Method for inducing human pluripotent stem cells to be differentiated into retinal pigment epithelial cells and application

Abstract

The invention provides a method for inducing human pluripotent stem cells to be differentiated into retinal pigment epithelial cells and application. The method comprises the following steps: performing adherent culture on pluripotent stem cells to serve as differentiation starting cells; the differentiation starting cells are cultured in stages with different culture media to obtain the retinal pigment epithelial cells, and the culture media in at least one stage comprise a WNT non-classical pathway activator. The invention further provides a composition and a culture medium for inducing the human pluripotent stem cells to be differentiated into the retinal pigment epithelial cells, the differentiation efficiency of the RPE can be remarkably improved, the cell harvesting purity is improved, and the composition and the culture medium are suitable for large-scale industrial production.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a method and application for inducing the differentiation of human pluripotent stem cells into retinal pigment epithelial cells. Background Art

[0002] Age-related macular degeneration (AMD) is the most common cause of blindness in people over 60 years old, affecting approximately 200 million people globally, mainly caused by the loss and death of retinal pigment epithelial (RPE) cells. The cell therapy for AMD aims to prevent the loss of photoreceptors and restore vision by transplanting healthy RPE cells into the macula, thereby preventing or reversing the progression of the disease. Transplantation of RPE cells derived from pluripotent stem cells (PSCs) is considered a promising therapeutic method for regenerating cell function and vision.

[0003] To date, most clinical trials have generated hPSC-RPE cells by spontaneous differentiation to minimize the risk of patient exposure to exogenous substances. However, spontaneous differentiation requires manual picking of clones, is inefficient, and varies greatly among hPSC lines. Directed differentiation protocols can significantly increase the yield of hPSC-RPE cells. However, the most effective differentiation methods are still not applicable to all hPSC lines, and there is also the addition of uncertain animal-derived components. Therefore, for hPSC-RPE clinical-grade cells, there is still a need for a robust and effective protocol that can obtain hPSC-RPE cells through a simple, efficient, and animal-component-free RPE differentiation method and can be used for large-scale industrial production. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present application has studied the RPE differentiation method and developed a new culture medium, which can significantly improve the differentiation efficiency of RPE, shorten the differentiation cycle, and increase the cell harvest purity, and is suitable for large-scale production. Specifically, the present application relates to the following aspects:

[0005] 1. A method for preparing retinal pigment epithelial cells, comprising:

[0006] Pre-culturing pluripotent stem cells to obtain differentiation starting cells;

[0007] Culturing the differentiation starting cells in different culture media in stages to obtain retinal pigment epithelial cells,

[0008] wherein, at least one stage of the culture medium comprises a WNT non-canonical pathway activator.

[0009] 2. The preparation method according to item 1, wherein the staged culture at least includes: culturing the differentiation starting cells in a first differentiation medium for a first differentiation stage, in a second differentiation medium for a second differentiation stage, and in a third differentiation medium for a third differentiation stage in sequence.

[0010] 3. The preparation method according to item 1, wherein the pluripotent stem cells are human pluripotent stem cells.

[0011] Preferably, the pluripotent stem cells are human pluripotent stem cells that have been passaged more than 10 times.

[0012] More preferably, the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

[0013] 4. The preparation method according to item 1, wherein the pre-culture is to perform adherent culture of the pluripotent stem cells in a maintenance medium.

[0014] Preferably, the maintenance medium is selected from E8 or TeSR-AOF medium.

[0015] 5. The preparation method according to item 1, wherein the pluripotent stem cells are pre-cultured until the cell confluence degree ≥ 70% and then subjected to staged culture.

[0016] 6. The preparation method according to item 2, wherein the first differentiation medium, the second differentiation medium, and the third differentiation medium all contain RPEM basal medium.

[0017] Preferably, the first differentiation medium further includes a TGFβ inhibitor, a BMP inhibitor, and a WNT inhibitor, and / or the second differentiation medium further includes a TGFβ activator, a WNT canonical pathway activator, and a WNT non-canonical pathway activator, and / or the third differentiation medium further includes a ROCK inhibitor.

[0018] 7. The preparation method according to item 6, wherein the RPEM basal medium contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2-Me.

[0019] 8. The preparation method according to item 6, wherein the TGFβ inhibitor is selected from at least one of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276 and A83-01, preferably A83-01, and / or the BMP inhibitor is selected from at least one of Chordin, Noggin, LDN193189, Follistatin and Dorsomorphin, preferably Dorsomorphin, and / or the WNT inhibitor is selected from at least one of DKK-1, D4476, CKI-7, IWRI and Cerberus protein, preferably IWRI.

[0020] 9. The preparation method according to item 6, wherein the TGFβ activator is selected from at least one of TGFB-1, TGFB-2, TGFB-3, IDE-1 / 2 and Activin A, preferably Activin A, and / or the WNT canonical pathway activator is selected from CHIR99021, and / or the WNT non-canonical pathway activator is selected from PY-60.

[0021] 10. The preparation method according to item 6, wherein the ROCK inhibitor is selected from at least one of Y-27632, CEPT and Blebbistatin, preferably Y-27632.

[0022] 11. The preparation method according to item 6, wherein in the staged culture, the differentiation starting cells are successively cultured in the first differentiation medium for the first differentiation stage for 5-7 days, in the second differentiation medium for the second differentiation stage for 7-9 days, and in the third differentiation medium for the third differentiation stage for 4-6 days.

[0023] Preferably, the culture medium is changed every day during the staged culture.

[0024] 12. The preparation method according to item 11, wherein the first differentiation medium is RPEM basal medium supplemented with 20 nM - 500 nM Dorsomorphin, 1 μM - 25 μM IWRI, 0.1 μM - 2.5 μM A83 - 01, and 2 mM - 50 mM NIC; the second differentiation medium is RPEM basal medium supplemented with 0.6 μM - 15 μM CHIR99021, 20 ng / mL - 500 ng / mL Activin A, and 2 μM - 50 μM PY - 60; and the third differentiation medium is RPEM basal medium.

[0025] The RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2 - Me.

[0026] 13. The preparation method according to item 12, wherein 10 μM ROCK inhibitor Y - 27632 is further added to the third differentiation medium used on the first day of the third differentiation stage culture.

[0027] 14. The preparation method according to item 11, wherein the staged culture further includes: after the third differentiation stage culture of the differentiation - starting cells, culturing with a maturation - promoting medium for 5 - 7 days.

[0028] Preferably, the maturation - promoting medium is a medium containing 60% - 80% DMEM Basic culture solution, 20% - 40% F12, and 1% B27 additive.

[0029] 15. A medium for inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells, which includes: a TGFβ activator, a WNT canonical pathway activator, and a WNT non - canonical pathway activator.

[0030] 16. The medium according to item 15, which further includes RPEM basal medium, and the RPEM basal medium contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2 - Me.

[0031] Preferably, the RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2 - Me.

[0032] 17. The culture medium according to item 15, wherein the TGFβ activator is selected from at least one of TGFB-1, TGFB-2, TGFB-3, IDE-1 / 2, and Activin A, preferably Activin A, and / or the WNT canonical pathway activator is selected from CHIR99021, and / or the WNT non-canonical pathway activator is selected from PY-60,

[0033] Preferably, in the culture medium, the concentration of the TGFβ activator is 20 ng / mL - 500 ng / mL, and / or the concentration of the WNT canonical pathway activator is 0.6 μM - 15 μM, and / or the concentration of the WNT non-canonical pathway activator is 2 μM - 50 μM.

[0034] 18. A culture medium for inducing the differentiation of induced pluripotent stem cells into retinal pigment epithelial cells, which comprises: a TGFβ inhibitor, a BMP inhibitor, and a WNT inhibitor.

[0035] 19. The culture medium according to item 18, which further comprises an RPEM basal medium, and the RPEM basal medium contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2-Me,

[0036] Preferably, the RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2-Me.

[0037] 20. The culture medium according to item 18, wherein the TGFβ inhibitor is selected from at least one of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, and A83-01, preferably A83-01, and / or the BMP inhibitor is selected from at least one of Chordin, Noggin, LDN193189, Follistatin, and Dorsomorphin, preferably Dorsomorphin, and / or the WNT inhibitor is selected from at least one of DKK-1, D4476, CKI-7, IWRI, and Cerberus protein, preferably IWRI,

[0038] Preferably, in the culture medium, the concentration of the TGFβ inhibitor is 0.1 μM - 2.5 μM, and / or the concentration of the BMP inhibitor is 20 nM - 500 nM, and / or the concentration of the WNT inhibitor is 1 μM - 25 μM.

[0039] 21. A culture medium composition for inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells, which comprises the culture medium according to any one of items 15 - 17, and the culture medium according to any one of items 18 - 20.

[0040] 22. Use of the composition according to any one of items 15 - 20 or the culture medium composition according to item 21 in the preparation of a drug for treating diseases related to retinal pigment epithelial cells. Preferably, the disease is age-related macular degeneration (AMD).

[0041] Advantages of the Invention

[0042] It is found through research in this application that the non-canonical WNT pathway may play an important role in the differentiation process of RPE. Further, it is found that after differentiating and culturing pluripotent stem cells with a culture medium added with the small molecule PY-60, an activator of the relevant pathway, the differentiation purity of RPE is significantly improved. Thus, a better RPE differentiation culture medium and culture protocol are developed, which can significantly improve the harvesting purity of RPE, increase the RPE differentiation efficiency, and reduce production costs. This protocol can be used in the treatment of age-related macular degeneration (AMD) and other RPE-related diseases, providing a promising treatment method for regenerating cell function and vision. Brief Description of the Drawings

[0043] Figure 1 It is a schematic diagram of a retinal pigment epithelial cell differentiation culture process provided by this application.

[0044] Figure 2 It is a microscopic bright-field observation of RPE cells in each group (scale bar: 100 μm).

[0045] Figure 3 It is a schematic diagram of the results of flow cytometry detection of RPE cells in each group.

[0046] Figure 4 It is a schematic diagram of the results of qPCR detection of RPE cells in each group. Detailed Description of the Embodiments

[0047] The following further illustrates this application with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain this application, and are not used to limit this application.

[0048] Unless otherwise defined, the technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including its definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and not restrictive. The present application will be further described below in conjunction with specific embodiments, but the scope of the present application is not limited thereby.

[0049] Definition

[0050] In this specification, the term "about" is used to indicate that a value includes the inherent variations of the error of the device, the method used to determine the value, or the variations existing among the subjects under study.

[0051] In this specification, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0052] In this specification, the term "pluripotent stem cells (PSCs)" generally refers to cells with the ability of unlimited proliferation and multi-germ layer differentiation. Different from the single-germ layer differentiation ability of adult stem cells, the said pluripotent stem cells have the ability to differentiate into ectoderm, mesoderm, and endoderm cells. Specifically, the said pluripotent stem cells can include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are highly similar in function and structure but differ in origin.

[0053] In this specification, the term "retinal pigment epithelial cells" refers to "RPE (retinal pigment epithelium)" cells, which are a layer of regularly shaped and neatly arranged hexagonal columnar pigment cells located between the photoreceptor cell layer and the choroidal capillaries. Retinal pigment epithelial cells play an important role in maintaining the normal physiological metabolism and functions of the retina. They can differentiate into epithelial cells with secretory functions, provide a cell layer for structural and nutritional support to the retina, and are of great significance for maintaining the functions of photoreceptor cells. Each eye has approximately 4.2 - 6.1 million retinal pigment epithelial cells. Retinal pigment epithelial cells have no regenerative ability and are not replaced after cell death. Abnormal functions of retinal pigment epithelial cells are closely related to the occurrence and development of retinal degenerative diseases. Retinal pigment epithelial lesions are a group of diseases that affect the retinal pigment epithelium and may cause visual impairment. Common causes include macular degeneration, pigment epithelial detachment, cataract, retinal pigment epitheliitis, central retinal pigment epithelial lesions, etc.

[0054] In this specification, the term "WNT signaling pathway" includes the canonical WNT pathway and the non-canonical WNT pathway. Extracellular Wnt signal stimulation can trigger several intracellular signal transduction cascades, including the Wnt / β-catenin-dependent (canonical) pathway and the β-catenin-independent (non-canonical) pathway, and the latter can be further divided into the planar cell polarity (PCP) and Wnt / Ca 2+ pathway.

[0055] The "WNT inhibitor" in this specification refers to an inhibitor of the signal of the canonical WNT canonical pathway (Wnt / β-catenin-dependent pathway). The activation of typical Wnt signals can be inhibited by some Wnt protein inhibitors, such as sFRP, Dkk, WIF, Wise / SOST, Cerberus, Tiki1, etc. Their common feature is to antagonize Wnt signal transduction by preventing ligand-receptor interaction or Wnt receptor maturation.

[0056] The "WNT non-canonical pathway activator" in this specification refers to an activator of the WNT non-canonical pathway (Wnt / PCP and Wnt / Ca 2+ dependent pathways). In the WNT / Ca 2+In this pathway, WNT proteins mainly consist of WNT1, WNT5A, and WNT11, bind to Frizzled transmembrane receptors on the cell surface, and participate in several cellular processes that involve stimulating heterotrimeric G proteins, thereby further activating PLC (phospholipase-C). In the Wnt / PCP pathway, Wnt proteins bind to Frizzled transmembrane receptors on the cell surface and then activate Rho / Rac small GTPases and JNK (Jun N-terminal kinase) through Dsh, subsequently assisting in the regulation of cytoskeletal organization and gene expression.

[0057] In this specification, "TGFβ activator" refers to a small molecule or protein that can promote the conversion of transforming growth factor (TGF-β) from its latent form to its active form. TGF-β is a multifunctional cytokine belonging to the transforming growth factor superfamily, and its active form within the cell needs to undergo a specific activation process to exert its biological functions. Integrin is one of the currently known factors that activate and mediate the TGF-β signaling pathway.

[0058] In this specification, "TGFβ inhibitor" is a drug or compound that can inhibit the function or signal transduction of transforming growth factor (TGF-β), and it can achieve the inhibition of the TGFβ signaling pathway through mechanisms such as inhibiting the expression of TGFβ and its receptors, blocking the binding of TGFβ to the receptors, or interfering with receptor kinase signal transduction.

[0059] In this specification, "BMP inhibitor" is a compound that can inhibit the signal transduction of bone morphogenetic protein (BMP), and it mainly inhibits the transmission of BMP signals by binding to BMP receptors.

[0060] In this specification, the term "DMEM / F12" is a mixture of two culture media, Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Medium, and it can be obtained commercially.

[0061] In this specification, the term "NEAA" refers to a non-essential amino acid solution, which is a cell culture medium additive that helps cell proliferation and growth, and it can be obtained commercially.

[0062] In this specification, the term "TeSR-AOF medium" is a serum-free medium that can expand and scale undifferentiated human embryonic stem cells and human induced pluripotent stem cell aggregates.

[0063] In this specification, "KSR", namely Knock Out Serum Replacement, is a serum-free cell culture substitute designed to provide the necessary components for cell growth while avoiding many of the disadvantages of using animal serum. KSR consists of a series of carefully screened and formulated chemical substances, including nutrients (such as amino acids, sugars, vitamins, etc.), growth factors (such as EGF, etc.), hormones, small molecule signaling molecules, and buffers.

[0064] In this specification, "Glutamax" refers to the stable form of L-glutamine, GlutaMAX-I. GlutaMAX-I is a dipeptide of L-alanine and L-glutamine, which is a derivative of L-glutamine with its unstable α-amino group protected by L-alanine.

[0065] In this specification, "2-Me" refers to 2-mercaptoethanol, which is a reducing organic sulfur compound with the ability to scavenge oxygen free radicals. Therefore, it is often used as a reducing agent and deoxidizer in biological and biochemical research, and can be used to inactivate IgM antibodies in highly inactivated plasma and serum samples. Its chemical properties are stable and can protect biomolecules from oxidation under certain conditions.

[0066] In this specification, "F12", namely Ham's F-12 nutrient medium, is a special medium specifically used for animal cell culture. The composition of F12 medium is complex, containing a variety of trace elements, non-essential amino acids, a wide range of vitamins, as well as inorganic salts and metabolic additives (such as nucleotides), which together provide comprehensive nutritional support for cells.

[0067] In this specification, "B27" is a serum-free additive, and its main components include antioxidants, growth factors required by nerve cells, vitamins (excluding vitamin A), and fatty acids required by neurons.

[0068] In a first aspect, the present application provides a method for preparing retinal pigment epithelial cells, the preparation method comprising:

[0069] Pre-culturing pluripotent stem cells and using them as differentiation starting cells;

[0070] Culturing the differentiation starting cells in different media in stages to obtain retinal pigment epithelial cells,

[0071] wherein, at least one stage of the medium includes a WNT non-canonical pathway activator.

[0072] In the embodiments of the present application, there is no specific limitation on the pluripotent stem cells used, which can be pluripotent stem cells that can be used for organoid and tissue construction in the art. For example, they can be pluripotent stem cells directly obtained through commercial purchase, or pluripotent stem cells prepared according to the existing technologies in the art. In a specific embodiment, the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

[0073] In a specific embodiment, the pluripotent stem cells can be pre-cultured by conventional methods in the art so that they can be used in subsequent differentiation stages.

[0074] In a specific embodiment, the pre-culture is to perform adherent culture of the pluripotent stem cells in a maintenance medium.

[0075] In a specific embodiment, the maintenance medium is selected from E8 or TeSR-AOF medium.

[0076] In a specific embodiment, the pluripotent stem cells need to be cultured for at least 5 passages or more before being used as differentiation starting cells. In some preferred embodiments, they need to be cultured for 10 passages or more.

[0077] In a specific embodiment, the pluripotent stem cells need to be cultured until the cell confluence ≥ 70% and then used as differentiation starting cells for subsequent staged culture.

[0078] In a specific embodiment, the differentiation starting cells are obtained by the following method:

[0079] First, resuscitate and culture hiPSC cells, passage and culture them for at least 5 passages or more, preferably 10 passages or more, and then seed the hiPSC at 1 - 2×10 4 cells / cm 2 onto a cell culture plate coated with Laminin521 (coating conditions: dilute with DPBS (containing calcium and magnesium) to 10 μg / mL, add 500 μL / well for 12-well plates and 1000 μL / well for 6-well plates, incubate the coating solution at 37 °C for at least 2 hours), and maintain the cells in TeSR-AOF complete medium until the cell confluence reaches about 70%, thus obtaining the differentiation starting cells that can be used for differentiation culture.

[0080] In the embodiments of the present application, after obtaining the differentiation starting cells, they are successively subjected to staged culture with different media to obtain retinal pigment epithelial cells.

[0081] In some specific embodiments, the staged culture at least includes the following stages:

[0082] Perform the first differentiation stage culture with the first differentiation medium;

[0083] Perform the second differentiation stage culture with the second differentiation medium; and

[0084] Perform the third differentiation stage culture with the third differentiation medium.

[0085] In some specific embodiments, the staged culture further includes:

[0086] After the third differentiation stage culture, continue to perform the maturation promotion culture with the maturation promotion medium.

[0087] In a specific embodiment, the first differentiation medium, the second differentiation medium, and the third differentiation medium all contain the RPEM basal medium.

[0088] In some specific embodiments, the RPEM basal medium contains DMEM / F12 (Gibco, C11330500BT), KSR (Gibco, 10828-028), sodium pyruvate (Gibco, 11360-070), NEAA (Sigma, M7145-100mL), Glutamax (Gibco, 35050061), and 2-Me (Gibco, 21985-023).

[0089] In a specific embodiment, the composition of the RPEM basal medium is: DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%.

[0090] In some specific embodiments, the first differentiation medium is the medium obtained by adding a TGFβ inhibitor, a BMP inhibitor, and a WNT inhibitor to the RPEM basal medium.

[0091] In a specific embodiment, the TGFβ inhibitor can be selected from at least one of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, and A83-01. In some preferred embodiments, the TGFβ inhibitor is A83-01.

[0092] In a specific embodiment, the BMP inhibitor can be selected from at least one of Chordin, Noggin, LDN193189, Follistatin, and Dorsomorphin. In some preferred embodiments, the BMP inhibitor is Dorsomorphin.

[0093] In a specific embodiment, the WNT inhibitor is selected from at least one of DKK-1, D4476, CKI-7, IWRI, and Cerberus protein. In some preferred embodiments, the WNT inhibitor is IWRI.

[0094] In some specific embodiments, the second differentiation medium is a medium obtained by adding a TGFβ activator, a WNT canonical pathway activator, and a WNT non-canonical pathway activator to the RPEM basal medium.

[0095] In a specific embodiment, the TGFβ activator is selected from at least one of TGFB-1, TGFB-2, TGFB-3, IDE-1 / 2, and Activin A. In some preferred embodiments, the TGFβ activator is Activin A. In a specific embodiment, the WNT canonical pathway activator is selected from CHIR99021.

[0096] In a specific embodiment, the WNT non-canonical pathway activator is selected from PY-60.

[0097] In some specific embodiments, the third differentiation medium is the RPEM basal medium.

[0098] In some other specific embodiments, the third differentiation medium is a medium obtained by adding a ROCK inhibitor to the RPEM basal medium.

[0099] In a specific embodiment, the ROCK inhibitor is selected from at least one of Y-27632, CEPT, and Blebbistatin. In some preferred embodiments, the ROCK inhibitor is Y-27632.

[0100] In a specific embodiment, the maturation-promoting medium comprises DMEM Basic culture solution (Gibco, C11995500BT), F12 (Gibco, C11765500BT), and B27 additive (Gibco, 17504-044).

[0101] In some specific embodiments, the maturation-promoting medium is a medium comprising 60%-80% DMEM Basic culture solution, 20%-40% F12, and 1% B27 additive.

[0102] In a specific embodiment, the composition of the maturation-promoting medium is as follows: 69% DMEM Basic culture medium, 30% F12, and 1% B27 additive. In a specific embodiment, the following method is used for the staged culture of the differentiation starting cells:

[0103] Perform the first differentiation stage culture with the first differentiation medium for 5 - 7 days,

[0104] Perform the second differentiation stage culture with the second differentiation medium for 7 - 9 days,

[0105] Perform the third differentiation stage culture with the third differentiation medium for 4 - 6 days,

[0106] Optionally, perform the maturation-promoting culture with the maturation-promoting medium for 5 - 7 days.

[0107] In a specific embodiment, the following method is used for the staged culture of the differentiation starting cells:

[0108] Day 0: Wash the differentiation starting cells once with DPBS (without calcium and magnesium), and start the culture with the first differentiation medium. Thereafter, change the medium every day. Among them, the first differentiation medium is: Add Dorsomorphin (100 nM, MCE), IWR1 (2 μM, Sigma), A83 - 01 (500 nM, Selleck), and NIC (10 mM, Sigma) to the RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%) according to the prepared volume;

[0109] Day 6: Aspirate the original medium, wash once with DPBS (without calcium and magnesium), and start the culture with the second differentiation medium. Thereafter, change the medium every day. Among them, the second differentiation medium is: Add CHIR99021 (3 μM, Sigma), Activin A (100 ng / mL, MCE), and PY - 60 (10 μM, MCE) to the RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%) according to the prepared volume;

[0110] Day 14: Aspirate the original medium, wash once with DPBS (without calcium and magnesium), and culture with the third differentiation medium. Among them, the third differentiation medium is the RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%);

[0111] Day 15: After aspirating the original medium, wash once with DPBS (calcium- and magnesium-free), digest with Accutase for 15 min, gently pipette the cells to resuspend, transfer to a 15 mL centrifuge tube, centrifuge at 300 g for 5 min, and seed the cells at a density of 1×10 6 cells / cm 2 onto a cell culture plate coated with Laminin521 (10 μg / mL), and culture with the third differentiation medium. The third differentiation medium is RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%) supplemented with Y-27632 (10 μM) according to the configured volume.

[0112] Day 16: After aspirating the original medium, wash once with DPBS (calcium- and magnesium-free), and culture with the third differentiation medium. Thereafter, change the medium daily. The third differentiation medium is RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%).

[0113] Day 19: After aspirating the original medium, wash once with DPBS (calcium- and magnesium-free), and culture with the maturation-promoting medium. Thereafter, change the medium daily. The composition of the maturation-promoting medium is: 69% DMEM Basic medium, 30% F12, and 1% B27 supplement.

[0114] Day 25: The cells are mature, and the differentiated RPE cells are harvested.

[0115] In a second aspect, the present application provides a medium and a medium composition for inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells.

[0116] Specifically, a medium for inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells (the second differentiation medium) includes a TGFβ activator, a WNT canonical pathway activator, and a WNT non-canonical pathway activator.

[0117] In a specific embodiment, the medium includes RPEM basal medium.

[0118] In some specific embodiments, the RPEM basal medium contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2-Me.

[0119] In some preferred embodiments, the RPEM basal medium is a medium comprising 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2-Me.

[0120] In a specific embodiment, the TGFβ activator can be selected from at least one of TGFB-1, TGFB-2, TGFB-3, IDE-1 / 2, and Activin A. In some preferred embodiments, the TGFβ activator is Activin A.

[0121] In a specific embodiment, the WNT canonical pathway activator is selected from CHIR99021.

[0122] In a specific embodiment, the WNT non-canonical pathway activator is selected from PY-60.

[0123] In some specific embodiments, the concentration of the TGFβ activator in the medium is 20 ng / mL - 500 ng / mL, such as 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL, etc.

[0124] In some specific embodiments, the concentration of the WNT canonical pathway activator in the medium is 0.6 μM - 15 μM, such as 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM.

[0125] In some specific embodiments, the concentration of the WNT non-canonical pathway activator in the medium is 2 μM - 50 μM, such as 2 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM.

[0126] In a specific embodiment, the medium comprises RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1%, and 2-Me 0.1%) and CHIR99021 (3 μM, Sigma), IDE-1 (250 nM, MCE), and PY-60 (10 μM, MCE) added according to the prepared volume.

[0127] Another medium (the first differentiation medium) for inducing the differentiation of induced pluripotent stem cells into retinal pigment epithelial cells includes a TGFβ inhibitor, a BMP inhibitor, and a WNT inhibitor.

[0128] In a specific embodiment, the medium includes an RPEM basal medium.

[0129] In some specific embodiments, the RPEM basal medium contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2-Me.

[0130] In some preferred embodiments, the RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2-Me.

[0131] In a specific embodiment, the TGFβ inhibitor can be selected from at least one of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, and A83-01. In some preferred embodiments, the TGFβ inhibitor is A83-01.

[0132] In a specific embodiment, the BMP inhibitor can be selected from at least one of Chordin, Noggin, LDN193189, Follistatin, and Dorsomorphin. In some preferred embodiments, the BMP inhibitor is Dorsomorphin.

[0133] In a specific embodiment, the WNT inhibitor can be selected from at least one of DKK-1, D4476, CKI-7, IWRI, and Cerberus protein. In some preferred embodiments, the WNT inhibitor is IWRI.

[0134] In some specific embodiments, the concentration of the TGFβ inhibitor in the medium is 0.1 μM - 2.5 μM, such as 0.1 μM, 0.2 μM, 0.5 μM, 0.8 μM, 1 μM, 1.2 μM, 1.8 μM, 1.5 μM, 2 μM, 2.5 μM.

[0135] In some specific embodiments, the concentration of the BMP inhibitor in the medium is 20 nM - 500 nM, such as 20 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM.

[0136] In some specific embodiments, the concentration of the WNT inhibitor in the medium is 1 μM - 25 μM, such as 1 μM, 2 μM, 5 μM, 10 μM, 12 μM, 15 μM, 18 μM, 20 μM, 22 μM, 25 μM.

[0137] In a specific embodiment, the medium comprises RPEM basal medium (DMEM / F12 76.9%, KSR 20%, sodium pyruvate 1%, NEAA 1%, Glutamax 1% and 2-Me 0.1%) and Dorsomorphin (100 nM, MCE), IWR1 (2 μM, Sigma), A83-01 (500 nμM, Selleck) and NIC (10 mM, Sigma) added according to the prepared volume.

[0138] The present application also provides a culture medium composition for inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells, which comprises the above-mentioned first differentiation medium and second differentiation medium.

[0139] In a third aspect, the present application provides the use of the above-mentioned medium or medium composition in the preparation of a drug for treating diseases related to retinal pigment epithelial cells. In a specific embodiment, the disease is age-related macular degeneration (AMD). Through the preparation method and medium provided by the present application, differentiated RPE cells can be obtained efficiently, the cell harvest purity can be improved, and it is suitable for large-scale production. These RPE cells can be used for cell transplantation in the treatment of AMD to prevent or reverse the progression of the disease, and have broad application prospects.

[0140] Examples

[0141] The following will illustrate the content of the present application with specific examples, but the scope of the present application is not limited thereto. If not otherwise specified, the reagents and instruments used in the following examples are conventional reagents and instruments in the art and can be obtained through commercial purchase. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the above-mentioned solutions and obtain corresponding results according to the content of the examples.

[0142] Example 1 Cultivation and preparation of retinal pigment epithelial cells (RPE)

[0143] Differentiated culture is carried out according to the following steps to obtain retinal pigment epithelial cells:

[0144] 1. Resuscitation and culture of hiPSC cells: After resuscitation, the self-established hiPSC cells are passaged and cultured for at least 5 generations, preferably more than 10 generations, for hiRPE differentiation.

[0145] 2. Differentiation of iPSC-RPE

[0146] The RPE differentiation process can be represented by Figure 1 and specifically includes the following steps:

[0147] 2.1 Passage the resuscitated hiPSC at a density of 1 - 2×10 4 cells / cm 2 onto a cell culture plate coated with Laminin521 (coating condition: dilute with DPBS (containing calcium and magnesium) to 10 μg / mL, add 500 μL / well to 12-well plates and 1000 μL / well to 6-well plates, incubate with the coating solution at 37 °C for at least 2 hours), and maintain the culture in TeSR-AOF complete medium (StemCell, 100 - 0401) until the cell confluence reaches about 70% and then start differentiation.

[0148] 2.2 Differentiation culture process

[0149] D0: Aspirate the AOF complete medium, wash once with DPBS (without calcium and magnesium), and change the medium to the first differentiation medium (RPEM-1), and then change the medium every day;

[0150] Among them, the preparation method of RPEM-1 is shown in Table 1:

[0151] Table 1

[0152]

[0153] D6: Aspirate the original medium, wash once with DPBS (without calcium and magnesium), and change the medium to the second differentiation medium (RPEM-2), and then change the medium every day;

[0154] Among them, the preparation method of RPEM-2 is shown in Table 2:

[0155] Table 2

[0156]

[0157] D14: Aspirate the original medium, wash once with DPBS (without calcium and magnesium), and change the medium to the third differentiation medium (RPEM-3);

[0158] Among them, the preparation method of RPEM-3 is shown in Table 3:

[0159] Table 3

[0160]

[0161] D15: Aspirate the original culture medium, wash once with DPBS (without calcium and magnesium), digest with Accutase for 15 min, gently pipette the cells, transfer them to a 15 mL centrifuge tube, centrifuge at 300 g for 5 min, and seed the cells at 1×10 6 cells / cm 2 onto a cell culture plate coated with Laminin521 (10 μg / mL). Add ROCK inhibitor Y-27632 (10 μM) additionally to the third differentiation medium used on day D15, and then change the medium daily;

[0162] D19: Aspirate the original culture medium, wash once with DPBS (without calcium and magnesium), change the medium to the maturation-promoting medium (RPEM-4), and then change the medium daily thereafter;

[0163] The preparation method of RPEM-4 is shown in Table 4:

[0164] Table 4

[0165]

[0166] D25: The cells are mature, harvest the cells, which are the differentiated retinal pigment epithelial cells, and use them for subsequent experiments.

[0167] Example 2 Verification of RPE Differentiation Efficiency

[0168] Perform relevant experiments on the RPE cells obtained by the method of Example 1 to verify their technical effects such as differentiation efficiency. At the same time, set up a Control group and a GA-017 group as controls:

[0169] (1) PY-60 group: RPE cells obtained by culturing in the manner of Example 1;

[0170] (2) Control group: Different from the PY-60 group in that PY-60 is not added to RPEM-2, and other culture methods are the same as in Example 1;

[0171] (3) GA-017 group: Different from the PY-60 group in that PY-60 in RPEM-2 is replaced with GA-017, and other culture methods are the same as in Example 1.

[0172] 1. Microscopic bright-field observation experiment:

[0173] Observe the three groups of RPE cells (all cells differentiated to D25) under the microscope bright field, and the results are as Figure 2As shown (scale bar: 100 μm). It can be seen that the cell purity of the Control group and the GA-017 group is relatively low, with approximately 50% of the cells having a hexagonal morphology, while the characteristic hexagonal structure of the cells in the PY-60 group is obvious under the microscope, and the purity is approximately 90%.

[0174] 2. Flow cytometry detection experiment:

[0175] Main instruments: Flow cytometer (BECKMAN), tabletop centrifuge, pipette, automatic cell fluorescence analyzer;

[0176] Main reagents: eBioscience TM Foxp3 / Transcription Factor Flow Cytometry Fixation and Permeabilization Buffer (Invitrogen, 00-5523-00);

[0177] Experimental method:

[0178] (1) Resuspend 2E5 - 5E5 samples in 100 μL of DPBS, one tube for each of the control group and the experimental group. Add 1 mL of Foxp3 fixation / permeabilization working solution, gently pipette up and down to resuspend 2 - 3 times. Incubate at 2 - 8 °C or room temperature for 30 - 60 minutes, protected from light.

[0179] (2) Add 2 mL of 1× permeabilization solution to each tube, centrifuge at 400 - 600 x g for 5 minutes at room temperature, and discard the supernatant. Resuspend the pellet in the remaining volume of 1× permeabilization solution, usually 100 μL / tube.

[0180] (3) Directly add 5 μL of Human TruStain FcX to the cells for blocking, and incubate at room temperature for 15 minutes. Without washing, add 1 μL of PMEL17 antibody to the experimental group and 1.5 μL of FITC-Isotype to the control group, mix well, and incubate at room temperature for 30 - 45 minutes or more, protected from light.

[0181] (4) Add 2 mL of 1× permeabilization solution to each tube, centrifuge at 400 - 600 x g for 5 minutes at room temperature, and discard the supernatant.

[0182] (5) Resuspend the stained cells in each tube in 300 μL of DPBS.

[0183] (6) Turn on the flow cytometer to detect the samples.

[0184] The three groups of cells (all differentiated to D25 cells) were detected using the above method, and the marker for detection was the RPE-specific marker PMEL17 (NOVUS, NBP2-34638G). The results are as Figure 3 shown.

[0185] Figure 3The Control group was the group without PY-60 added, and the GA-017 group was the group with GA-017 replacing PY-60. The cell differentiation purity of these two groups was relatively low, and the expression levels of the RPE characteristic marker PMEL17 were relatively low, being 55.0% and 59.7% respectively; the cell differentiation purity of the group with PY-60 added increased significantly, and the positive rate of PMEL17 was 97.2%.

[0186] 3. qPCR experiment:

[0187] Main instruments: Fluorescent quantitative PCR instrument (ABI7500)

[0188] Main reagents: RNA extraction kit, RNA fast200 (Shanghai Feijie, 220011), HiScript lll All-in-one RT SuperMix Perfect for Qpcr (Novoprotein, R333-01), Taq Pro Universal SYBR qPCR Master Mix (Novoprotein, Q712-02)

[0189] Experimental steps:

[0190] (1) Samples of iPSC and RPE at differentiation days 14 and 40 were collected;

[0191] (2) RNA extraction and reverse transcription were carried out according to the experimental steps in the RNA extraction kit and the RT SuperMix Perfect for Qpcr kit;

[0192] (3) qPCR sample loading and on-machine detection were carried out according to the experimental steps in the Taq Pro Universal SYBR qPCR Master Mix kit.

[0193] qPCR detection was carried out on the three groups of cells at different differentiation stages (D14 and D25), and the detection results were compared, as Figure 4 shown. It can be seen that the transcriptome expression levels of the RPE characteristic markers PMEL17, BEST1, and RPE65 in the cells of the PY-60 group increased significantly at Day14 and Day25. The expression level of the group with PY-60 added was significantly higher than that of the other two groups, indicating that the differentiation efficiency of adding PY-60 to RPE was significantly improved.

[0194] As described above, it is only the preferred embodiment of the present application, and it does not limit the present application in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present application without departing from the technical solution content of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A method for preparing retinal pigment epithelial cells, comprising: Pre-culturing pluripotent stem cells to obtain differentiation starting cells; Culturing the differentiation starting cells in different culture media in stages to obtain retinal pigment epithelial cells, wherein, the culture medium in at least one stage includes a WNT non-canonical pathway activator.

2. The preparation method according to claim 1, wherein, The staged culture at least includes: culturing the differentiation starting cells in a first differentiation medium for a first differentiation stage, in a second differentiation medium for a second differentiation stage, and in a third differentiation medium for a third differentiation stage in sequence.

3. The preparation method according to claim 2, wherein, The first differentiation medium, the second differentiation medium, and the third differentiation medium all contain RPEM basal medium, Preferably, the first differentiation medium further includes a TGFβ inhibitor, a BMP inhibitor, and a WNT inhibitor, and / or the second differentiation medium further includes a TGFβ activator, a WNT canonical pathway activator, and a WNT non-canonical pathway activator, and / or the third differentiation medium further includes a ROCK inhibitor, More preferably, the RPEM basal medium contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2-Me.

4. The preparation method according to claim 3, wherein The TGFβ inhibitor is selected from at least one of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, and A83-01, preferably A83-01, and / or the BMP inhibitor is selected from at least one of Chordin, Noggin, LDN193189, Follistatin, and Dorsomorphin, preferably Dorsomorphin, and / or the WNT inhibitor is selected from at least one of DKK-1, D4476, CKI-7, IWRI, and Cerberus protein, preferably IWRI, the TGFβ activator is selected from at least one of TGFB-1, TGFB-2, TGFB-3, IDE-1 / 2, and ActivinA, preferably Activin A, and / or the WNT canonical pathway activator is selected from CHIR99021, and / or the WNT non-canonical pathway activator is selected from PY-60.

5. The preparation method according to claim 3, wherein, The first differentiation medium is RPEM basal medium supplemented with 20 nM - 500 nM Dorsomorphin, 1 μM - 25 μM IWRI, 0.1 μM - 2.5 μM A83 - 01, and 2 mM - 50 mM NIC. The second differentiation medium is RPEM basal medium supplemented with 0.6 μM - 15 μM CHIR99021, 20 ng / mL - 500 ng / mL Activin A, and 2 μM - 50 μM PY - 60. The third differentiation medium is RPEM basal medium. The RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2 - Me.

6. A culture medium for inducing the differentiation of induced pluripotent stem cells into retinal pigment epithelial cells, comprising: TGFβ activator, WNT canonical pathway activator, and WNT non - canonical pathway activator. Preferably, it further includes RPEM basal medium, which contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2 - Me. More preferably, the RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2 - Me.

7. The culture medium according to claim 6, wherein, The TGFβ activator is selected from at least one of TGFB - 1, TGFB - 2, TGFB - 3, IDE - 1 / 2, and Activin A, preferably Activin A, and / or the WNT canonical pathway activator is selected from CHIR99021, and / or the WNT non - canonical pathway activator is selected from PY - 60. More preferably, in the medium, the concentration of the TGFβ activator is 20 ng / mL - 500 ng / mL, and / or the concentration of the WNT canonical pathway activator is 0.6 μM - 15 μM, and / or the concentration of the WNT non - canonical pathway activator is 2 μM - 50 μM.

8. A culture medium for inducing the differentiation of induced pluripotent stem cells into retinal pigment epithelial cells, comprising: TGFβ inhibitor, BMP inhibitor, and WNT inhibitor. Preferably, it further includes RPEM basal medium, which contains DMEM / F12, KSR, sodium pyruvate, NEAA, Glutamax, and 2 - Me. More preferably, the RPEM basal medium is a medium containing 76.9% DMEM / F12 medium, 20% KSR, 1% sodium pyruvate, 1% NEAA, 1% Glutamax, and 0.1% 2 - Me.

9. The culture medium according to claim 8, wherein, The TGFβ inhibitor is selected from at least one of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276 and A83-01, preferably A83-01, and / or the BMP inhibitor is selected from at least one of Chordin, Noggin, LDN193189, Follistatin and Dorsomorphin, preferably Dorsomorphin, and / or the WNT inhibitor is selected from at least one of DKK-1, D4476, CKI-7, IWRI and Cerberus protein, preferably IWRI. More preferably, in the medium, the concentration of the TGFβ inhibitor is 0.1 μM - 2.5 μM, and / or the concentration of the BMP inhibitor is 20 nM - 500 nM, and / or the concentration of the WNT inhibitor is 1 μM - 25 μM.

10. A culture medium composition for inducing the differentiation of induced pluripotent stem cells into retinal pigment epithelial cells, comprising the culture medium according to claim 6 or 7, and the culture medium according to claim 8 or 9.

Images