0.5% (v / v) to about 1.8% (v / v). / v), about 0.5% (v / v) to about 1.7% (v / v), about 0.5% (v / v) to about 1.6% (v / v), about 0.5% (v / v) to about 1.5% (v / v), about 0.5% (v / v) to about 1.4% (v / v), about 0.5% (v / v) to about 1.3% (v / v), about 0.5% (v / v) to about 1.2% (v / v), about 0.5% (v / v) to about 1.1% (v / v) or about 0.5% (v / v) to about 1% (v / v). For example, the concentration of the L-glutamine may be at least about 0.5% (v / v), at least about 0.6% (v / v), at least about 0.7% (v / v), at least about 0.8% (v / v), at least about 0.9% (v / v), at least about 1.0% (v / v), at least about 1.1% (v / v), at least about 1.2% (v / v), at least about 1.3% (v / v), at least about 1.4% (v / v), at least about 1.5% (v / v), at least about 1.6% (v / v), at least about 1.7% (v / v), at least about 1.8% (v / v), at least about 1.9% (v / v), or at least about 2.0% (v / v).

[0092] In this application, in the first culture medium, the GlutaMAX TM The concentration can be from about 0.5% (v / v) to about 2% (v / v). For example, the GlutaMAX... TMThe concentration can be approximately 0.5% (v / v) to approximately 2% (v / v), approximately 0.6% (v / v) to approximately 2% (v / v), approximately 0.7% (v / v) to approximately 2% (v / v), approximately 0.8% (v / v) to approximately 2% (v / v), approximately 0.9% (v / v) to approximately 2% (v / v), approximately 1.0% (v / v) to approximately 2% (v / v), approximately 0.5% (v / v) to approximately 1.9% (v / v), approximately 0.5% (v / v) to approximately 1.8% (v / v), approximately 0. 0.5% (v / v) - about 1.7% (v / v), about 0.5% (v / v) - about 1.6% (v / v), about 0.5% (v / v) - about 1.5% (v / v), about 0.5% (v / v) - about 1.4% (v / v), about 0.5% (v / v) - about 1.3% (v / v), about 0.5% (v / v) - about 1.2% (v / v), about 0.5% (v / v) - about 1.1% (v / v), or about 0.5% (v / v) - about 1% (v / v). For example, the GlutaMAX... TM The concentration may be at least about 0.5% (v / v), at least about 0.6% (v / v), at least about 0.7% (v / v), at least about 0.8% (v / v), at least about 0.9% (v / v), at least about 1.0% (v / v), at least about 1.1% (v / v), at least about 1.2% (v / v), at least about 1.3% (v / v), at least about 1.4% (v / v), at least about 1.5% (v / v), at least about 1.6% (v / v), at least about 1.7% (v / v), at least about 1.8% (v / v), at least about 1.9% (v / v), or at least about 2.0% (v / v).

[0093] For example, the first culture medium may contain DMEM medium, about 10% (v / v) to about 30% (v / v) serum, about 0.1% (v / v) to about 1% (v / v) β-mercaptoethanol, about 0.5% (v / v) to about 2% (v / v) non-essential amino acids (NEAA), and about 0.5% (v / v) to about 2% (v / v) GlutaMAX. TM .

[0094] For example, the first culture medium may contain DMEM medium, approximately 20% (v / v) serum, approximately 1% (v / v) β-mercaptoethanol, approximately 1% (v / v) non-essential amino acids (NEAA), and approximately 1% (v / v) GlutaMAX. TM .

[0095] For example, the first culture medium may consist of DMEM medium, approximately 20% (v / v) serum, approximately 1% (v / v) β-mercaptoethanol, approximately 1% (v / v) non-essential amino acids (NEAA), and approximately 1% (v / v) GlutaMAX. TMcomposition.

[0096] In this application, the culture can be carried out in the first culture medium for about 5 to about 15 days. For example, it can be carried out in the first culture medium for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days or more.

[0097] In this application, the first culture medium may further contain activin A.

[0098] In this application, the concentration of Activin A can be from about 50 ng / mL to about 200 ng / mL. For example, the Activin The concentration of A can be approximately 50 ng / mL to approximately 200 ng / mL, approximately 60 ng / mL to approximately 200 ng / mL, approximately 70 ng / mL to approximately 200 ng / mL, approximately 80 ng / mL to approximately 200 ng / mL, approximately 90 ng / mL to approximately 200 ng / mL, approximately 100 ng / mL to approximately 200 ng / mL, approximately 50 ng / mL to approximately 190 ng / mL, approximately 50 ng / mL to approximately 180 ng / mL, approximately 50 ng / mL to approximately 170 ng / mL, approximately 50 ng / mL to approximately 160 ng / mL, approximately 50 ng / mL to approximately 150 ng / mL, approximately 50 ng / mL to approximately 140 ng / mL, approximately 50 ng / mL to approximately 130 ng / mL, approximately 50 ng / mL to approximately 120 ng / mL, approximately 50 ng / mL to approximately 110 ng / mL, or approximately 50 ng / mL to approximately 100 ng / mL. For example, the concentration of Activin A may be at least about 50 ng / mL, at least about 60 ng / mL, at least about 70 ng / mL, at least about 80 ng / mL, at least about 90 ng / mL, at least about 100 ng / mL, at least about 110 ng / mL, at least about 120 ng / mL, at least about 130 ng / mL, at least about 140 ng / mL, at least about 150 ng / mL, at least about 160 ng / mL, at least about 170 ng / mL, at least about 180 ng / mL, at least about 190 ng / mL, or at least about 200 ng / mL.

[0099] In this application, the first culture medium may also contain SU5402.

[0100] In this application, the concentration of SU5402 can be from about 5 μM to about 20 μM. For example, the concentration of SU5402 can be from about 5 μM to about 20 μM, from about 6 μM to about 20 μM, from about 7 μM to about 20 μM, from about 8 μM to about 20 μM, from about 9 μM to about 20 μM, from about 10 μM to about 20 μM, from about 5 μM to about 19 μM, from about 5 μM to about 18 μM, from about 5 μM to about 17 μM, from about 5 μM to about 16 μM, from about 5 μM to about 15 μM, from about 5 μM to about 14 μM, from about 5 μM to about 13 μM, from about 5 μM to about 12 μM, from about 5 μM to about 11 μM, or from about 5 μM to about 10 μM. For example, the concentration of SU5402 may be at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 11 μM, at least about 12 μM, at least about 13 μM, at least about 14 μM, at least about 15 μM, at least about 16 μM, at least about 17 μM, at least about 18 μM, at least about 19 μM, or at least about 20 μM.

[0101] For example, the first culture medium may contain DMEM medium, approximately 10% (v / v) to approximately 30% (v / v) serum, approximately 0.1% (v / v) to approximately 1% (v / v) β-mercaptoethanol, approximately 0.5% (v / v) to approximately 2% (v / v) non-essential amino acids (NEAA), and approximately 0.5% (v / v) to approximately 2% (v / v) GlutaMAX. TM Approximately 50 ng / mL to approximately 200 ng / mL Activin A and approximately 5 μM to approximately 20 μM U5402.

[0102] For example, the first culture medium may contain DMEM medium, approximately 20% (v / v) serum, approximately 1% (v / v) β-mercaptoethanol, approximately 1% (v / v) non-essential amino acids (NEAA), and approximately 1% (v / v) GlutaMAX. TM Approximately 100 ng / mL Activin A and approximately 10 μM SU5402.

[0103] For example, the first culture medium may consist of DMEM medium, approximately 20% (v / v) serum, approximately 1% (v / v) β-mercaptoethanol, approximately 1% (v / v) non-essential amino acids (NEAA), and approximately 1% (v / v) GlutaMAX. TM It consists of approximately 100 ng / mL Activin A and approximately 10 μM SU5402.

[0104] In this application, the stem cells can be cultured in the first culture medium for about 5 to about 15 days. For example, they can be cultured in the first culture medium for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, or more.

[0105] In this application, the method includes processing the first culture medium (composed of DMEM medium, about 20% (v / v) serum, about 1% (v / v) β-mercaptoethanol, about 1% (v / v) non-essential amino acids (NEAA) and about 1% (v / v) GlutaMAX). TM Cultured in DMEM medium for approximately 1–10 days, followed by culture in the first medium-1 (composed of DMEM medium, approximately 20% (v / v) serum, approximately 1% (v / v) β-mercaptoethanol, approximately 1% (v / v) non-essential amino acids (NEAA), and approximately 1% (v / v) Glutamax. TM It is cultured in an atmosphere containing approximately 100 ng / mL Activin A and approximately 10 μM SU5402 for approximately 1 to 5 days.

[0106] In this application, the cell state decay may include at least about 20% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more) of the cells exhibiting a state selected from the group consisting of: cell shrinkage, cell shedding, and cell apoptosis.

[0107] In this application, apoptosis can be a self-initiated, orderly cell death. Cell detachment can refer to cells falling off the wall of the culture medium container. Cell shrinkage can be a change in cell morphology, such as a decrease in size, deformation, rounding, and / or intact cell membrane but bubbling. The proportion can be obtained by calculating the ratio of the number of cells exhibiting the cell state degradation to the total number of cells in the same field of view.

[0108] In this application, the cell state decline can occur from about day 6 to about day 15 after the stem cells have been cultured (e.g., about day 6, about day 7, about day 8, about day 9, about day 10, about day 11, about day 12, about day 13, about day 14 or about day 15 after the cells have been cultured).

[0109] In this application, prior to cell culture, the cell fusion rate of the stem cells can reach approximately 70% (e.g., at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or higher).

[0110] In this application, the stem cells may be digested before cell culture. The digestion may use digestive enzymes or reagents conventional in the art, as long as they do not affect the survival and / or biological activity of the stem cells.

[0111] In this application, after digestion, the stem cells can be seeded at a ratio of about 1:2 to about 1:4 (for example, about 1:2, about 1:2.5, about 1:3, about 1:3.5 or about 1:4).

[0112] In this application, the seeding can be placed in Fibronectin or Cell superior.

[0113] In this application, the cell culture can be carried out in a second culture medium, which may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, and non-essential amino acids (NEAA). For example, the second culture medium may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, and 1×NEAA. For example, the second culture medium may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, and 1×NEAA.

[0114] For example, after the digestion, the stem cells can be cultured in the second culture medium described above. For example, they can be cultured for at least about 12 hours.

[0115] In this application, the cell culture can be carried out in a second culture medium-1, wherein the second culture medium-1 may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), human cephalin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), and nicotinamide (NIC); or, the second culture medium-1 may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), human cephalin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), and 3-aminobenzamide (3ABA). For example, the second culture medium-1 may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 50 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1 and 10 mM NIC; or, the second culture medium-1 may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 50 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1 and 5 mM 3ABA. For example, the second culture medium-1 may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 50 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1 and 10 mM NIC; or, the second culture medium-1 may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 50 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1 and 5 mM 3ABA.

[0116] In this application, the cell culture can be carried out in the second culture medium-1 for about 1-2 days. The second culture medium-1 can be replaced daily.

[0117] In this application, the cell culture can be further carried out in a second culture medium-2, wherein the second culture medium-2 may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), human cephalin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and nicotinamide (NIC); or, the second culture medium-2 may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), human cephalin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and 3-aminobenzamide (3ABA). For example, the second culture medium-2 may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 10 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1, 5 ng / mL bFGF and 10 mM NIC; or, the second culture medium-2 may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 10 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1, 5 ng / mL bFGF and 5 mM 3ABA. For example, the second culture medium-2 may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 10 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1, 5 ng / mL bFGF and 10 mM NIC; or, the second culture medium-2 may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 10 ng / mL Noggin, 10 ng / mL DKK1, 10 ng / mL IGF1, 5 ng / mL bFGF and 5 mM 3ABA.

[0118] In this application, the cell culture can continue to be carried out in the second culture medium-2 for about 1-2 days. The second culture medium-2 can be replaced daily.

[0119] In this application, the cell culture can be further carried out in a second culture medium-3, which may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), DKK1 protein, insulin-like growth factor-1 (IGF-1), and activin A. For example, the second culture medium-3 may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 10 ng / mL DKK1, 10 ng / mL IGF1, and 100 ng / mL Activin A. For example, the second culture medium-3 may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 10 ng / mL DKK1, 10 ng / mL IGF1, and 100 ng / mL Activin A.

[0120] In this application, the cell culture can continue in the second culture medium-3 for approximately 1-2 days. The second culture medium-3 can be replaced daily.

[0121] In some cases, the cell culture can be continued in a second culture medium-4, which may contain DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), activin A, and SU5402. For example, the second culture medium-3 may contain DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 100 ng / mL Activin A, and 10 μM SU5402. For example, the second culture medium-3 may consist of DMEM medium, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×NEAA, 100 ng / mL Activin A, and 10 μM SU5402.

[0122] In this application, the cell culture can continue to be carried out in the second culture medium-4 for about 1-2 days. The second culture medium-4 can be replaced daily.

[0123] In this application, the method may further include the following step: digesting cells cultured with the RPE morphology. For example, TrypLE Express can be used for digestion, followed by single-cell filtration to remove cells that do not have the RPE morphology.

[0124] In this application, the method may further include the following step: enriching cells with RPE morphology cultured from the cells.

[0125] In this application, the enrichment is performed using methods selected from the group consisting of flow cytometry (FACS) and immunomagnetic bead cell sorting (MACS).

[0126] In this application, the method may further include the step of: continuing to culture cells with the RPE morphology. The continued culture may use the first culture medium. During the continued culture, the first culture medium may be replaced every two days.

[0127] In this application, the stem cells may include embryonic stem cells and / or induced stem cells.

[0128] In this application, the induced stem cells (iPS) can be stem cells induced from somatic cells, which have developmental pluripotency similar to embryonic stem cells.

[0129] In this application, the stem cells may include human cells.

[0130] In this application, the stem cells may be human embryonic stem cells. For example, the stem cells can be obtained without destroying a human embryo.

[0131] On the other hand, this application provides a method for generating RPE cells, which includes the following steps:

[0132] a) Obtaining stem cells;

[0133] b) According to the method described in this application, the stem cells of step a) are induced to differentiate into RPE cells.

[0134] On the other hand, this application provides the use of the method described in this application in the preparation of a medicament for treating retinal diseases.

[0135] The method described in this application can efficiently obtain large quantities of RPE cells. These RPE cells can be used to prepare drugs for treating retinal diseases. For example, the RPE cells can be used to treat retinal degenerative diseases.

[0136] This application also relates to the following implementation methods:

[0137] 1. A method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE), comprising the following steps:

[0138] Before the stem cells show signs of cell deterioration after cell culture, the stem cells are placed in a first culture medium for culture.

[0139] The first culture medium contains DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA), and L-glutamine.

[0140] 2. The method according to Embodiment 1, wherein the first culture medium comprises DMEM medium, serum, β-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX. TM .

[0141] 3. The method according to any one of embodiments 1-2, wherein the DMEM culture medium contains KnockOut TM DMEM and / or DMEM / F-12.

[0142] 4. The method according to any one of embodiments 1-3, wherein the serum comprises a clinical-grade serum substitute.

[0143] 5. The method according to any one of embodiments 1-4, wherein the serum contains KnockOut TM Serum substitutes.

[0144] 6. The method according to any one of embodiments 1-5, wherein the NEAA comprises L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and / or glycine.

[0145] 7. The method according to any one of embodiments 1-6, wherein the concentration of the serum in the first culture medium is about 10% (v / v) to about 30% (v / v).

[0146] 8. The method according to any one of embodiments 1-7, wherein the concentration of β-mercaptoethanol in the first culture medium is 0.1% (v / v) to about 1% (v / v).

[0147] 9. The method according to any one of embodiments 1-8, wherein the concentration of NEAA in the first culture medium is 0.5% (v / v) to about 2% (v / v).

[0148] 10. The method according to any one of embodiments 1-9, wherein the concentration of L-glutamine in the first culture medium is 0.5% (v / v) to about 2% (v / v).

[0149] 11. The method according to any one of embodiments 2-10, wherein in the first culture medium, the GlutaMAX TM The concentration is 0.5% (v / v) to about 2% (v / v).

[0150] 12. The method according to any one of embodiments 1-11, wherein the first culture medium further comprises activin A.

[0151] 13. The method according to embodiment 12, wherein the concentration of Activin A is about 50 ng / mL to about 200 ng / mL.

[0152] 14. The method according to any one of embodiments 1-13, wherein the first culture medium further comprises SU5402.

[0153] 15. The method according to embodiment 14, wherein the concentration of SU5402 is about 5 μM to about 20 μM.

[0154] 16. The method according to any one of embodiments 1-15, wherein the stem cells are cultured in the first culture medium for about 5 to about 15 days.

[0155] 17. The method according to any one of embodiments 1-16, wherein the cell state deterioration includes at least about 20% of the cells exhibiting a state selected from the group consisting of: cell shrinkage, cell shedding, and cell apoptosis.

[0156] 18. The method according to any one of embodiments 1-17, wherein the cell state decline occurs approximately 6 to approximately 15 days after the stem cells have been cultured.

[0157] 19. The method according to any one of embodiments 1-18, wherein the cell fusion rate of the stem cells reaches about 70% before the cell culture.

[0158] 20. The method according to any one of embodiments 1-19, wherein the stem cells are digested prior to the cell culture.

[0159] 21. The method according to embodiment 20, wherein after digestion, the stem cells are seeded at a ratio of about 1:2 to about 1:4.

[0160] 22. The method according to embodiment 21, wherein the seeding is placed in Fibronectin or Cell superior.

[0161] 23. The method according to any one of embodiments 1-22, wherein the cell culture is performed in a second culture medium, wherein the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive and non-essential amino acids (NEAA).

[0162] 24. The method according to any one of embodiments 1-23, wherein the cell culture is performed in a second culture medium, wherein the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), and nicotinamide (NIC); or, the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), and 3-aminobenzamide (3ABA).

[0163] 25. The method according to embodiment 24, wherein the cell culture is carried out in the second culture medium for about 2 days.

[0164] 26. The method according to any one of embodiments 1-25, wherein the cell culture is performed in a second culture medium, wherein the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and nicotinamide (NIC); or, the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and 3-aminobenzamide (3ABA).

[0165] 27. The method according to embodiment 26, wherein the cell culture continues to be cultured in the second culture medium for about 2 days.

[0166] 28. The method according to any one of embodiments 1-27, wherein the cell culture is performed in a second culture medium, wherein the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), DKK1 protein, insulin-like growth factor-1 (IGF-1) and activin A.

[0167] 29. The method according to embodiment 28, wherein the cell culture continues to be cultured in the second culture medium for about 2 days.

[0168] 30. The method according to any one of embodiments 1-29, wherein the cell culture is performed in a second culture medium, wherein the second culture medium comprises DMEM medium, B27 cell culture additive, N2 cell culture additive, non-essential amino acids (NEAA), activin A, and SU5402.

[0169] 31. The method according to embodiment 30, wherein the cell culture continues to be cultured in the second culture medium for about 1 day.

[0170] 32. The method according to any one of embodiments 1-31 further includes the step of digesting cells with RPE morphology cultured by the said cells.

[0171] 33. The method according to any one of embodiments 1-32 further includes the step of enriching cells with RPE morphology cultured by the said cells.

[0172] 34. The method according to embodiment 33, wherein the enrichment is performed using a method selected from the group consisting of flow cytometry (FACS) and immunomagnetic bead cell sorting (MACS).

[0173] 35. The method according to any one of embodiments 1-34, wherein the stem cells comprise: embryonic stem cells and / or pluripotent stem cells.

[0174] 36. The method according to any one of embodiments 1-35, wherein the stem cells comprise human cells.

[0175] 37. The method according to any one of embodiments 1-36, wherein the stem cells are human embryonic stem cells.

[0176] 38. The method according to any one of embodiments 1-37, wherein the stem cells are obtained by means of receiving a human embryo without destroying it.

[0177] 39. A method for producing RPE cells, comprising the following steps:

[0178] a) Obtaining stem cells;

[0179] b) Inducing the stem cells of step a) to differentiate into RPE cells according to any one of embodiments 1-38.

[0180] 40. The application of the method described in any one of embodiments 1-39 in the preparation of a medicament for treating retinal diseases.

[0181] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0182] Example

[0183] Example 1: Inducing stem cell differentiation into RPE

[0184] 1. One day before cell culture: When the stem cells (iPS) are cultured in a 3.5cm dish to a cell confluence rate of about 70%, the iPS cells (iPS cells obtained by reprogramming UCs (renal epithelial cells) extracted from human urine or PBMCs (peripheral blood mononuclear cells) isolated from human whole blood) are digested into small clumps using Accutase / TryPLE (purchased from Invitrogen). After centrifugation, the cells are seeded onto Matrigel-coated culture plates at a ratio of 1:2 to 1:4.

[0185] Add the second culture medium: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive and 1×non-essential amino acids (NEAA), and incubate overnight in an incubator.

[0186] 2. Day 0-1 of cell culture: Remove cell supernatant and replace with the second culture medium-1: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×non-essential amino acid (NEAA), 50 ng / mL human cephalin (Noggin), 10 ng / mL DKK1 protein, 10 ng / mL insulin-like growth factor-1 (IGF-1) and 10 mM nicotinamide (NIC) / or 5 mM 3ABA. Culture for two days, changing the culture medium daily.

[0187] 3. Days 2-3 of cell culture: Remove cell supernatant and replace with medium 2: DMEM / F12, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×Non-essential amino acids (NEAA), 10 ng / mL Noggin, 10 ng / mL DKK1 protein, 10 ng / mL insulin-like growth factor-1 (IGF-1), 5 ng / mL bFGF and 10 mM nicotinamide (NIC) / or 5 mM 3ABA; culture for two days, changing the culture medium daily.

[0188] 4. Days 4-5 of cell culture: Remove cell supernatant and replace with second culture medium-3: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×non-essential amino acids (NEAA), 10 ng / mL DKK1 protein, 10 ng / mL insulin-like growth factor-1 (IGF-1) and 100 ng / mL Activin A. Culture for two days, changing the culture medium daily.

[0189] 5. Cell culture days 6-15:

[0190] Replace the second culture medium-3 with the first culture medium:

[0191] KnockOut TM DMEM, 20% KnockOut TM Serum substitute, 0.5% β-mercaptoethanol, 1% NEAA, 1% L-glutamine.

[0192] After culturing in the first culture medium for 6-10 days, add 100 ng / mL Activin A and 10 μM SU5402 to the first culture medium.

[0193] 6. Cell culture days 21-28: Digest cells into single cells using TrypLE Express (purchased from Invitrogen). Pass the cells through a 40μm single-cell filter to remove non-RPE cell morphology cells to achieve RPE cell purification.

[0194] Cells were seeded onto Matrigel-coated tissue culture plates or Transwell membranes. The purified cells were cultured in the first culture medium, with the culture medium changed every two days. Mature RPE cells with high purity and typical morphology were observed around day 27 of cell culture.

[0195] The differentiated RPE cells were photographed at different time points during cell culture, as detailed in the following figures. Figures 1-9 .in Figures 1-9 They were displayed respectively Figure 1 After 9 days of cultivation, the field of vision is magnified 4 times; Figure 2 After 9 days of cultivation, the field of vision is magnified 10 times; Figure 3 After 9 days of cultivation, the field of vision is magnified 20 times; Figure 4 After 25 days of cultivation, the field of vision is magnified 4 times; Figure 5 After 25 days of cultivation, the field of vision is magnified 10 times; Figure 6 After 25 days of cultivation, the field of vision is magnified 20 times; Figure 7 After 32 days of cultivation, the field of vision is magnified 20 times; Figure 8 After 32 days of cultivation, the field of vision is magnified 40 times; Figure 9 Observations after 45 days of cultivation, magnified 40 times.

[0196] Example 2: Identification of retinal pigment epithelial (RPE) markers (qPCR method)

[0197] The expression of the marker genes CRALBP, RPE65, and iPS marker gene OCT4 at the mRNA level in retinal pigment epithelial cells induced at each stage was examined.

[0198] Experimental methods

[0199] The qPCR method was used, based on the literature Rezania, A., et al., Reversal of diabetes withinsulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol, 2014, 32(11): p.1121-33.

[0200] The equipment involved

[0201]

[0202] Reagents and primers involved

[0203]

[0204]

[0205] This embodiment also involves the use of 10μl pipette tips, 200μl pipette tips, and 1000μl pipette tips.

[0206] Specific steps

[0207] Collect the cells obtained by culturing according to the method described in Example 1, and wash them twice by centrifugation with DPBS.

[0208] Total RNA was extracted according to the instructions of the "High Purity Total RNA Rapid Extraction Kit (Centrifuge Column Type)".

[0209] Reverse transcription was performed according to the instructions in "TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix" to obtain cDNA.

[0210] Reagent names in the kit Add volume R-Mix 10μl E-Mix 1μl Oligdf 1μl Remove 1μl RNA+water 7μl

[0211] According to the instructions in "TransStart Top Green qPCR SuperMix", primers and template were added to a 20 μl system on ice.

[0212] Template + Water 9.2μl Mix 10μl forward primer 0.4μl reverse primer 0.4μl

[0213] In the qPCR instrument, set the program parameters, select the FAM and VIC fluorescence channels, and set the reaction conditions as follows:

[0214] Pre-variation 94℃ 30s transsexual 94℃ 5s annealing 60℃ 15s extend 72℃ 34s

[0215] Meanwhile, cells obtained by culturing according to the control method were collected, and the expression levels of retinal pigment epithelial cell marker genes CRALBP, RPE65, and iPS marker gene OCT4 at the mRNA level were measured according to the above method.

[0216] The steps for using the comparison method are as follows:

[0217] Day 0 of cell culture: iPS cells were cultured at a rate of 30,000 cells / cm³. 2 The cells were seeded at a density of 90%-100% in T25 flasks and cultured until the cell density reached 90%-100%.

[0218] Day 1 of cell culture: Aspirate the culture medium and add a 1:1 E8:TLP medium. The TLP medium contains KnockOut... TM DMEM, 20% KnockOut TM Serum substitutes, 0.5% β-mercaptoethanol, 1% NEAA and 1% L-glutamine.

[0219] Cell culture days 2-70: On day 2, replace with complete TLP medium, 7 mL / flask. Change the medium every 3 days thereafter. After approximately 6 weeks, determine whether to proceed to the next stage based on pigment cell yield.

[0220] Days 71-113 of cell culture: Pigmented cell clusters were manually collected into DPBS. After centrifugation and removal of supernatant, 1 mL of Accutase (Sigma) was added, and the cells were digested at 37°C for 1 hour. The cells were then dispersed by pipette tip. Digestion was terminated by adding 5 volumes of TLP medium. Cells were passed through a 70 μm cell sieve (Corning), washed with TLP medium, and counted. Cells were counted at a density of greater than 5 cells / cm². 2 Seeds were planted at a density in 12-well or 24-well plates (the plates were pre-coated with Matrigel diluted 1:100). The medium was changed every 3 days for about 6 weeks.

[0221] The results are as follows Figures 10A-10C As shown. Figures 10A-10C The expression levels of iPS marker gene OCT4, RPE marker gene RPE65, and CRALBP are shown sequentially.

[0222] The results in Figure 10 show that, compared with the control method, the cells obtained using the method described in Example 1 showed a significant decrease in the expression level of the iPS marker gene OCT4 and a significant increase in the expression levels of the RPE marker genes RPE65 and CRALBP. This indicates that, compared with the control method, the method described in Example 1 can significantly promote the differentiation of iPS cells into RPE.

[0223] Example 3: RPE Immunofluorescence Identification Experiment

[0224] 1. Wash the RPE cells prepared by culturing for 30 days using the method described in Example 1 three times with DPBS, fix with 4% PFA at room temperature for 20 min, and protect from light.

[0225] 2. Wash three times with DPBS.

[0226] 3. 0.2% Triton X-100 (dissolved in DPBS), perforate for 15 min.

[0227] 4. Block with “5% BSA blocking solution” + 0.1% Triton X-100 overnight at 4°C.

[0228] 5. PBST-1 (for washing): DPBS + 0.1% Triton X-100;

[0229] 6. PBST-2 (for dissolving primary antibody (ZO-1, rabbit source) and secondary antibody (586, goat anti-rabbit)): DPBS + 0.1% Triton X-100 + 1% BSA.

[0230] 7. Add the primary antibody, primary antibody:PBST-2 = 1:200, incubate overnight at 4°C.

[0231] 8. Recover the primary antibody solution and wash three times with PBST-1 for 10 minutes each time.

[0232] 9. Add secondary antibody, secondary antibody:PBST-2 = 1:500, incubate overnight at 4℃, protected from light.

[0233] 10. Wash three times with PBST-1, 10 minutes each time.

[0234] 11. Dilute DAPI to 1000× and incubate for 2-3 minutes in the dark. Wash with DPBS 1-2 times.

[0235] 12. Cover with 90% glycerin and take a picture to observe directly.

[0236] The results are as follows Figure 11A-11B As shown. Figure 11A-11B The observations were shown sequentially after adding the primary antibody ZO-1 at 20x magnification, after adding the primary antibody ZO-1 at 40x magnification, and after adding the secondary antibody ES at 40x magnification.

[0237] The results indicate that the ZO-1 protein is located between adjacent cells, thus exhibiting the hexagonal outline of RPE cells.

[0238] Example 4: RPE phagocytosis experiment

[0239] RPE cells can phagocytose detached photoreceptor cell segments, preventing excessive accumulation of segments from affecting the stability of the subretinal space environment. To verify whether the RPE cells obtained using the method described in Example 1 have the function of phagocytizing photoreceptor cell segments, GFP-labeled magnetic beads were used to replace the photoreceptor cell segments and were co-cultured with RPE cells to observe the phagocytosis.

[0240] 1. Dilute the magnetic beads (latex balls) to 4.8 × 10⁻⁶ using RPE differentiation medium TLP. 4 Up to 4.0 × 10 7 / mL.

[0241] 2. Remove the culture medium from the culture dish to be tested and wash it once with DPBS.

[0242] 3. Add the diluted culture medium containing magnetic beads to the dish, shake well, and culture the RPE cells prepared by the method described in Example 1 for 30 days in a 37℃ / 5% CO2 cell culture incubator for 12-24 hours.

[0243] 4. Remove the culture medium from the dish, wash thoroughly with DPBS at least 5 times, and observe under a fluorescence microscope to see if it is clean. After confirming that it is clean, add RPE differentiation medium.

[0244] 5. Observe and photograph under a fluorescence microscope.

[0245] The results are as follows Figure 12A-12B As shown. Figure 12A-12B The results show the fluorescence signal magnified 20 times and the combined fluorescence signal and white light under these conditions. Figure 12 shows that green fluorescence signals exist within the differentiated RPE cells, all located in the cytoplasm. No green fluorescence signals are observed in the nucleus or between cells, indicating that latex globules are phagocytosed into the cell interior rather than adhering to the cell surface. Furthermore, the green fluorescence signals are mainly concentrated in more mature RPE cells with greater pigment accumulation. Therefore, the RPE cells prepared using the method described in Example 1 possess the function of phagocytizing photoreceptor outer segments.

[0246] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents. sequence list <110> Beijing Zhongyin Technology Co., Ltd. <120> Methods to induce stem cells to differentiate into retinal pigment epithelial cells <130> 0138-PA-017 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> OCT3 / 4 forward primers <400> 1 gcaaaacccg gaggaggagt c 21 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> OCT3 / 4 reverse primers <400> 2 ccacatcggc ctgtgtatat c 21 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> CRALBP forward primer <400> 3 agatctcagg aagatggtgg ac 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRALBP reverse primer <400> 4 gaagtggatg gctttgaacc 20 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> RPE65 forward primer <400> 5 caatgggttt ctgattgtgg a 21 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> RPE65 reverse primer <400> 6 ccagttctca cgtaaattgg cta 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 7 accacagtcc atgccatcac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 8 tccaccaccc tgttgctgta 20

Claims

1. A method for inducing stem cells to differentiate into retinal pigment epithelial cells (RPE), comprising the following steps: 1) One day before cell culture: When the iPS cells reach 70% confluence, digest the iPS cells into small clumps, centrifuge them, and seed them onto Matrigel-coated culture plates at a ratio of 1:2 to 1:

4. Add the second culture medium: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive and 1×non-essential amino acids (NEAA), and incubate overnight in an incubator; 2) Day 0-1 of cell culture: Remove cell supernatant and replace with second culture medium-1: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×non-essential amino acids (NEAA), 50 ng / mL human cephalin (Noggin), 10 ng / mL DKK1 protein, 10 ng / mL insulin-like growth factor-1 (IGF-1) and 10 mM nicotinamide (NIC) / or 5 mM 3-aminobenzamide (3ABA), culture for two days, changing the culture medium daily; 3) Days 2-3 of cell culture: Remove cell supernatant and replace with second culture medium-2: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×non-essential amino acids (NEAA), 10 ng / mL human cephalin (Noggin), 10 ng / mL DKK1 protein, 10 ng / mL insulin-like growth factor-1 (IGF-1), 5 ng / ml bFGF and 10 mM nicotinamide (NIC) / or 5 mM 3ABA. Culture for two days, changing the culture medium daily. 4) Days 4-5 of cell culture: Remove cell supernatant and replace with second culture medium-3: DMEM medium / F12, 1×B27 cell culture additive, 1×N2 cell culture additive, 1×non-essential amino acids (NEAA), 10 ng / mL DKK1 protein, 10 ng / mL insulin-like growth factor-1 (IGF-1) and 100 ng / mL Activin A. Culture for two days, changing the culture medium daily. 5) Days 6-15 of cell culture: Change second medium-3 to first medium: KnockOut TM DMEM, 20% KnockOut TM serum replacement, 0.5% beta-mercaptoethanol, 1% NEAA, 1% L-glutamine, and further additions to the first medium after 6-10 days of culture with the first medium: 100 ng / mL Activin A and 10 μM SU5402; 6) Cell culture days 21-28: Digest the cells into single cells, remove non-RPECells morphology cells through a single-cell filter to purify RPE cells, seed the cells onto Matrigel-coated tissue culture plates or Transwell membranes, and culture the purified cells in the first culture medium, changing the culture medium every two days. On day 27 of cell culture, mature RPE cells with high purity and typical morphology can be seen.

Images