An in vitro culture method for achieving cell differentiation, and cell populations obtained thereby and uses thereof

Abstract

The present application specifically relates to a method for obtaining corneal endothelial cells, and the initial cells can be pluripotent stem cell-derived or neural crest cell-derived. Specifically, the present application discloses that pluripotent stem cells are efficiently induced to generate corneal endothelial cells by using a culture medium with completely clear chemical components. The present application also relates to CEC cells and their application in treating corneal endothelial diseases.

Description

TECHNICAL FIELD

[0001] The present application specifically relates to a method for obtaining corneal endothelial cells, the initial cells can be pluripotent stem cell-derived or neural crest cell-derived. Specifically, the present application discloses that pluripotent stem cells are efficiently induced to generate corneal endothelial cells using a culture medium with completely known chemical composition. The present application also relates to CEC cells and their application in treating corneal endothelial diseases. BACKGROUND

[0002] At present, the number of patients worldwide who need corneal transplantation due to corneal endothelial damage is as high as 6.5 million, while only 2% of them actually receive the transplantation, and the supply and demand of corneas is seriously imbalanced. In 2018, Kinoshita published a work in "New England" which showed that human primary corneal endothelial cells (CEC) cultured in vitro and transplanted into patients have a significant therapeutic effect. Corneal endothelial cells obtained by differentiation of pluripotent stem cells (PSC) do not rely on donated corneas and have great clinical application value in treating corneal endothelial diseases. Scientists have tried to differentiate corneal endothelial cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), mainly by simulating in vivo development into two stages: the first stage is to induce differentiation of hESCs or iPSCs into neural crest cells (NCCs); the second stage is to differentiate mature corneal endothelial cells from neural crest. From the published works, the first stage is relatively mature, but the differentiation methods in the second stage (including differentiation from pluripotent stem cells to corneal endothelial cells, although some articles do not mention neural crest cells, but also experience this intermediate process) are of mixed quality, mostly rely on co-culture, the culture medium composition is not clear, contains animal sources, and the involved differentiation pathways are not clear. SUMMARY

[0003] The present application provides:

[0004] 1. A composition for adding to a culture medium when inducing differentiation of pluripotent stem cells into neural crest cells, comprising: a TGF-β inhibitor, a WNT activator, and a TGF-β activator; optionally comprising a ROCK inhibitor.

[0005] 2. The composition of item 1, wherein,

[0006] the TGF-β inhibitor is an ALK4 receptor inhibitor selected from one or two or three of SB431542, SB505124, A83-01;

[0007] the WNT activator is selected from one or two or three of GSK3P inhibitor, Wnt3A, Wntl; the GSK3P inhibitor is selected from one or two or more than three of CHIR99021, GSK3P inhibitor IX, GSK3P inhibitor VII, Indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, SB216763;

[0008] the TGF-β activator is selected from one or two or more than three of TGF-βl, TGF-β2, TGF-β3, SRI-011381 (hydrochloride);

[0009] Preferably, the ROCK inhibitor is Y-27632.

[0010] 3. A composition for inducing differentiation of neural crest cells into corneal endothelial cells, comprising: a TGF-β inhibitor, a WNT inhibitor, and a bFGF inhibitor; optionally comprising a ROCK inhibitor.

[0011] 4. The composition of item 3, wherein,

[0012] the TGF-β inhibitor is an ALK4 receptor inhibitor selected from one, two or three of SB431542, SB505124, A83-01;

[0013] the WNT inhibitor is a DKK2 polypeptide or a WNT signaling pathway inhibitor selected from one or two of XAV-939, IWR-1;

[0014] the bFGF inhibitor is selected from one, two or three of a VEGFR inhibitor, a FGFRl inhibitor, and a PDGFRP inhibitor; preferably, the bFGF inhibitor is SU-5402;

[0015] Preferably, the ROCK inhibitor is Y-27632.

[0016] 5. A medium for inducing differentiation of pluripotent stem cells into neural crest cells, comprising: the composition of item 1 or 2; further comprising:

[0017] a) a basal medium; and

[0018] b) a serum replacement, glutamine or stabilized dipeptide of L-alanyl-L- glutamine, an insulin-transferrin-selenium additive (ITS), non-essential amino acids, a L-ascorbic acid and vitamin C cocktail (L-AA).

[0019] wherein the TGF-β inhibitor is added to the culture medium at a concentration of 1 μΜ - 100 mM; the WNT activator is added to the culture medium at a concentration of 10 nM - 100 μΜ; the TGF-β activator is added to the culture medium at a concentration of 0.1 ng / ml - 100 μg / ml; the ROCK inhibitor is added to the culture medium at a concentration of 10 nM - 100 μΜ.

[0020] 6. A culture medium for inducing differentiation of neural crest cells into corneal endothelial cells, comprising: the composition of item 3 or 4; further comprising:

[0021] a) a basal medium; and

[0022] b) a serum replacement, a stabilized dipeptide of glutamine or L-alanyl-L- glutamine, an insulin-transferrin-selenium additive (ITS), non-essential amino acids, a L-ascorbic acid and vitamin C cocktail (L-AA);

[0023] wherein the TGF-β inhibitor is added to the culture medium at a concentration of 1 μΜ - 100 mM; the WNT inhibitor is added to the culture medium at a concentration of 1 ng / ml - 100 μg / ml when the WNT inhibitor is a protein, and at a concentration of 10 nM - 10 μΜ when the WNT inhibitor is a small molecule compound; the bFGF inhibitor is added to the culture medium at a concentration of 10 nM - 10 μΜ; the ROCK inhibitor is added to the culture medium at a concentration of 10 nM - 100 μΜ.

[0024] 7. The culture medium of item 5 or 6, wherein the serum replacement is one or more selected from the group consisting of KOSR, MSC serum-free additive, Ultroser TM G.

[0025] 8. The culture medium of item 5 or 6, wherein the non-essential amino acids are one or more selected from the group consisting of glycine, L-alanine, L-asparagine, L- aspartic acid, L-glutamic acid, L-proline, L-serine.

[0026] 9. The culture medium of item 5 or 6, wherein the basal medium is one or more selected from the group consisting of KO-DMEM, KO-DMEM / F12, DMEM, α-MEM, F-12, MEM, BME, RPMI 1640, G-MEM.

[0027] 10. The medium of item 5 or 6, wherein the serum replacement is 1-50% of the basal medium; the glutamine or L-alanyl-L-glutamine-stabilized dipeptide is 1-20% of the basal medium; the ITS is 1-20% of the basal medium; the non-essential amino acids are 1-20% of the basal medium; and the vitamin cocktail (L-AA) is 0.1-10% of the basal medium.

[0028] 11. A method of differentiating pluripotent stem cells into neural crest cells (NCC) by in vitro culturing, comprising culturing pluripotent stem cells using the composition of item 1 or 2 or the medium of any one of items 5, and 7-10 when dependent on item 5.

[0029] 12. A method of differentiating neural crest cells (NCC) into corneal endothelial cells (CEC) by in vitro culturing, comprising culturing pluripotent stem cells using the composition of item 3 or 4 or the medium of any one of items 6, and 7-10 when dependent on item 6.

[0030] 13. A method of differentiating pluripotent stem cells into corneal endothelial cells (CEC) by in vitro culturing, wherein the method is a two-stage culturing method, first differentiating pluripotent stem cells into neural crest cells (NCC) in a first stage and then differentiating neural crest cells (NCC) into corneal endothelial cells (CEC) in a second stage, wherein the first stage uses the method of item 11 and the second stage uses the method of item 12.

[0031] 14. A corneal endothelial cell (CEC) expressing one or more of the following markers: DCN, LUM, SPARCL1, SERPINF1, SERPING1, CXCL3, AEBP1, RARRES2, PCOLCE, HTRA1, FTL, ANGPTL7, LOX, PMP22, VMO1, EMP3, ARNT, SDC2, COLEC12, ANXA5, JUN, SQSTM1, CTSZ, PLPP3, CTSL, LMNA, GADD45A, ABCA6, TSC22D1, and TIMP3.

[0032] 15. A pharmaceutical composition comprising the CEC cell of item 14 and a pharmaceutically acceptable excipient.

[0033] 16. Use of the CEC cell of item 14 or the composition of item 15 in the manufacture of a medicament for treating a disease associated with corneal endothelial cells.

[0034] 17. The use according to item 16, wherein the diseases associated with corneal endothelial cells include: Fuchs corneal endothelial dystrophy, iridocorneal endothelial syndrome, posterior polymorphic corneal dystrophy, congenital hereditary corneal endothelial dystrophy, and secondary diseases requiring corneal endothelial transplantation.

[0035] Beneficial technical effects achieved by this application

[0036] This application establishes a differentiation method for corneal endothelial cells with clear components, no animal origin, no need for co-culture, and induced only by small molecules or factors, and describes new markers for the cells, providing a basis for the treatment of corneal endothelial diseases and better solving the global cornea shortage dilemma. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A schematic diagram of the differentiation of human embryonic stem cells into corneal endothelial cells is shown, with the expression of cell surface markers at each stage of differentiation indicated.

[0038] Figure 2 Shows the differentiation of human embryonic stem cells into neural crest cells; A: optimized neural crest cell differentiation system; B: morphological characteristics of D10 neural crest cells; C: flow cytometry analysis of D10 neural crest cell markers; D: immunofluorescence staining analysis of D10 neural crest cell markers.

[0039] Figure 3 Differentiation of neural crest cells into corneal endothelial cells is shown.

[0040] Figure 4 The identification of differentiated corneal endothelial cell markers is shown; A: flow cytometry analysis of the expression ratio of ZO-1; B: immunofluorescence staining analysis of the expression of ZO-1, Na+ / K+ATPase, FOXC1, PITX2, and N-Cadherin.

[0041] Figure 5 RNA-seq data analysis of different time points of corneal endothelial cell differentiation is shown; Figure 5 A shows the results of cluster analysis and PCA analysis: corneal endothelial cells at different differentiation time points were compared with the data of primary corneal endothelial cells in published literature. The results showed that corneal endothelial cells differentiated at D23 days were more similar to primary corneal endothelial cells; Figure 5 B shows the corneal endothelial gene heat map results.

[0042] Figure 6This demonstrates the important role of SU-5402 in corneal endothelial cell differentiation. Figure 6A shows that when SU-5402 is added to the corneal endothelial cell differentiation system, the expression ratio of the marker Na+ / K+ ATPase is high; however, corneal endothelial cells without SU-5402 do not express Na+ / K+ ATPase. Figure 6 B shows that the relative expression of specific genes was the highest when corneal endothelial cells were differentiated using 100 nM SU-5402; Figure 6 In C, q-PCR results also demonstrated that the relative expression of specific genes in corneal endothelial cells differentiated with high concentration (10 μM) SU-5402 was not significantly different from that with 100 nM SU-5402. Figure 6 D shows that the expression ratio of the marker Na+ / K+ATPase is the highest, and PITX2 appears to be expressed.

[0043] Figure 7 The effect of adding TGFβ2 during the neural crest differentiation stage on the differentiation efficiency of corneal endothelial cells is shown.

[0044] Figure 8 The figure is an in vivo functional evaluation of corneal endothelial cells; A is the corneal transparency at different time points after surgery; B is the corneal thickness and corneal transparency statistical chart at different time points after surgery; C is the slit lamp examination, corneal endothelial confocal and optical coherence tomography imaging at D28 after surgery; D is the corneal sampling at D28 after surgery and immunofluorescence staining identification after frozen section. DETAILED DESCRIPTION

[0045] Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0046] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the present invention shall be as defined in the attached claims.

[0047] As used herein, "substantially free of," with respect to a particular component, is used herein to mean that the particular component is not deliberately formulated into the composition and / or is only present as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any inadvertent contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferably, compositions wherein the amount of the particular component is not detectable by standard analytical methods.

[0048] As used in this specification, "a" or "an" can mean one or more. As used in this specification, the word "or" when used in a list of two or more items indicates that any one of the items can be present, or combinations of any of the items can be present.

[0049] The term "or" is used in the claims to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0050] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects over the course of the study.

[0051] The routes described in the examples for obtaining the various biomaterials are only provided as a way to experimentally obtain the specific disclosed purposes and should not be considered as limiting the source of the biomaterials of the present application. In fact, the source of the biomaterials used is broad and any biomaterial that can be obtained without violating the law and the ethical-moral code can be used as a replacement according to the indications given in the examples.

[0052] The practice of the present application can employ, unless otherwise indicated, conventional techniques and descriptions in organic chemistry, polymer technology, molecular biology, cell biology, biochemistry and immunology, which are within the skill of the art. Specific illustrations of suitable techniques can be had by reference to the examples herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Davis et al., Basic Methods in Molecular Biology, Academic Press; Miller and Calos, Gene Transfer Vectors for Mammalian Cells, Cold Spring Harbor; and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Wiley & Sons; Current Protocols in Protein Science, J.E. Colligan et al., eds., Wiley & Sons; Current Protocols in Cell Biology, J.S. Bonifacino et al., Wiley & Sons; and Current Protocols in Immunology, J.E. Colligan et al., eds., Wiley & Sons; Culture of Animal Cells: A Manual of Basic Technique, R.I. Freshney, ed., Wiley & Sons; General Techniques of Cell Culture, M.A. Harrison and I.F. Rae, Cambridge Univ. Press; and Embryonic Stem Cells: Methods and Protocols, K. Turksen, ed., Humana Press. Other references of interest include Culture Is Our Business, M. McLuhan, Ballantine Books, 1970); and Understanding Media, M. McLuhan, Signet, 1970; all of which are incorporated herein by reference in their entirety for all purposes.

[0053] The present application provides, in a first aspect, a differentiation inducing composition (pluripotent stem cells to NCCs).

[0054] In one embodiment, a composition for adding to a culture medium when inducing pluripotent stem cells to differentiate into neural crest cells is provided, comprising: a TGF-β inhibitor, a WNT activator, and a TGF-β activator; optionally comprising a ROCK inhibitor.

[0055] In the context of the present specification, the term "pluripotent stem cell" refers to a pluripotent stem cell derived from a human or non-human animal, which can be an embryonic stem cell or an induced pluripotent stem cell. The embryonic stem cell can be derived from a human or non-human animal. In the context of the present specification, the terms "neural crest cell" and "NCC" are used interchangeably to refer to a transitional cell during embryonic development, which can be induced to differentiate into corneal endothelial cells. NCCs can be identified by existing markers, for example, SOX10, AP2, HNK1, PAX3, PAX7, and P75 (NGFR), and low or no expression of PAX6.

[0056] In the context of the present specification, the TGF-β activator is specifically TGFβ2, which is specifically added to the culture medium in the second half of the process of inducing pluripotent stem cells to differentiate into neural crest cells, which facilitates the generation of CECs more smoothly in the subsequent process of differentiating neural crest cells (NCCs) into corneal endothelial cells (CECs).

[0057] In yet another embodiment, a composition is provided, which can be added to a culture medium for the purpose of inducing differentiation, wherein the TGF-β inhibitor is an ALK4 receptor inhibitor selected from one or two or three of SB431542, SB505124, A83-01; the TGF-β inhibitor is added to the culture medium at a concentration of 1 μM-100 mM; specifically, the concentration can be 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM.

[0058] The WNT activator is selected from one or two or three of GSK3 inhibitor, Wnt3A, Wnt1; the GSK3 inhibitor is selected from one or two or more than three of CHIR99021, GSK3 inhibitor IX, GSK3 inhibitor VII, Indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, SB216763; the WNT activator is added to the culture medium at a concentration of 10 nM-100 μM; in particular, the concentration can be 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM.

[0059] The TGF-β activator is selected from one or two or more than three of TGF-β1, TGF-β2, TGF-β3, SRI-011381 (hydrochloride); the TGF-β activator is added to the culture medium at a concentration of 0.1 ng / ml-100 μg / ml; in particular, the concentration can be 0.1 ng / ml, 0.2 ng / ml, 0.5 ng / ml, 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 500 ng / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml, 100 μg / ml.

[0060] Preferably, the ROCK inhibitor is Y-27632, and the ROCK inhibitor is added to the culture medium at a concentration of 10 nM-100 μM; in particular, the concentration can be 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM.

[0061] In the context of the present specification, the GSK3 inhibitor IX is also known as 6-bromoindirubin-3'-oxime, BIO (BIO is the abbreviation of GSK3 inhibitor IX); the GSK3 inhibitor VII is also known as 4-dibromoacetophenone.

[0062] In the context of the present specification, the "addition concentration" refers to the mass concentration or molar concentration of the macromolecular or small molecular substance mentioned in the corresponding culture medium.

[0063] The present application relates in a second aspect to a differentiation-inducing composition (NCC to CEC).

[0064] In one embodiment, a composition for adding to a culture medium when inducing differentiation of neural crest cells into corneal endothelial cells is provided, comprising: a TGF-β inhibitor, a WNT inhibitor, and a bFGF inhibitor; optionally comprising a ROCK inhibitor.

[0065] As used herein, the terms "corneal endothelial cell" and "CEC" are used interchangeably, also known as the anterior chamber endothelium, the posterior corneal epithelium, is a single layer of cells that lies immediately posterior to the Descemet's membrane. Exemplary markers thereof include, but are not limited to: Na+ / K+ ATPase, ZO-1, KLF13, AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2 / SCL4A2, SLC16A1, CA12, CA4, and FOXC1.

[0066] In yet another embodiment, a composition for adding to a culture medium for the purpose of inducing differentiation is provided, wherein the TGF-β inhibitor is an ALK4 receptor inhibitor selected from one, two or three of SB431542, SB505124, A83-01; the TGF-β inhibitor is added to the culture medium at a concentration of 1 μΜ to 100 mM; in particular, the concentration can be 1 μΜ, 2 μΜ, 5 μΜ, 10 μΜ, 20 μΜ, 50 μΜ, 100 μΜ, 200 μΜ, 500 μΜ, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM.

[0067] The WNT inhibitor is a DKK2 polypeptide or a WNT signaling pathway inhibitor selected from one or both of XAV-939, IWR-1; the WNT inhibitor is added to the culture medium at a concentration of 1 ng / ml to 100 μg / ml (protein), 10 nM to 10 μΜ (small molecule compound); in particular, when the WNT inhibitor is a protein, the concentration can be 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 500 ng / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml, 100 μg / ml; in particular, when the WNT inhibitor is a small molecule compound, the concentration can be 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μΜ, 2 μΜ, 5 μΜ, 10 μΜ.

[0068] The bFGF inhibitor is one, two or three selected from the group consisting of: a VEGFR inhibitor, a FGFRl inhibitor and a PDGFRbeta inhibitor; preferably, the bFGF inhibitor is SU-5402; the bFGF inhibitor is added to the culture medium at a concentration of 10 nM to 10 μΜ; in particular, the concentration can be 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μΜ, 2 μΜ, 5 μΜ, 10 μΜ.

[0069] Preferably, the ROCK inhibitor is Y-27632, the ROCK inhibitor is added to the culture medium at a concentration of 10 nM to 100 μΜ; in particular, the concentration can be 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μΜ, 2 μΜ, 5 μΜ, 10 μΜ, 20 μΜ, 50 μΜ, 100 μΜ.

[0070] The present application relates in a third aspect to an induction differentiation medium (pluripotent stem cells to NCCs).

[0071] In one embodiment, a medium for inducing differentiation of pluripotent stem cells into neural crest cells is provided, comprising: a composition as described in the first aspect; further comprising:

[0072] a) a basal medium; and

[0073] b) a serum replacement, glutamine or stabilized dipeptide of L-alanyl-L-glutamine, an insulin-transferrin-selenium supplement (ITS), non-essential amino acids, a L-ascorbic acid and vitamin C cocktail (L-AA).

[0074] As used herein, the term "basal medium" refers to any known cell culture medium known in the art for supporting the growth of cells in vitro, generally, it is a medium comprising a defined basal solution that contains salts, sugars, amino acids, and any other nutrients required to maintain cells in culture in a viable state. Non-limiting examples of commercially available basal media that can be used in accordance with the present application include, but are not limited to, MEM, a-MEM, RPMI-1640, DMEM, DMEM / F12 (DF12), Neurobasal, and knockout DMEM (KODMEM). The basal medium can be supplemented with various reagents known in the art for the treatment of cell cultures.

[0075] The present application relates in a fourth aspect to an induction differentiation medium (NCCs to CECs).

[0076] In one embodiment, there is provided a medium for inducing differentiation of neural crest cells into corneal endothelial cells, comprising: a composition according to the fourth aspect; further comprising:

[0077] a) a basal medium; and

[0078] b) a serum substitute, glutamine or stabilized dipeptide of L-alanyl-L-glutamine, an insulin-transferrin-selenium additive (ITS), non-essential amino acids, L-ascorbic acid and a cocktail of vitamins (L-AA).

[0079] The medium of the third and fourth aspects described above further has the following features:

[0080] In one embodiment, the serum substitute is one or more selected from the group consisting of KOSR, MSC serum-free additive, Ultroser™ G.

[0081] In yet another embodiment, the non-essential amino acids are one or more selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartate, L-glutamate, L-proline, L-serine.

[0082] In another embodiment, the basal medium is one or more selected from the group consisting of KO-DMEM, KO-DMEM / F12, DMEM, a-MEM, F-12, MEM, BME, RPMI 1640, G-MEM.

[0083] In one embodiment, the serum substitute is 1% to 50% of the basal medium; the glutamine or stabilized dipeptide of L-alanyl-L-glutamine is 1% to 20% of the basal medium; the ITS is 1% to 20% of the basal medium; the non-essential amino acids are 1% to 20% of the basal medium; and the cocktail of vitamins (L-AA) is 0.1% to 10% of the basal medium.

[0084] The present application relates in a fifth aspect to a method of inducing differentiation of cells.

[0085] In one embodiment, there is provided a method of differentiating pluripotent stem cells into neural crest cells (NCC) by in vitro culture, comprising: culturing the pluripotent stem cells using the composition according to the first aspect or the medium according to the third aspect, and adding TGFβ2 only in the second half of the process of inducing differentiation of the pluripotent stem cells into neural crest cells.

[0086] In the context of the present specification, "in the second half of the process of inducing pluripotent stem cells to differentiate into neural crest cells" specifically refers to any period of time between the sixth day of the neural crest differentiation stage and the end of the differentiation stage; for example, TGFβ2 is added only from day 6 to day 10 of the neural crest differentiation stage; or TGFβ2 is added only from day 7 to day 10; or TGFβ2 is added only from day 8 to day 10; or TGFβ2 is added only from day 9 to day 10.

[0087] In yet another specific embodiment, there is provided a method of differentiating neural crest cells (NCCs) into corneal endothelial cells (CECs) by in vitro culturing, comprising culturing pluripotent stem cells using the composition of the second aspect or the medium of the fourth aspect.

[0088] In yet another specific embodiment, there is provided a method of differentiating pluripotent stem cells into corneal endothelial cells (CECs) by in vitro culturing, wherein the method is a two-stage culturing method, first differentiating pluripotent stem cells into neural crest cells (NCCs) in a first stage, and then differentiating neural crest cells (NCCs) into corneal endothelial cells (CECs) in a second stage.

[0089] The present application relates in a sixth aspect to a corneal endothelial cell.

[0090] In one specific embodiment there is provided a corneal endothelial cell (CEC) expressing one or more of the following markers: DCN, LUM, SPARCL1, SERPINF1, SERPING1, CXCL3, AEBP1, RARRES2, PCOLCE, HTRA1, FTL, ANGPTL7, LOX, PMP22, VMO1, EMP3, ARNT, SDC2, COLEC12, ANXA5, JUN, SQSTM1, CTSZ, PLPP3, CTSL, LMNA, GADD45A, ABCA6, TSC22D1 and TIMP3.

[0091] The present application relates in a seventh aspect to a pharmaceutical composition.

[0092] In one specific embodiment there is provided a pharmaceutical composition comprising the above-mentioned CEC cell and a pharmaceutically acceptable excipient.

[0093] The present application relates in an eighth aspect to the use of a CEC cell.

[0094] In one specific embodiment there is provided the use of the above-mentioned CEC cell or the above-mentioned pharmaceutical composition in the manufacture of a medicament for treating a disease associated with corneal endothelial cells.

[0095] In yet another embodiment, the use described above is provided, wherein the disease related to corneal endothelial cells comprises Fuchs corneal endothelial dystrophy, iridocorneal endothelial syndrome, posterior polymorphous corneal dystrophy, congenital hereditary corneal endothelial dystrophy, and secondary diseases requiring corneal endothelial transplantation.

[0096] Example 1 Corneal endothelial cell differentiation experiment

[0097] Experimental materials:

[0098] Human embryonic stem cells, 6-well plate;

[0099] Reagents:

[0100] Essential 6, Knockout DMEM Medium, KOSR, CTS-GlutaMax, ITS, NEAA, L-Ascorbic acid (L-AA), SB431542, CHIR 99021, DKK2, SU-5402;

[0101] Instrument equipment:

[0102] Biological safety cabinet, CO2 incubator, flow cytometer, fluorescence confocal microscope.

[0103] NCC differentiation basic medium

[0104] Reagent Proportion 50mL added amount

[0105] Essential 6 Medium 99% 49.5ml

[0106] CTS-GlutaMax 1% 0.5ml

[0107] CEC differentiation basic medium

[0108]

[0109] First stage:

[0110] NCC differentiation (referring to Barber, K., et al. (2019). “Derivation of enteric neural lineages from human pluripotent stem cells.” Nat Protoc 14(4): 1261-1279.)

[0111] D0-D10: Essential 6 + 1% GlutaMax + 10 μΜ SB431542;

[0112] D0-D1 : +0.6 μΜ CHIR99021 ;

[0113] D2-D5: +1.5 μΜ CHIR99021 ;

[0114] D6-D10: +1.5 μΜ CHIR99021 + 10 ng / ml TGFβ2.

[0115] Second stage: CEC differentiation

[0116] D11-D23: KODMEM + 15% KOSR + 1% GlutaMax + 1% ITS + 1% NEAA + 0.1% L-AA + 10 μΜ SB431542 + 10 ng / ml DKK2 + 100 nM SU-5402;

[0117] D11 : +10 μΜ Y-27632;

[0118] D12-D23: only basal medium.

[0119] The specific operation steps are as follows:

[0120] D0: On the first two days of D0, ESCs were plated in 6-well plates at a density of 6 x 10 4 / cm 2 D0: On the first two days of D0, ESCs were plated in 6-well plates at a density of 6 x 10

[0121] D1 : Discard D0 medium and add 3 ml medium Essential 6 + 1% GlutaMax + 10 μΜ SB431542 + 0.6 μΜ CHIR99021 per well;

[0122] D2-D5: Discard medium from the previous day each time, add 3.5 ml medium Essential 6 + 1% GlutaMax + 10 μΜ SB431542 + 1.5 μΜ CHIR99021 per well;

[0123] D6-D10: Discard medium from the previous day each time, add 3.5 ml medium Essential 6 + 1% GlutaMax + 10 μΜ SB431542 + 1.5 μΜ CHIR99021 + 10 ng / ml TGFβ2 per well;

[0124] D11: Differentiated mature NCCs were digested into single cells and cultured at 1×10 5 / cm 2 The cells were seeded into 6-well plates at a density of 100 μg / ml and 2 ml of culture medium KODMEM+15% KOSR+1% GlutaMax+1% ITS+1% NEAA+0.1% L-AA+10 μM SB431542+10 ng / ml DKK2+100 nM SU-5402+10 μM Y-27632 was added to each well;

[0125] D12-D23: The culture medium of the previous day was discarded, and 2 ml of culture medium KODMEM + 1% GlutaMax + 1% NEAA + 0.1% L-AA + 10 μM SB431542 + 10 ng / ml DKK2 + 100 nM SU-5402 was added to each well, and the medium was changed every other day.

[0126] RNA-seq sequencing: Corneal endothelial cells were subjected to RNA-seq at days 15, 19, and 23 of differentiation. Highly expressed non-housekeeping genes not previously reported in human or mouse corneal endothelial cells were selected for further qPCR validation.

[0127] qPCR verification: Primers were designed for the genes found in RNA-seq sequencing, and qPCR was performed using differentiated corneal endothelial cells as the experimental group and fibroblasts or pluripotent stem cells as controls. The CT value and relative expression level were used to verify that the gene was indeed expressed in corneal endothelial cells.

[0128] Experimental results

[0129] In the first stage, human embryonic stem cells differentiate into neural crest cells. Figure 2 A shows the optimized neural crest cell differentiation system. Using only two small molecules, neural crest cells can be differentiated and mature after 10-11 days of differentiation. The basal medium used for differentiation from D0 to D10 was Essential 6, 1% GlutaMax, and 10μM SB431542. From D0 to D1, 0.6μM CHIR99021 was added to the basal medium, and from D2 to D10, 1.5μM CHIR99021 was added to the basal medium. Cells at D10 of differentiation were dense and uniform in morphology. Figure 2 B), and the markers of the cells at differentiation D10 were detected by flow cytometry ( Figure 2 C) and immunofluorescence identification ( Figure 2D), the results showed that the proportion of neural crest cells expressing each specific marker was high after differentiation D10. Obtaining a high proportion of neural crest cells can provide a good basis for the differentiation of corneal endothelial cells. Among them, flow cytometry analysis showed that the positive rate of neural crest cell marker P75 was as high as more than 95%, and the positive rate of HNK1 was as high as more than 99%; the results of immunofluorescence identification showed that the expression proportion of neural crest cell markers P75, NESTIN, SOX10 and AP2α was high, indicating that we obtained high-purity neural crest cells.

[0130] In the second stage, in order to obtain mature corneal endothelial cells from neural crest cells, we refer to the published literature and conduct a large number of pathway and small molecule screening to establish a differentiation system of corneal endothelial cells Figure 3 A). First, based on the first stage D2-D10 medium, 10 ng / ml TGFβ2 was added from D6 to D10; the base medium used for differentiation from D11 to D23 was KODMEM, 15% KOSR, 1% GlutaMax, 1% ITS, 1% NEAA, 0.1% L-AA, 10 μM SB431542, 10 ng / ml DKK2, 100 nM SU-5402; D11 inoculated cells need to add 10 μM Y-27632 based on the base medium, and D12-D23 can be added only with the base medium. The morphology of corneal endothelial cells differentiated D23 was hexagonal, and the morphology observed under 10x objective was like paving stones, and the cell morphology observed under 20x and 40x objective was clear hexagonal Figure 3 B), which shows that we have differentiated to obtain corneal endothelial cells similar in morphology to primary corneal endothelial cells. At the same time, we have conducted flow detection Figure 4 A) and immunofluorescence identification Figure 4 B) of the surface markers of corneal endothelial cells, among which the expression proportion of ZO-1 and Na+ / K+ATPase was analyzed by flow cytometry, and the expression of ZO-1, Na+ / K+ATPase, N-Cadherin and Aquaporin 1 was analyzed by immunofluorescence staining. The results showed that corneal endothelial cells differentiated D23 can express their markers ZO-1, Na+ / K+ATPase, N-Cadherin and Aquaporin 1, with a positive rate of ZO-1 as high as more than 98%, a positive rate of Na+ / K+ATPase as high as more than 99%, and a double positive rate of both as high as more than 99%, indicating that we have obtained mature corneal endothelial cells.

[0131] At the same time, we selected different time points for sampling cells during the differentiation of corneal endothelial cells, conducted RNA-seq data analysis, and compared with the data of primary corneal endothelial cells reported in the literatureFigure 5 ). Wherein Figure 5 A shows the results of cluster analysis and PCA analysis. The differentiated corneal endothelial cells at different time points were compared with the data of primary corneal endothelial cells in published literature, and the results showed that the differentiated corneal endothelial cells at D23 were more similar to the primary corneal endothelial cells. Figure 5 B shows the results of corneal endothelial gene heat map. The gene expression pattern of the differentiated corneal endothelial cells at D23 is more similar to that of the primary corneal endothelial cells. And from the RNA-seq data, we also found new markers of corneal endothelial cells DCN, LUM, SPARCL1, SERPINF1, SERPING1, CXCL3, AEBP1, RARRES2, PCOLCE, HTRA1, etc. (see Table 1).

[0132] DCN LUM SPARCL1 SERPINF1 SERPING1 CXCL3 AEBP1 RARRES2 PCOLCE HTRA1 FTL ANGPTL7 LOX PMP22 VMO1 EMP3 ARNT SDC2 COLEC12 ANXA5 JUN SQSTM1 CTSZ PLPP3 CTSL LMNA GADD45A ABCA6 TSC22D1 TIMP3

[0133] Table 1 Marker of corneal endothelial cells obtained according to the method of the present application

[0134] Example 2 Effect of additives in culture medium on differentiation of corneal endothelial cells

[0135] 1. Effect of SU-5402 on differentiation of corneal endothelial cells

[0136] According to the method in Reference Example 1, ESCs were induced and cultured. The concentration of SU-5402 in the medium at D11-D23 was adjusted to evaluate its effect on the differentiation of corneal endothelial cells, and the results are shown in Figure 6 .

[0137] The results show that the proportion of marker Na+ / K+ ATPase expression is high in the corneal endothelial cell differentiation system with the addition of SU-5402, while the corneal endothelial cells without the addition of SU-5402 do not express Na+ / K+ ATPase( Figure 6 A).

[0138] The relative expression amount of specific genes (Figure 6B) is the highest, and the proportion of marker Na+ / K+ ATPase expression is the highest, and PITX2 appears to be expressed( Figure 6 D) when the corneal endothelial cells are differentiated using 100 nM SU-5402. At the same time, the q-PCR results also prove that the relative expression amount of specific genes is not significantly different compared with 100 nM SU-5402( Figure 6 C) when the corneal endothelial cells are differentiated using high concentration (10 μM) SU-5402.

[0139] 2. Effect of TGFβ2 on the differentiation efficiency of corneal endothelial cells.

[0140] The ESCs were induced and cultured according to the method in Example 1. The concentration of TGFβ2 in the D6-D10 culture medium was adjusted to evaluate its effect on the differentiation efficiency of corneal endothelial cells, and the results are shown in Table 1. Figure 7 .

[0141] The results show that, during the neural crest differentiation stage, the corneal endothelial cells were differentiated after the neural crest cells were treated with different concentrations of TGFβ2 during D6-D10. The q-PCR results show that, when the neural crest cells were treated with 10 ng / ml of TGFβ2, the relative expression of corneal endothelial cell-specific genes was high.

[0142]

[0143] Table 2 Summary of compositions used for cell induction and differentiation in the “two-stage method”

[0144] Example 3 Experimental results of corneal endothelial cells treating corneal endotheliopathy

[0145] To evaluate the in vivo function of the corneal endothelial cells, rabbits were selected as animal models, and after the corneal endothelial cells of the rabbits were mechanically scraped off, the differentiated corneal endothelial cells were labeled with DiI (a red cell membrane probe) and transplanted into the eyes of the rabbits. On D7 after the operation, it was observed that the corneas of the transplanted group began to recover transparency, and on D28, the corneas of the transplanted group recovered transparency significantly, while the corneas of the control group remained severely turbid (Fig. 8A). At the same time, the corneal thickness and corneal transparency of the animals in the two groups were statistically analyzed at different time points, and the results show that, on D21 after the operation, the corneal thickness of the transplanted group was significantly different from that of the control group, and the corneal transparency of the transplanted group was always higher than that of the control group Figure 8 B). On D28 after the operation, the corneas of the animals in the two groups were examined, and the results of slit lamp observation (SL) show that the corneal transparency of the transplanted group was higher than that of the control group; the results of corneal endothelial confocal (HRT3) show that a large number of corneal endothelial cells were observed in the transplanted group, while no corneal endothelial cells were observed in the control group; the results of optical coherence tomography (OCT) show that the corneal thickness of the transplanted group was significantly lower than that of the control group Figure 8 C). After the in vivo observation, the corneas of the animals in the transplanted group and the control group were taken, and after the frozen sections were prepared, immunofluorescence staining was performed to identify the results, which show that the corneal endothelial cell marker (ZO-1), the cell membrane marker (DiI), and the human cell marker (Stem121) were co-expressed Figure 8 D). This indicates that the corneal endothelial cells have a certain therapeutic effect on corneal endotheliopathy.

[0146] Although the embodiments of the present application have been described above with reference to the accompanying drawings, the present application is not limited to the above-described specific embodiments and application fields, and the above-described specific embodiments are merely illustrative and instructive, but are not restrictive. Those skilled in the art can make various forms under the guidance of the present specification and without departing from the scope of the claims of the present application, and these all belong to the protection of the present application.

Claims

1. A method of differentiating pluripotent stem cells into corneal endothelial cells (CECs) by in vitro culture, wherein, The method is a two-stage culture method, first differentiating pluripotent stem cells into neural crest cells (NCC) in a first stage, and then differentiating neural crest cells (NCC) into corneal endothelial cells (CEC) in a second stage; In the first stage, the pluripotent stem cells are cultured using a medium comprising SB431542, CHIR99021 and TGFβ2, the medium further comprising: a) a basal medium, and b) glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine; the SB431542 is at a concentration of 1 μM-100 mM in the medium, the CHIR99021 is at a concentration of 10 nM-100 μM in the medium, and the TGFβ2 is at a concentration of 0.1 ng / ml-100 μg / ml in the medium; In the second stage, the neural crest cells are cultured using a medium comprising SB431542, a DKK2 polypeptide, SU-5402 and Y-27632, the medium further comprising: a) a basal medium, and b) a serum substitute, glutamine or a stabilizing dipeptide of L-alanyl-L-glutamine, an insulin-transferrin-selenium supplement (ITS), non-essential amino acids, L-ascorbic acid and a vitamin C cocktail (L-AA); the SB431542 is at a concentration of 1 μM-100 mM in the medium, the DKK2 polypeptide is at a concentration of 1 ng / ml-100 μg / ml in the medium, the SU-5402 is at a concentration of 10 nM-10 μM in the medium, and the Y-27632 is at a concentration of 10 nM-100 μM in the medium; In the first stage, the TGFβ2 is added after day 6.

2. The method of claim 1, wherein, The serum substitute is one or more selected from the group consisting of KOSR, MSC serum-free supplement, Ultroser™ G.

3. The method of claim 1 or 2, wherein, The non-essential amino acids are one or more selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartate, L-glutamate, L-proline, L-serine.

4. The method of any one of claims 1-3, wherein, The basal medium is one or more selected from the group consisting of KO-DMEM, KO-DMEM / F12, DMEM, α-MEM, F-12, MEM, BME, RPMI 1640, G-MEM, Essential 6.

5. The method of any one of claims 1-4, wherein, The serum substitute is at a concentration of 1%-50% of the basal medium; the glutamine or stabilizing dipeptide of L-alanyl-L-glutamine is at a concentration of 1%-20% of the basal medium; the ITS is at a concentration of 1%-20% of the basal medium; the non-essential amino acids are at a concentration of 1%-20% of the basal medium; and the L-AA is at a concentration of 0.1%-10% of the basal medium.

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