CN115305239A

CN115305239A - Method for inducing stem cells to differentiate into retinal pigment epithelial cells

Info

Publication number: CN115305239A
Application number: CN202110493801.6A
Authority: CN
Inventors: 张凡; 曾露颖; 乔静; 陈邵宏; 赵宇
Original assignee: Beijing Chinagene Tech Co ltd
Current assignee: Beijing Chinagene Tech Co ltd
Priority date: 2021-05-07
Filing date: 2021-05-07
Publication date: 2022-11-08

Abstract

The present application relates to a method of inducing stem cell differentiation into retinal pigment epithelial cells (RPEs), comprising the steps of: before the cell state of the pluripotent stem cells is declined through cell culture, the pluripotent stem cells are placed in a first culture medium to be cultured, wherein the first culture medium comprises DMEM medium, serum, beta-mercaptoethanol, non-essential amino acid (NEAA) and L-glutamine. The methods described herein can significantly improve the efficiency of differentiation into RPE.

Description

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

Technical Field

The application relates to the field of biomedicine, in particular to a method for inducing stem cells to differentiate into retinal pigment epithelial cells.

Background

Retinal degenerative diseases are the leading cause of blindness worldwide. In this disease, damage and dysfunction of the retinal pigment epithelial cells (RPEs) leads to degeneration of the photoreceptor cells, ultimately affecting the patient's vision until blindness. Due to the non-regenerative nature of RPE and photoreceptor cells, there is a clinical lack of effective therapeutic measures for such diseases to date.

Because the stem cells have the capacity of unlimited proliferation and differentiation into various cells, somatic cells can be obtained from a mutant patient, are reprogrammed to iPS cells and then are induced into RPE cells, and on one hand, the stem cells can be used as disease models for drug screening, and on the other hand, the stem cells can be used as ideal sources of transplanted cells after the RPE cells after gene editing recover functions. The feasibility and safety of the application of the RPE derived from stem cells to the clinical research of human retinal degenerative diseases have been reported, although the requirements of patients on postoperative vision and normal life are greatly different.

During the last decade, various methods have been reported to control the differentiation of stem cells into RPE cells, but these methods still suffer from several drawbacks such as long time consumption, cumbersome methods, large batch-to-batch variation and low cell yield.

Therefore, a method for efficiently inducing differentiation of stem cells into RPE cells is required.

Disclosure of Invention

The present application provides a method of inducing stem cell differentiation into retinal pigment epithelial cells (RPEs). Mature RPE cells can be obtained using the methods described herein. Further, the method described herein has at least the beneficial effects selected from the group consisting of: high differentiation efficiency, short differentiation period, strong repeatability, no need of exogenous components and clinical application.

In one aspect, the present application provides a method of inducing stem cell differentiation into retinal pigment epithelial cells (RPEs), comprising the steps of: culturing stem cells in a first medium before the stem cells have a decline in cell status due to cell culture, wherein the first medium comprises DMEM medium, serum, beta-mercaptoethanol, a non-essential amino acid (NEAA), and L-glutamine.

In certain embodiments, the first medium comprises DMEM medium, serum, beta-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX ^TM 。

In certain embodiments, the DMEM medium comprises KnockOut ^TM DMEM and/or DMEM/F-12.

In certain embodiments, the serum comprises a clinical grade serum replacement.

In certain embodiments, the serum comprises KnockOut ^TM A serum replacement.

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

In certain embodiments, the serum is at a concentration of about 10% (v/v) to about 30% (v/v) in the first culture medium.

In certain embodiments, the concentration of the beta-mercaptoethanol in the first culture medium is from about 0.1% (v/v) to about 1% (v/v).

In certain embodiments, the concentration of the NEAA is from about 0.5% (v/v) to about 2% (v/v) in the first culture medium.

In certain embodiments, the concentration of L-glutamine in the first medium is from about 0.5% (v/v) to about 2% (v/v).

In certain embodiments, in the first medium, the GlutaMAX ^TM Is in the range of about 0.5% (v/v) to about 2% (v/v).

In certain embodiments, the first medium further comprises Activin a (Activin a).

In certain embodiments, the concentration of Activin A is from about 50ng/mL to about 200ng/mL.

In certain embodiments, the first medium further comprises SU5402.

In certain embodiments, the concentration of SU5402 is from about 5 μ Μ to about 20 μ Μ.

In certain embodiments, the stem cells are cultured in the first medium for about 5 to about 15 days.

In certain embodiments, the decline in cellular state comprises at least about 20% of the number of cells exhibiting a state selected from the group consisting of: cell shrinkage, cell shedding and apoptosis.

In certain embodiments, the decline in cell state occurs from about day 6 to about day 15 after the stem cells are cultured with the cells.

In certain embodiments, the stem cells achieve a cell fusion rate of about 70% prior to the cell culture.

In certain embodiments, the stem cells are digested prior to culturing the cells.

In certain embodiments, following said digestion, said stem cells are plated in a ratio of about 1:2 to about 1: 4.

In certain embodiments, the seed is placed in

Fibronectin or Cell

The above.

In certain embodiments, the cell culture is performed in a second medium, wherein the second medium comprises DMEM medium, B27 cell culture supplements, N2 cell culture supplements, and non-essential amino acids (NEAA).

In certain embodiments, the cell culture is performed in a second medium, wherein the second medium comprises DMEM medium, B27 cell culture supplements, N2 cell culture supplements, non-essential amino acids (NEAA), human Noggin, DKK1 protein, insulin-like growth factor-1 (IGF-1), and Nicotinamide (NIC); alternatively, the second medium comprises DMEM medium, B27 cell culture supplement, N2 cell culture supplement, non-essential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), and 3-aminobenzamide (3 ABA).

In certain embodiments, the cell culture is cultured in the second medium for about 2 days.

In certain embodiments, the cell culture is performed in a second medium, wherein the second medium comprises DMEM medium, B27 cell culture supplements, N2 cell culture supplements, non-essential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and Nicotinamide (NIC); alternatively, the second medium comprises DMEM medium, B27 cell culture supplement, N2 cell culture supplement, non-essential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and 3-aminobenzamide (3 ABA).

In certain embodiments, the cell culture is continued in the second medium for about 2 days.

In certain embodiments, the cell culture is performed in a second medium, wherein the second medium comprises DMEM medium, B27 cell culture supplements, N2 cell culture supplements, non-essential amino acids (NEAA), DKK1 protein, insulin-like growth factor-1 (IGF-1), and Activin a (Activin a).

In certain embodiments, the cell culture is performed in a second medium, wherein the second medium comprises DMEM medium, B27 cell culture additives, N2 cell culture additives, non-essential amino acids (NEAA), activin a (Activin a), and SU5402.

In certain embodiments, the cell culture is continued in the second medium for about 1 day.

In certain embodiments, the method further comprises the steps of: digesting the cells having RPE morphology cultured with said cells.

In certain embodiments, the method further comprises the steps of: enriching cells having a morphology of RPE cultured with said cells.

In certain embodiments, the enriching employs a method selected from the group consisting of: flow cytometry (FACS) and immunomagnetic bead cell sorting (MACS).

In certain embodiments, the stem cell comprises: embryonic stem cells and/or induced stem cells.

In certain embodiments, the stem cell comprises a human cell.

In certain embodiments, the stem cell is a human embryonic stem cell.

In certain embodiments, the stem cells are obtained by harvesting without disruption of the human embryo.

In another aspect, the present application provides a method of producing RPE cells, comprising the steps of:

a) Obtaining stem cells;

b) Inducing the stem cells of step a) to differentiate into RPE cells according to the methods described herein.

In another aspect, the present application provides a use of the method described herein for the manufacture of a medicament for the treatment of a retinal disease.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

Specific features of the invention to which this application relates are set forth in the following claims. The features and advantages of the invention to which the present application relates will be better understood by reference to the exemplary embodiments and drawings described in detail below. Brief description of the drawingsthe following:

FIGS. 1-9 show photographs of differentiated RPE cells at various time periods of cell culture using the methods described herein.

FIGS. 10A-10C show the expression of iPS marker gene and RPE marker gene by cells obtained using the methods described herein.

FIGS. 11A-11B show results of immunofluorescence identification assays using the methods described herein on differentiated RPE cells at various time periods in cell culture.

FIGS. 12A-12B show the results of phagocytosis assays on differentiated RPE cells at various time periods of cell culture using the methods described herein.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "stem cell" generally refers to an undifferentiated cell that has the ability to self-renew while retaining different potentials to form differentiated cells and tissues. The stem cells can proliferate and produce cells capable of producing large numbers of cells that can in turn produce differentiated or differentiable daughter cells. In the present application, the stem cells may include totipotent stem cells and/or pluripotent stem cells. The totipotent stem cells generally refer to stem cells that can differentiate into embryonic and extra-embryonic cell types. For example, the pluripotent stem cells may comprise embryonic stem cells (ES). Such embryonic stem cells may include embryos at a pre-embryonic stage, embryos produced artificially, i.e., by in vitro fertilization, and the like, which are capable of dividing in culture for extended periods of time without differentiation, and/or have the ability to develop into cells and or tissues of the three primitive germ layers, ectoderm, mesoderm, and endoderm. In the present application, the stem cells may be obtained by methods that do not involve the destruction of human embryos. In the present application, the stem cells may be vertebrate stem cells, for example may be mammalian stem cells, for example may be human-, primate-or rodent-derived stem cells. For example, the pluripotent cells may be human stem cells.

In the present application, the term "pluripotent stem cell" generally refers to a cell that has the ability to self-replicate and produce all types of cells in a subject. The pluripotent stem cells can differentiate into cells having a cell type of three germ layers (for example, a cell type capable of differentiating into ectoderm, mesoderm, and endoderm) under appropriate conditions. In the present application, the pluripotent cells can also be maintained in an undifferentiated state for an extended period of time by in vitro culture. . For example, the pluripotent stem cells may include hED cells, hiPS cells, hEG cells, and/or hEC cells.

In this application, the term "retinal pigment epithelial cells (RPE)" generally refers to a layer of pigmented cells that lie immediately adjacent to and outside the sensory nerve of the retina. The retinal pigment epithelium consists of a single layer of hexagonal cells containing dense pigment particles. The Retinal Pigment Epithelium (RPE), which is closely associated with the underlying choroid and the overlying retinal nerve cells, may have major functions including: control the fluid and nutrients in the subretinal space, functioning as a blood-retinal barrier; synthesizing growth factors to adjust local structure; absorbing light, and adjusting electric balance; regeneration and synthesis of visual pigment; phagocytosis and digestion of photoreceptor outer segments; maintaining the attachment of the retina; regeneration and repair after injury. RPE is generally considered to be an important tissue for maintaining photoreceptor function, and is also affected by many pathologies of the choroid and retina.

In the present application, the term "nonessential amino acids (NEAA)" generally refers to amino acids that can be synthesized in vivo, and do not need to be supplemented from the outside as a nutrient source. For example, most cells can synthesize the NEAA by glutaminolysis, glycolysis, or the TCA cycle. In the present application, the NEAA may include L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine and/or glycine. For example, the NEAA may include L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine.

In this application, the term "GlutaMAX ^TM "generally refers to L-alanyl-L-glutamine. It may be a dipeptide substitute for L-glutamine. GlutaMAX ^TM Can eliminate the problem related to the spontaneous decomposition of L-glutamine in the process of cell incubation culture. GlutaMAX ^TM Can be dissolved in water. GlutaMAX ^TM Has thermal stability.

In the present application, the term "DMEM medium" may include DMEM/F12 medium. The DMEM/F12 culture medium can be added into an F12 culture medium on the basis of a DMEM culture mediumAnd (c) separating the resulting culture medium. The DMEM/F12 medium can be used for serum-free culture. In the present application, the DMEM medium may include KnockOut ^TM DMEM。

In this application, the term "KnockOut ^TM DMEM "generally refers to a basic medium for undifferentiated embryonic stem cells and for inducing the growth of stem cells. KnockOut ^TM DMEM may not contain L-glutamine. KnockOut ^TM DMEM can meet the requirements of cGMP. KnockOut ^TM DMEM may not contain serum.

In the present application, the term "clinical-grade serum replacement" generally refers to a replacement that avoids the disadvantages that serum may have (e.g., cell differentiation, and/or heat inactivation), and that may include certain desirable components of serum (e.g., some cytokines). The clinical-grade serum replacement can be directly used for clinical-level tests due to the avoidance of the risk of viral contamination.

In this application, the term "KnockOut ^TM The serum replacement is usually a serum-free additive, and is suitable for culturing human and mouse pluripotent stem cells. KnockOut ^TM The serum replacement may be used for stem cell culture and/or differentiation, e.g., may be used for differentiation of iPS.

In the present application, the term "Activin a" generally refers to a secreted protein belonging to the Transforming Growth Factor (TGF) β family. The activin a can be a protein having a dimeric structure. The activin a can be produced in bone marrow cells and immune cells within the bone marrow, can have a Follicle Stimulating Hormone (FSH) regulating effect, can trigger hemoglobin production, can signal dendritic cells to initiate an immune response, and can play a role in vascular cell growth. In the present application, the activin a may be human activin a.

In this application, the term "SU5402" generally refers to a multi-target receptor kinase inhibitor. For example, the SU5402 can inhibit VEGFR2, FGFR1, and PDGF-R β. For example, the IC of SU5402 for VEGFR2, FGFR1 and PDGF-R β ₅₀ May be 20nM,30nM and 510nM, respectively.

In the present application, the term "decline of the cellular state" generally refers to a process in which the proliferation, differentiation ability and physiological functions of cells gradually decline in the cell cycle.

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

In the present application, the term "N2 cell culture additive" generally refers to an additive based on the N1 formulation of bottens's, which may not contain serum. The N2 cell culture additive can be used for growth and expression of postmitotic neurons in neuroblastoma and primary cell cultures of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The N2 cell culture supplement can replace the N1 formulation of bottens's.

In the present application, the term "human Noggin" refers generally to NOG, which may be a homodimer consisting of two non-glycosylated polypeptide chains. In the present application, the gene number of the recombinant human noggin in NCBI may be 9241, and the accession number of the protein thereof may be Q13253.

In the present application, the term "DKK1 protein" generally refers to a member of a dickkopf-related protein family. The DDK1 protein can inhibit the WNT signaling pathway from participating in embryonic development, bind LRP6 with high affinity, and prevent Frizzled-WNT-LRP6 complex formation in response to WNT. The DDK1 protein can affect 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 Pdgfr α and E-cadherin expression. Human DKK1 protein may have an accession number in GenBank of 22943.

In the present application, the term "insulin-like growth factor-1 (IGF-1)" generally refers to a broad class of growth promoting factors that have chemical structures similar to insulin. The IGF-1 has important promotion effects in cell differentiation, proliferation, and growth and development of individuals. Human IGF-1 has an accession number of 3479 in GenBank.

In the present application, the term "Nicotinamide (NIC)" generally refers to amide compounds of nicotinic acid. It may have the following chemical formula:

in the present application, the term "3-aminobenzamide (3 ABA)" generally refers to a PARP inhibitor. It may have the following chemical formula:

in the present application, the term "basic fibroblast growth factor (bFGF)" generally refers to basic fibroblast growth factor, or heparin-binding growth factor, which is a member of the FGF superfamily. The bFGF can play an important role in processes such as embryogenesis, tissue regeneration, wound healing, central nervous system development, angiogenesis, tumor development and the like. The bFGF can promote the division of mesodermal and neuroectodermal cells.

In the present application, the term "retinal disease" generally refers to atrophy of the Retinal Pigment Epithelium (RPE) or crystalline retinal degeneration. The Retinal Pigment Epithelium (RPE) atrophy is generally referred to as degenerative changes in the Retinal Pigment Epithelium (RPE) manifested as cell death or dysfunction. Age-related macular degeneration or retinal pigment degeneration (RP) is usually accompanied by atrophy of the retinal pigment epithelium. Retinitis Pigmentosa (RP), also known as Retinitis Pigmentosa, is a generic ophthalmic disorder. The inheritance modes of the gene are autosomal recessive, dominant and X-linked, and the gene is also inherited by double genes and mitochondria. The common initial symptoms are night blindness, narrow visual field, and the vision can see the scenery right in front but not the slightly left or right visual field, and then the vision will disappear gradually. RP may include primary pigmentary degeneration of the single eye, quadrant, central or paracentric, leucovorin, crystalloid, varicose, arteriolar peripigmentary, periarteritic, preserved retinal pigmentary degeneration, leber congenital amaurosis, and retinary degeneration in other syndromes. The main symptoms of the crystalline retinal degeneration may include crystals in the cornea (clear covering), fine, yellow or white crystalline deposits deposited in the light-sensitive tissues of the retina, and progressive atrophy of the retina, choroidal capillaries and choroid. Crystalline retinal degeneration may include diseases caused by mutations in the CYP4V2 gene.

In the present application, the term "comprising" or "comprises" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 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 the stated value.

Detailed Description

In one aspect, the present application provides a method that can induce differentiation of stem cells into retinal pigment epithelial cells (RPEs). The method has the advantages that the appropriate cell culture medium is used in the culture stage of the appropriate stem cells, so that the efficiency of obtaining RPE through differentiation of the stem cells is obviously improved. The method has good repeatability and simple and convenient operation, and the obtained RPE can be directly used for clinical treatment of patients, and/or the cell number and/or cell activity of the obtained RPE are obviously improved.

In the present application, the first medium may comprise DMEM medium, serum, beta-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX ^TM 。

The components involved in the first culture medium may all be components that meet clinical requirements (e.g. meet GMP standards). The first culture medium described herein and/or the method described herein may meet clinical requirements. The RPE cells obtained by the first culture medium described herein and/or the method described herein may also meet clinical requirements.

In the present application, the DMEM medium may not contain serum. For example, the DMEM medium may include KnockOut ^TM DMEM and/or DMEM/F-12.

In the present application, the serum may comprise a clinical grade serum replacement. For example, the serum may comprise KnockOut ^TM A serum replacement.

In the present 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 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acids.

In the present application, the serum may be at a concentration of about 10% (v/v) to about 30% (v/v) in the first medium. For example, the serum can be at a concentration of about 10% (v/v) to about 30% (v/v), about 11% (v/v) to about 30% (v/v), about 12% (v/v) to about 30% (v/v), about 13% (v/v) to about 30% (v/v), about 14% (v/v) to about 30% (v/v), about 15% (v/v) to about 30% (v/v), about 16% (v/v) to about 30% (v/v), about 17% (v/v) to about 30% (v/v), about 18% (v/v) to about 30% (v/v), about 19% (v/v) to about 30% (v/v), about 20% (v/v) to about 30% (v/v), about 10% (v/v) to about 28% (v/v), about 10% (v/v) to about 26% (v/v), about 10% (v/v) to about 24% (v/v), about 10% (v/v) to about 22% (v/v), about 10% (v/v) to about 20% (v/v), or about 15% (v/v) to about 25% (v/v). For example, the serum may be at a concentration of 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).

In the present application, the concentration of the beta-mercaptoethanol in the first culture medium may be from about 0.1% (v/v) to about 1% (v/v). For example, the concentration of the beta-mercaptoethanol can be from about 0.1% (v/v) to about 1% (v/v), from about 0.2% (v/v) to about 1% (v/v), from about 0.4% (v/v) to about 1% (v/v), from about 0.5% (v/v) to about 1% (v/v), from about 0.6% (v/v) to about 1% (v/v), from about 0.7% (v/v) to about 1% (v/v), from about 0.8% (v/v) to about 1% (v/v), from about 0.1% (v/v) to about 0.9% (v/v), from about 0.2% (v/v) to about 0.9% (v/v), from about 0.4% (v/v) to about 0.9% (v/v), or from about 0.5% (v/v) to about 0.9% (v/v). For example, the concentration of the beta-mercaptoethanol can 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).

In the present application, the concentration of the NEAA may be from about 0.5% (v/v) to about 2% (v/v) in the first medium. For example, the concentration of the NEAA may 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), from about 0.5% (v/v) to about 1.8% (v/v), from about 0.5% (v/v) to about 1.7% (v/v), from about 0.5% (v/v) to about 1.6% (v/v), from about 0.5% (v/v) to about 1.5% (v/v), from about 0.5% (v/v) to about 1.4% (v/v), from about 0.5% (v/v) to about 1.5% (v/v), from about 1.5% (v/v) to about 1.5% (v/v), or from about 1.5% (v/v) to about 1.5% (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).

In the present application, the concentration of the L-glutamine in the first medium may be about 0.5% (v/v) to about 2% (v/v). For example, the L-glutamine can be at a concentration of about 0.5% (v/v) to about 2% (v/v), about 0.6% (v/v) to about 2% (v/v), about 0.7% (v/v) to about 2% (v/v), about 0.8% (v/v) to about 2% (v/v), about 0.9% (v/v) to about 2% (v/v), about 1.0% (v/v) to about 2% (v/v), about 0.5% (v/v) to about 1.9% (v/v), about 0.5% (v/v) to about 1.8% (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 L-glutamine can 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).

In the present application, the GlutaMAX is present in the first medium ^TM Can be from about 0.5% (v/v) to about 2% (v/v). For example, the GlutaMAX ^TM The concentration of (b) may be about 0.5% (v/v) to about 2% (v/v), about 0.6% (v/v) to about 2% (v/v), about 0.7% (v/v) to about 2% (v/v), about 0.8% (v/v) to about 2% (v/v), about 0.9% (v/v) to about 2% (v/v), about 1.0% (v/v) to about 2% (v/v), about 0.5% (v/v) to about 1.9% (v/v), about 0.5% (v/v) to about 1.8% (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 GlutaMAX ^TM 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).

For example, the first medium may comprise DMEM medium, about 10% (v/v) to about 30% (v/v) serum, about 0.1% (v/v) to about 1% (v/v) beta-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 。

For example, the first medium may comprise DMEM medium, about 20% (v/v) serum, about 1% (v/v) beta-mercaptoethanol, about 1% (v/v) nonessential amino acids (NEAA), and about 1% (v/v) GlutaMAX ^TM 。

For example, the first medium may consist of DMEM medium, about 20% (v/v) serum, about 1% (v/v) beta-mercaptoethanol, about 1% (v/v) nonessential amino acid (NEAA), and about 1% (v/v) GlutaMAX ^TM And (4) forming.

In the present application, the first medium may be cultured for about 5 to about 15 days. For example, the culture can be carried out in the first medium described above 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.

In the present application, the first medium may further comprise Activin a (Activin a).

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

In the present application, the first medium may further comprise SU5402.

In the present application, the concentration of SU5402 can be from about 5. Mu.M to about 20. Mu.M. For example, the concentration of SU5402 can be about 5 μ Μ to about 20 μ Μ, about 6 μ Μ to about 20 μ Μ, about 7 μ Μ to about 20 μ Μ, about 8 μ Μ to about 20 μ Μ, about 9 μ Μ to about 20 μ Μ, about 10 μ Μ to about 20 μ Μ, about 5 μ Μ to about 19 μ Μ, about 5 μ Μ to about 18 μ Μ, about 5 μ Μ to about 17 μ Μ, about 5 μ Μ to about 16 μ Μ, about 5 μ Μ to about 15 μ Μ, about 5 μ Μ to about 14 μ Μ, about 5 μ Μ to about 13 μ Μ, about 5 μ Μ to about 12 μ Μ, about 5 μ Μ to about 11 μ Μ or about 5 μ Μ to about 10 μ Μ. For example, the concentration of SU5402 can be at least about 5 μ Μ, at least about 6 μ Μ, at least about 7 μ Μ, at least about 8 μ Μ, at least about 9 μ Μ, at least about 10 μ Μ, at least about 11 μ Μ, at least about 12 μ Μ, at least about 13 μ Μ, at least about 14 μ Μ, at least about 15 μ Μ, at least about 16 μ Μ, at least about 17 μ Μ, at least about 18 μ Μ, at least about 19 μ Μ or at least about 20 μ Μ.

For example, the first medium may comprise DMEM medium, about 10% (v/v) to about 30% (v/v) serum, about 0.1% (v/v) to about 1% (v/v) beta-mercaptoethanol, about 0.5% (v/v) to about 2% (v/v) nonessential amino acid (NEAA), about 0.5% (v/v) to about 2% (v/v) GlutaMAX ^TM About 50ng/mL to about 200ng/mL Activin A, and about 5. Mu.M to about 20. Mu.M SU5402.

For example, the first medium may comprise DMEM medium, about 20% (v/v) serum, about 1% (v/v) beta-mercaptoethanol, about 1% (v/v) nonessential amino acids (NEAA), about 1% (v/v) GlutaMAX ^TM About 100ng/mL Activin A, and about 10. Mu.M SU5402.

For example, the first medium may consist of DMEM medium, about 20% (v/v) serum, about 1% (v/v) beta-mercaptoethanol, about 1% (v/v) nonessential amino acids (NEAA), about 1% (v/v) GlutaMAX ^TM About 100ng/mL Activin A, and about 10. Mu.M SU5402.

In the present application, the stem cells may be cultured in the first medium for about 5 to about 15 days. For example, the culture can be 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.

In the present application, the method includes culturing the microorganism in the first medium (consisting of DMEM medium, about 20% (v/v) serum, about 1% (v/v) beta-mercaptoethanol, about 1% (v/v) nonessential amino acid (NEAA), and about 1% (v/v) GlutaMAX ^TM Composition) for about 1 to about 10 days, followed by culturing in said first medium-1 (consisting of DMEM medium, about 20% (v/v) serum, about 1% (v/v) beta-mercaptoethanol, about 1% (v/v) non-essential amino acid (NEAA), about 1% (v/v) GlutaMAX ^TM About 100ng/mL Activin a, and about 10 μ M SU 5402) for about 1 to about 5 days.

In the present application, the decline in cellular state may comprise at least about 20% (e.g., may be 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 number of cells exhibiting a state selected from the group consisting of: cell shrinkage, cell shedding and apoptosis.

In the present application, the apoptosis may be an autonomous, orderly death of the cell. The cell shedding may refer to shedding of cells from the wall of the culture medium vessel. The cell shrinkage may be a change in the morphology of the cell, for example, it may be manifested as a reduction in volume, deformation, rounding off and/or an intact cell membrane but foaming. The ratio can be obtained by calculating the ratio of the number of cells showing the decline of the cell state to the total number of all cells in the field in the same field.

In the present application, the decline in cell status may occur from about day 6 to about day 15 after the stem cells are cultured with the cells (e.g., may occur from 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 are cultured).

In the present application, the stem cells may have a cell fusion rate of up to about 70% (e.g., up to at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more) prior to culturing the cells.

In the present application, the stem cells may be digested prior to performing the cell culture. The digestion may be with digestive enzymes or digestive reagents conventional in the art, so long as the survival and/or biological activity of the stem cells is not affected.

In the present application, the stem cells can be plated at a ratio of about 1:2 to about 1:4 (e.g., can be about 1:2, about 1: 2.5, about 1: 3, about 1: 3.5, or about 1: 4) after the digestion.

In the present application, the bedding may be placed in

Fibronectin or Cell

The above.

In the present application, the cell culture may be performed in a second medium, wherein the second medium may comprise DMEM medium, B27 cell culture supplements, N2 cell culture supplements, and non-essential amino acids (NEAA). For example, the second medium may comprise DMEM medium, 1 xb 27 cell culture supplement, 1 xn 2 cell culture supplement, and 1 xneaa. For example, the second medium may consist of DMEM medium, 1 xb 27 cell culture supplement, 1 xn 2 cell culture supplement, and 1 xneaa.

For example, the stem cells may be cultured in the second medium described above after the digestion is performed. For example, the culture may be incubated for at least about 12 hours.

In the present application, the cell culture may be continued in a second medium-1, wherein the second medium-1 may comprise DMEM medium, B27 cell culture additives, N2 cell culture additives, nonessential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), and Nicotinamide (NIC); alternatively, the second medium-1 may comprise DMEM medium, B27 cell culture additives, N2 cell culture additives, non-essential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), and 3-aminobenzamide (3 ABA). For example, the second medium-1 can comprise DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 50ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, and 10mM NIC; alternatively, the second medium-1 may comprise DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 50ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, and 5mM 3ABA. For example, the second medium-1 may consist of DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 50ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, and 10mM NIC; alternatively, the second medium-1 may consist of DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 50ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, and 5mM 3ABA.

In the present application, the cell culture may be cultured in the second medium-1 for about 1 to 2 days. The cell culture may be changed daily by changing the second medium-1.

In the present application, the cell culture may be continued in a second medium-2, wherein the second medium-2 may comprise DMEM medium, B27 cell culture additives, N2 cell culture additives, nonessential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and Nicotinamide (NIC); alternatively, the second medium-2 may comprise DMEM medium, B27 cell culture additives, N2 cell culture additives, non-essential amino acids (NEAA), human Noggin (Noggin), DKK1 protein, insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), and 3-aminobenzamide (3 ABA). For example, the second medium-2 can comprise DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 10ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, 5ng/mL bFGF, and 10mM NIC; alternatively, the second medium-2 can comprise DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 10ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, 5ng/mL bFGF, and 5mM 3ABA. For example, the second medium-2 may consist of DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 10ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, 5ng/mL bFGF and 10mM NIC; alternatively, the second medium-2 may consist of DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 10ng/mL Noggin, 10ng/mL DKK1, 10ng/mL IGF1, 5ng/mL bFGF and 5mM 3ABA.

In the present application, the cell culture may be continued in the second medium-2 for about 1-2 days. The cell culture may be changed daily by changing the second medium-2.

In the present application, the cell culture may be continued in a second medium-3, wherein the second medium-3 may comprise DMEM medium, B27 cell culture additives, N2 cell culture additives, non-essential amino acids (NEAA), DKK1 protein, insulin-like growth factor-1 (IGF-1), and Activin a (Activin a). For example, the second medium-3 can comprise DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 10ng/mL DKK1, 10ng/mL IGF1, and 100ng/mL Activin A. For example, the second medium-3 can consist of DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 10ng/mL DKK1, 10ng/mL IGF1, and 100ng/mL Activin A.

In the present application, the cell culture may be continued in the second medium-3 for about 1-2 days. The cell culture may be changed daily by changing the second medium-3.

In certain instances, the cell culture can be continued in a second medium-4, wherein the second medium-4 can comprise DMEM medium, B27 cell culture additives, N2 cell culture additives, non-essential amino acids (NEAA), activin a (Activin a), and SU5402. For example, the second medium-3 can comprise DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 100ng/mL Activin A, and 10 μ M SU5402. For example, the second medium-3 can consist of DMEM medium, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 XNEAA, 100ng/mL Activin A, and 10 μ M SU5402.

In the present application, the cell culture may be continued in the second medium-4 for about 1-2 days. The cell culture may be changed daily with the second medium-4.

In this application, the method may further comprise the steps of: digesting the cells cultured with the cells having the RPE morphology. For example, trypLE Express can be used for digestion and then single cell filtration to remove cells that do not have RPE morphology.

In the present application, the method may further comprise the steps of: enriching the cells cultured with said cells for RPE morphology.

In the present application, the enrichment is performed by a method selected from the group consisting of: flow cytometry (FACS) and immunomagnetic bead cell sorting (MACS).

In the present application, the method may further comprise the steps of: the cells with RPE morphology were continued to be cultured. The continuous culture may use the first medium. During the continued culturing, the first medium may be replaced every other day.

In the present application, the stem cells may include: embryonic stem cells and/or induced stem cells.

In the present application, the induced stem cell (iPS) may be a stem cell induced from a somatic cell, and has development multipotency similar to that of an embryonic stem cell.

In the present application, the stem cells may include human cells.

In the present application, the stem cells may be human embryonic stem cells. For example, the stem cells can be obtained by harvesting without destruction of the human embryo.

a) Obtaining stem cells;

The method disclosed by the application can be used for efficiently obtaining a large batch of RPE cells. The RPE cells can be used for preparing medicaments for treating retinal diseases. For example, the RPE cells may be used to treat retinal degenerative diseases.

The present application also relates to the following embodiments:

1. a method of inducing stem cell differentiation into retinal pigment epithelial cells (RPEs), comprising the steps of:

before the cell state of the stem cells is declined through cell culture, the stem cells are placed in a first culture medium for culture,

wherein the first medium comprises DMEM medium, serum, beta-mercaptoethanol, non-essential amino acids (NEAA), and L-glutamine.

2. The method of embodiment 1, wherein the first medium comprises DMEM medium, serum, beta-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX ^TM 。

3. The method according to any one of embodiments 1-2, wherein the DMEM medium comprises KnockOut ^TM DMEM and/or DMEM/F-12.

4. The method of any one of embodiments 1-3, wherein the serum comprises a clinical-grade serum replacement.

5. The method according to any one of embodiments 1-4, wherein the serum comprises KnockOut ^TM A serum replacement.

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.

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

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

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

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

11. The method according to any one of embodiments 2-10, wherein in said first medium, said GlutaMAX ^TM Is in the range of 0.5% (v/v) to about 2% (v/v).

12. The method according to any one of embodiments 1-11, wherein the first medium further comprises Activin a (Activin a).

13. The method of embodiment 12, wherein said Activin A is at a concentration of about 50ng/mL to about 200ng/mL.

14. The method of any one of embodiments 1-13, wherein the first medium further comprises SU5402.

15. The method of embodiment 14, wherein the concentration of SU5402 is about 5 μ Μ to about 20 μ Μ.

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

17. The method according to any one of embodiments 1-16, wherein the decline in cellular state comprises at least about 20% of the number of cells exhibiting a state selected from the group consisting of: cell shrinkage, cell shedding and apoptosis.

18. The method according to any one of embodiments 1-17, wherein the decline in cell state occurs from about day 6 to about day 15 after the stem cells are cultured with the cells.

19. The method of any one of embodiments 1-18, wherein the stem cells achieve a cell fusion rate of about 70% prior to the cell culture.

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

21. The method of embodiment 20, wherein said stem cells are plated in a ratio of about 1:2 to about 1:4 after said digesting.

22. The method of embodiment 21, wherein the plating is placed in

Fibronectin or Cell

The above.

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

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

25. The method of embodiment 24, wherein the cell culture is cultured in the second medium for about 2 days.

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

27. The method of embodiment 26, wherein the cell culture is continued in the second medium for about 2 days.

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

29. The method of embodiment 28, wherein the cell culture is continued in the second medium for about 2 days.

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

31. The method of embodiment 30, wherein the cell culture is continued in the second medium for about 1 day.

32. The method of any one of embodiments 1-31, further comprising the steps of: digesting the cells cultured with the cells having the RPE morphology.

33. The method of any one of embodiments 1-32, further comprising the steps of: enriching cells having a morphology of RPE cultured with said cells.

34. The method of embodiment 33, wherein said enriching employs a method selected from the group consisting of: flow cytometry (FACS) and immunomagnetic bead cell sorting (MACS).

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

36. The method of any one of embodiments 1-35, wherein the stem cells comprise human cells.

37. The method of any one of embodiments 1-36, wherein the stem cells are human embryonic stem cells.

38. The method of any one of embodiments 1-37, wherein the stem cells are obtained by harvest without disruption of human embryos.

39. A method of producing RPE cells, comprising the steps of:

a) Obtaining stem cells;

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

40. Use of the method of any one of embodiments 1-39 in the manufacture of a medicament for treating a retinal disease.

Without wishing to be bound by any theory, the following examples are only intended to illustrate various aspects of the invention of the present application and are not intended to limit the scope of the invention of the present application.

Examples

Example 1 Induction of differentiation of Stem cells into RPE

1.1 day before cell culture: when stem cells (iPS) were cultured in a 3.5cm dish to a cell fusion rate of about 70%, iPS cells (iPS cells obtained from reprogramming UCs (renal epithelial cells) extracted from human urine or PBMCs (peripheral blood mononuclear cells) isolated from human whole blood) were digested with Accutase/TryPLE (purchased from Invitrogen) into small clumps, and the cells were plated on Matrigel-coated culture plates in a ratio of 1.

Adding a second culture medium: DMEM medium/F12, 1 XB 27 cell culture additive, 1 XN 2 cell culture additive and 1 Xnonessential amino acid (NEAA) are placed in an incubator to be cultured overnight.

2. Cell culture days 0-1: removing cell supernatant, and replacing with a second culture medium-1: DMEM medium/F12, 1 XB 27 cell culture additive, 1 XN 2 cell culture additive, 1 Xnon-essential amino acids (NEAA), 50ng/mL human Noggin (Noggin), 10ng/mL DKK1 protein, 10ng/mL insulin-like growth factor-1 (IGF-1) and 10mM Nicotinamide (NIC)/or 5mM3ABA, incubated for two days with daily changes of the culture medium.

3. Cell culture days 2-3: the cell supernatant was removed and replaced with a second medium-2: DMEM medium/F12, 1 XB 27 cell culture additive, 1 XN 2 cell culture additive, 1 Xnon-essential amino acids (NEAA), 10ng/mL human Noggin (Noggin), 10ng/mL DKK1 protein, 10ng/mL insulin-like growth factor-1 (IGF-1), 5ng/mL bFGF and 10mM Nicotinamide (NIC)/or 5mM 3ABA; culturing for two days, and replacing culture solution every day.

4. Cell culture days 4-5: the cell supernatant was removed and replaced with a second medium-3: DMEM medium/F12, 1 XB 27 cell culture supplement, 1 XN 2 cell culture supplement, 1 Xnonessential amino acids (NEAA), 10ng/mL DKK1 protein, 10ng/mL insulin-like growth factor-1 (IGF-1) and 100ng/mL Activin A, incubated for two days with daily changes of the culture medium.

5. Cell culture days 6-15:

the second medium-3 was changed to the first medium:

KnockOut ^TM DMEM、20％KnockOut ^TM serum replacement, 0.5% β -mercaptoethanol, 1% NEAA, 1% L-glutamine.

After 6-10 days of culture using the first medium, further adding to the first medium: 100ng/mL Activin A and 10. Mu.M SU5402.

6. Cell culture days 21-28: the Cells were digested into single Cells with TrypLE Express (purchased from Invitrogen), and the Cells were passed through a 40 μm single cell filter to remove non-RPE Cells morphology Cells from the Cells to achieve RPE cell purification.

Inoculating the cells on a Matrigel-coated tissue culture plate or a Transwell membrane, culturing the purified cells in the first culture medium, changing the culture solution every two days, and culturing until mature RPE cells with high purity and typical morphology can be seen on about 27 days of the cell culture.

The RPE cells obtained from the differentiation were photographed at different periods of cell culture, see in particular fig. 1-9. Wherein FIGS. 1-9 show FIG. 1: culturing for 9 days, and magnifying the visual field by 4 times; FIG. 2 is a schematic diagram: culturing for 9 days, and magnifying the visual field by 10 times; FIG. 3: culturing for 9 days, and magnifying the visual field by 20 times; FIG. 4: culturing for 25 days, and magnifying the visual field by 4 times; FIG. 5: culturing for 25 days, and enlarging the visual field by 10 times; FIG. 6: culturing for 25 days, and magnifying the visual field by 20 times; FIG. 7: culturing for 32 days, and magnifying the visual field by 20 times; FIG. 8: culturing for 32 days, and magnifying the visual field by 40 times; FIG. 9: and culturing for 45 days, and observing the condition with a visual field magnification of 40 times.

Example 2 identification of retinal pigment epithelial cell (RPE) marker (qPCR method)

The retinal pigment epithelial cells induced at each stage are tested, and the expression of the marker genes CRALBP, RPE65 and iPS marker gene OCT4 on the mRNA level is tested.

Experimental method

qPCR was used, according to the literature Rezania, A., et al, reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent cells Nat Biotechnol,2014.32 (11): p.1121-33.

To an apparatus

Reagents and primers involved

This example also relates to the use of a 10. Mu.l lance tip, a 200. Mu.l lance tip and a 1000. Mu.l lance tip.

The concrete steps

The cells obtained by culturing according to the method described in example 1 were collected and washed twice by centrifugation with DPBS.

Total RNA was extracted according to the instruction manual of the high-purity total RNA rapid extraction kit (centrifugal column type).

The cDNA was obtained by reverse transcription according to the instructions of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix.

Reagent name in kit	Adding volume
		R-Mix	10μl
E-Mix	1μl
		Oligdf	1μl
Remove	1μl
		RNA + Water	7μl

Primers and templates were added to ice in a 20. Mu.l system according to the instructions of TransStart Top Green qPCR SuperMix.

Template + water	9.2μl
		Mix	10μl
Forward primer	0.4μl
		Reverse primer	0.4μl

Setting program parameters in a qPCR instrument, selecting FAM and VIC channels as fluorescence channels, and setting reaction conditions as follows:

pre-denaturation	94℃	30s
			Denaturation of the material	94℃	5s
Annealing	60℃	15s
			Extension	72℃	34s

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

The operating steps of the comparative method are as follows:

cell culture day 0: iPS cells were cultured at 3 ten thousand/cm ² The cells are planted in a T25 bottle and cultured until the cell density reaches 90 to 100 percent.

Cell culture day 1: the medium was aspirated and 1E 8: TLP medium was added. Wherein the TLP medium comprises KnockOut ^TM DMEM、20％KnockOut ^TM Serum replacement, 0.5% β -mercaptoethanol, 1% NEAA and 1% L-glutamine.

Cell culture days 2-70: day 2 was replaced with complete TLP medium, 7 mL/vial. The solution was changed every 3 days thereafter. After about 6 weeks, the pigment cell yield is determined whether to proceed to the next stage.

Cell culture days 71-113: the pigment cell pellet was collected in DPBS by hand and machine. The supernatant was centrifuged off, and 1mL of Accutase (from Sigma) was added and digested at 37 ℃ for 1 hour, followed by blow-off with a pipette tip. Digestion was stopped by adding 5 volumes of TLP medium. The cells were screened at 70 μm (purchased from Corning), rinsed with TLP medium and counted. According to the ratio of more than 5/cm ² In 12-well or 24-well plates (plates were coated with Matrigel diluted in 1. The solution was changed every 3 days for about 6 weeks.

The results are shown in FIGS. 10A to 10C. FIGS. 10A to 10C show expression levels of the iPS marker gene OCT4, the RPE marker gene RPE65, and CRALBP in this order.

The results in fig. 10 demonstrate that the cells obtained by the method described in example 1 had a significantly reduced expression level of OCT4, which is the iPS marker gene, and significantly improved expression levels of RPE marker genes RPE65 and CRALBP, as compared to the control method, which demonstrates that the method described in example 1 significantly promotes the differentiation of iPS cells into RPEs as compared to the control method.

Example 3 RPE immunofluorescence identification assay

1. RPE cells prepared to identify cells cultured for 30 days as described in example 1 were washed three times with DPBS, 4% PFA fixed at room temperature for 20min, protected from light.

And 2, washing the DPBS for three times.

3.0.2% TritonX-100 (dissolved using DPBS), punch for 15min.

4."5% BSA blocking solution" +0.1% TritonX-100,4 ℃ overnight blocking.

PBST-1 (for washing): DPBS +0.1% TritonX-100;

PBST-2 (for primary antibody (ZO-1, rabbit source), secondary antibody (586, goat anti-rabbit)): DPBS +0.1% TritonX-100+1% BSA.

7. Add the primary antibody, primary antibody: PBST-2= 1: 200,4 ℃ overnight.

8. The primary antibody solution was recovered and washed three times with PBST-1 for 10min each.

9. The secondary antibody is added, the secondary antibody is PBST-2= 1: 500, and the mixture is kept at 4 ℃ overnight and protected from light.

10. The wash was performed three times for 10min using PBST-1.

And 11, diluting the DAPI by 1000 times, and incubating for 2-3min in a dark place. And cleaning the DPBS for 1-2 times.

12. 90% glycerol was added for covering, and direct photographing was performed.

The results are shown in FIGS. 11A-11B. FIGS. 11A-11B show the observation of 20 times magnification after the addition of the primary anti-ZO-1 and 40 times magnification after the addition of the primary anti-ZO-1, in that order; and observations at 40-fold magnification after addition of secondary antibody ES.

The results indicate that the ZO-1 protein is located between adjacent cells, thereby exhibiting a hexagonal outline of RPE cells.

Example 4RPE phagocytosis assay

The RPE cells can phagocytose the outer segment of the shed photoreceptor cell, and the condition that the excessive accumulation of the outer segment influences the stability of the retinal lower cavity environment is prevented. In order to verify whether the RPE cells obtained by the method described in example 1 have a function of phagocytosing the photoreceptor outer segment, the RPE cells were co-cultured with GFP-fluorescently labeled magnetic beads instead of the photoreceptor outer segment to observe phagocytosis.

1. The magnetic beads (latex spheres) were diluted 4.8X 10 with RPE differentiation medium TLP ⁴ Doubling to 4.0X 10 ⁷ /mL。

2. The culture medium in the petri dish to be tested was aspirated and washed once with DPBS.

3. Adding diluted culture medium containing magnetic beads into dish, shaking thoroughly, and adding CO at 37 deg.C/5% ₂ The RPE cells prepared by culturing for 30 days using the method described in example 1 were cultured in a cell incubator for 12 to 24 hours.

4. And (3) absorbing the culture medium in the dish, fully cleaning the culture medium for at least 5 times by using DPBS, observing whether the culture medium is cleaned or not under a fluorescence microscope, and adding an RPE differentiation culture medium after the culture medium is confirmed to be cleaned.

5. And observing and photographing under a fluorescent microscope.

The results are shown in FIGS. 12A-12B. FIGS. 12A-12B show fluorescence signals at 20-fold magnification of the field of view and the results of combining fluorescence signals with white light under these conditions. The results in FIG. 12 indicate that the presence of green fluorescent signal in the differentiated RPE cells, the signal is in the cytoplasmic part of the cells, and the nucleus and the cells have no green fluorescent signal between the cells, which indicates that the latex ball is phagocytosed into the cell interior rather than adhered to the cell surface. Meanwhile, green fluorescence signals can be found to be mainly concentrated in RPE cells which are mature and have more pigment accumulation. RPE cells prepared using the method described in example 1 thus function to phagocytose photoreceptor outer segments.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Sequence listing

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Claims

1. A method of inducing stem cell differentiation into retinal pigment epithelial cells (RPEs) comprising the steps of:

culturing stem cells in a first medium before the stem cells have a decline in cell status due to cell culture, wherein the first medium comprises DMEM medium, serum, beta-mercaptoethanol, a non-essential amino acid (NEAA), and L-glutamine.

2. According to claimThe method of claim 1, wherein the first medium comprises DMEM medium, serum, beta-mercaptoethanol, non-essential amino acids (NEAA), and GlutaMAX ^TM 。

3. The method according to any of claims 1-2, wherein the DMEM medium comprises KnockOut ^TM DMEM and/or DMEM/F-12.

4. The method of any one of claims 1-3, wherein the serum is at a concentration of about 10% (v/v) to about 30% (v/v) in the first culture medium.

5. The method of any one of claims 1-4, wherein the concentration of β -mercaptoethanol in the first culture medium is from 0.1% (v/v) to about 1% (v/v).

6. The method according to any one of claims 1-5, wherein the concentration of the NEAA in the first culture medium is from 0.5% (v/v) to about 2% (v/v).

7. The method according to any one of claims 1-6, wherein the concentration of L-glutamine in the first medium is from 0.5% (v/v) to about 2% (v/v).

8. The method of any one of claims 2-7, wherein in the first medium, the GlutaMAX is ^TM Is in the range of 0.5% (v/v) to about 2% (v/v).

9. The method of any one of claims 1-8, wherein the stem cells are cultured in the first medium for about 5 to about 15 days.

10. The method of any one of claims 1-9, wherein the decline in cellular state comprises at least about 20% of the number of cells exhibiting a state selected from the group consisting of: cell shrinkage, cell shedding and apoptosis.