Method for obtaining or maintaining ABCG2-positive limbal corneal stem cells.

Abstract

This invention provides ABCG2-positive limbal corneal stem cells and methods for their production and maintenance. [Solution] The present invention provides a method for producing ABCG2-positive limbal stem cells by first inducing pluripotent stem cells into ophthalmic progenitor cells, and then differentiating these ophthalmic progenitor cells into ABCG2-positive limbal stem cells. The present invention also provides a method for maintaining the ABCG2-positive phenotype of limbal stem cells, such as primary limbal stem cells.

Description

Technical Field

[0001] This specification relates to the differentiation of pluripotent stem cells into ocular progenitor cells and further into ABCG2-positive limbal stem cells. These cells may be further differentiated, as desired, into more proliferative ΔNp63α-positive corneal limbal progenitor cells and ultimately into corneal epithelial cells. The present invention also relates to a method for maintaining the ABCG2-positive phenotype of limbal stem cells such as primary limbal stem cells or limbal stem cells obtained by the differentiation method.

Background Art

[0002] The human cornea is a multilayered, transparent connective tissue that functions as a protective barrier at the front of the eye and provides us with a clear field of vision. The outermost corneal epithelium has a rapid renewal cycle, which manifests through continuous cell loss and replacement on the eye surface. What enables this epithelial layer turnover is the limbal stem cell (LSC) that exists in a specific anatomical niche structure called the Palisades of Vogt and supplies new cells to the epithelium. Dysfunction or deficiency of LSC causes a clinical condition called "limbal stem cell deficiency (LSCD)", which may ultimately lead to corneal blindness. Unfortunately, due to the worldwide shortage of suitable donor tissue, the efficient treatment of LSCD by LSC transplantation is hampered. Therefore, several protocols have been developed aiming to provide an inexhaustible alternative cell source by differentiating LSC from human pluripotent stem cells (hPSCs) including human embryonic pluripotent stem cells and induced pluripotent stem cells (hESCs and hiPSCs, respectively). Importantly, although many laboratories still utilize animal-derived components, which hinder the clinical translation of these methods, some clinically relevant, feeder cell-free technologies have also been introduced (Hongisto et al., 2017 Stem Cells Res Ther; Mikhailova et al., 2014 Stem Cell Rep).

[0003] In addition to the wide variety of techniques, another important challenge in this field is identifying clinically relevant LSC populations. While several proteins are associated with cells suspected to be LSCs in the limbal basal epithelium of the cornea, no specific markers have been found to clearly identify these cells. p63α expression has been associated with successful clinical transplantation and is currently considered the most promising marker for indicating clinical relevance (Rama et al., 2010, N Engl J Med, 363:147-55).

[0004] International Publication No. 2018 / 037161 and Hongisto et al., 2017 Stem Cells Res Ther disclose a method for differentiating human pluripotent stem cells obtained from feeder-free culture into ophthalmic progenitor cells, and then into p63-positive limbal epithelial progenitor cells. This method includes an induction phase in which pluripotent stem cells are first cultured in the presence of a TGF-β inhibitor and fibroblast growth factor (FGF), and then cultured in the presence of bone morphogenetic protein 4 (BMP-4). Next, the ophthalmic progenitor cells thus obtained are differentiated into p63-positive corneal epithelial progenitor cells using a cell medium containing one or more supplements selected from the group consisting of epidermal growth factor (EGF), hydrocortisone, insulin, isoproterenol, and triiodothyronine. Optionally, the p63-positive corneal epithelial progenitor cells are then matured into mature corneal epithelial cells or corneal stratified epithelium.

[0005] In the cornea, expression of ATP-binding cassette subfamily G member 2 (ABCG2) is present only in the limbal basal layer and has been associated with a phenotype of immature, quiescent LSC subpopulations (SPs) that possess the ability to excrete Hoechst 33342 pigment. When cultured, these cells are more activated and exhibit greater growth potential compared to ABCG2-negative cells (De Paiva et al., 2007, Stem Cells).

[0006] Further understanding of the differentiation hierarchy and functional roles of the diverse proteins expressed in limbal basal epithelium is expected to significantly improve both the efficacy and safety of future LSC transplantation. Currently, LSC identification relies on the co-expression of several positive markers, combined with the non-expression of cytokeratin (CK)3 and cytokeratin 12, markers of terminally differentiated corneal epithelium (CE) (Schloetzer-Schrehardt and Kruse 2005 Exp Eye Res). The interrelationships of these markers and their precise functional positions are largely unknown, with the exception of a few studies.

[0007] Nevertheless, ABCG2 is recognized as a promising marker for the phenotype of potent stem cells in vivo, and therefore could be used to identify clinically relevant LSCs. Thus, there is a need for methods to obtain ABCG2-positive LSCs that may be used for the treatment and study of corneal epithelial conditions, diseases, and pathologies. In addition, such cells may be used in toxicological studies and drug development, particularly when stem cells are obtained from cell cultures lacking feeder cells. Furthermore, avoiding heterologous or undefined components is also an important objective to improve the potential clinical translation safety of these methods. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2018 / 037161 Brochure [Non-patent literature]

[0009] [Non-Patent Document 1] Hongisto et al., 2017 Stem Cells Res Ther [Non-Patent Document 2] Mikhailova et al., 2014 Stem Cell Rep [Non-Patent Document 3] Rama et al., 2010, N Engl J Med, 363:147-55 [Non-Patent Document 4] De Paiva et al., 2007, Stem Cells [Non-Patent Document 5] Schloetzer-Schrehardt and Kruse 2005 Exp Eye Res [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide ABCG2-positive limbal stem cells and methods for their production and maintenance, in order to overcome the challenges associated with the readily lost expression of ABCG2 under cell culture conditions. The object of the present invention is achieved by a method characterized by the content described in the independent claim. Preferred embodiments of the present invention are disclosed in the dependent claims. [Means for solving the problem]

[0011] Accordingly, the present invention provides a method for maintaining the ABCG2-positive phenotype of limbal stem cells, comprising the step of culturing ABCG2-positive limbal stem cells in a culture medium containing EGF and at least one Wnt activator. In some embodiments, the Wnt activator is selected from the group consisting of GSK3 inhibitors, preferably CHIR99021, and proteins of the R-spondin family, preferably R-spondin-1 or its supplement RS-246204. In some further embodiments, the culture medium may contain noggin or its supplement, for example LDN-193189.

[0012] In addition, the present invention also provides a method for producing ABCG2-positive limbal stem cells, which includes the following: A method for producing ABCG2-positive limbal stem cells, comprising: a) providing pluripotent stem cells; b) culturing the cells in a cell culture medium containing a TGF-β inhibitor and fibroblast growth factor (FGF), preferably basic FGF; c) removing the TGF-β inhibitor and FGF, culturing the cells obtained in step b) in a cell culture medium containing bone morphogenetic protein 4 (BMP-4) to generate ophthalmic progenitor cells; d) culturing the ophthalmic progenitor cells in a corneal differentiation medium to generate ABCG2-positive limbal stem cells; and e) culturing the ABCG2-positive limbal stem cells in a maintenance cell culture medium containing EGF and at least one Wnt activator to maintain the phenotype of the ABCG2-positive limbal stem cells obtained through steps a to d). In some embodiments, the Wnt activator is selected from the group consisting of GSK3 inhibitors, preferably CHIR99021, and proteins of the R-spongin family, preferably R-spongin g-1 or its supplement RS-246204. In some further embodiments, the culture medium may also include noggin or its supplement, such as LDN-193189.

[0013] Further aspects, specific embodiments, objectives, details, and advantages of the present invention are described below in the drawings, detailed description, and examples. [Brief explanation of the drawing]

[0014] The present invention will be described in more detail below with reference to the attached drawings, based on preferred embodiments.

[0015] [Figure 1] Schematic diagram of the method of the present invention. Figure 1A shows one embodiment of a method for producing ABCG2-positive limbal stem cells from pluripotent stem cells. Figure 1B shows one embodiment of a method for maintaining the ABCG2-positive phenotype of primary limbal stem cells. [Figure 2]Characterization of presumptive LSC marker expression during hPSC-LSC differentiation. (Figure 2A) Representative morphology and protein expression of hPSC-LSC cultures at selected time points, shown in IF. A scale bar of 100 μm is applied to all images in the same column. Cell nuclei are counterstained with DAPI and are shown in the upper right corner of each panel. (Figure 2B) Differences in marker expression in hPSC-LSC populations at day 10 and day 24. Five images per sample and a minimum of 1400 cells at each time point were analyzed for each marker from cytospin samples. Data are shown as the mean + SD of individual cell differentiations (n=2-3). (Figure 2C) Representative IF image of double staining for ΔNp63 and p63α in a cytospin sample at day 24, confirming the ΔNp63α-positive phenotype. A scale bar of 100 μm is applied to both C and D. (Figure 2D) Representative IF image of double staining for ABCG2 and p63α in a cytospin sample at day 10, showing the co-localization pattern. (Figure 2E) Expression and co-localization of p63α and ABCG2 in hPSC-LSCs at days 10 and 24, analyzed from cytospin samples. Five images per sample and a minimum of 3000 cells at each time point were analyzed from double-stained cytospin samples. Data are shown as the mean + SD of individual cell differentiation for n=3. (Figure 2F) Expression levels of ABCG2 protein in UD-hPSCs, hPSC-LSCs at days 10 and 24, analyzed using FACS from 10,000 recorded events in each sample. Data are shown as the mean + SD of individual cell differentiation for n≧3, and statistics were calculated using the Mann-Whitney U test. (Figure 2G) Relative ABCG2 mRNA expression levels in UD-hPSCs, hPSC-LESCs at day 10 and day 24, analyzed by qRT-PCR using three technical replicates for each sample. Data are shown as the mean + SD of individual cell differentiation for n=2. All representative data are shown for the hESC strain Regea08 / 017. [Figure 3]Effect of culture conditions on the morphology of hPSC-LSCs and the expression of p63α / ABCG2. Representative cell morphologies and the expression of p63α and ABCG2 proteins on day 21 (out of the total culture time) during continuous culture under corneal differentiation (CnT-30) conditions and ABCG2-positive LSC maintenance (Cnt-07+ENRC) conditions. (Figure 3A) The expression patterns of P63α and ABCG2 were maintained even after subculturing the cells under Cnt-07+ENRC conditions. (Figure 3B) Relative ABCG2 mRNA expression of hPSC-LSCs on day 21 (out of the total culture time) during continuous culture under corneal differentiation (CnT-30) conditions and ABCG2-positive LSC maintenance (Cnt-07+ENRC) conditions. (Figure 3C) All representative data are shown for the hESC line Regea08 / 017. The scale bar for all images is 100 μm. [Figure 4] Marker expression and proliferation ability of ABCG2-positive hPSC-LSCs during subculture. (Figure 4A) Human PSC-LSC colonies maintained their morphology and p63α / ABCG2 expression patterns even after subculture under CnT-07+ENRC conditions. The black scale bar is 200 μm and the white scale bar is 100 μm. As can also be seen from the stable population doubling time, population doublings of 20 and 20 were achieved during 5 subcultures, and no clear signs of culture exhaustion were observed (Figure 4B - Figure 4C, black dots). Cryopreservation of the cells at passage 3 also did not seem to affect the proliferation ability of hPSC-LSCs (Figure 4B - Figure 4C, white frames). All representative data are shown for the hESC line Regea11 / 013.

Mode for Carrying Out the Invention

[0016] The present invention provides a method for producing ABCG2-positive limbal stem cells by first inducing pluripotent stem cells into ocular progenitor cells (induction stage), then differentiating these ocular progenitor cells into ABCG2-positive limbal ring region stem cells (corneal epithelial stem cells) (differentiation stage), and then maintaining the expression of ABCG2 (maintenance stage). The present invention also provides a method for maintaining the ABCG2-positive phenotype of limbal stem cells, including primary limbal stem cells and limbal stem cells derived from pluripotent stem cells. Furthermore, the present invention relates to the therapeutic use of ABCG2-positive cells obtained or maintained according to the present invention.

[0017] cell As used herein, the term "limbal stem cell" refers to adult stem cells located in the basal epithelial layer of the limbal region that are responsible for the regeneration of the corneal epithelium. This term is interchangeable with the terms "limbal stem cell (LSC)", "limbal epithelial stem cell (LESC)", "corneal epithelial stem cell", and "corneal epithelial progenitor cell". As used herein, the terms "precursor" and "progenitor cell" may be used interchangeably unless otherwise specified.

[0018] As used herein, the term "primary limbal stem cell" refers to limbal stem cells directly collected from biological tissues. Means and methods for obtaining primary limbal stem cells are readily available in the art.

[0019] As used herein, the term "limbal stem cell derived from pluripotent stem cells" refers to limbal stem cells derived from pluripotent stem cells. In some embodiments, the present invention provides a method for producing ABCG2-positive limbal stem cells by first inducing pluripotent stem cells into ocular progenitor cells (induction stage), then further differentiating these ocular progenitor cells into highly proliferative ΔNp63α-positive limbal epithelial progenitor cells as desired, and finally differentiating them into ABCG2-positive limbal stem cells that can be further differentiated into mature corneal epithelial cells (differentiation stage).

[0020] As used herein, the term “pluripotent stem cell” refers to any stem cell that has the potential to differentiate into all cell types of the human or animal body, excluding extraembryonic tissues. These stem cells include both embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs). Accordingly, cells suitable for use in the present invention include stem cells selected from iPSCs and ESCs. Human pluripotent stem cells (hPSCs) are preferred, and these include human iPSCs (hiPSCs) and human ESCs (hESCs).

[0021] ESCs, particularly hESCs, are of great therapeutic interest because they can proliferate indefinitely during culture and therefore can supply cells and tissues that can replace faulty or defective human tissue. However, producing ophthalmic progenitor cells from human embryonic stem cells may face ethical challenges. According to one embodiment of the present invention, human embryonic stem cells may be used, provided that the method itself or any related actions do not involve the destruction of human embryos.

[0022] Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs, are a type of pluripotent stem cell artificially induced from non-pluripotent cells, typically adult somatic cells, by inducing the forced expression of specific genes using means and methods well known in the art. The advantage of using iPS cells is that ethical issues can be avoided because there is no need to use embryonic cells. A further advantage is that employing iPSC technology makes it possible to generate patient-specific cells without the problem of immune rejection. Therefore, according to one embodiment of the present invention, the use of iPS cells is preferred. For clinical applications, hiPS cells are preferred.

[0023] Induced pluripotent stem cells (IPS cells) are similar in many respects to naturally occurring pluripotent stem cells, such as embryonic stem cells. Specific aspects include the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, efficacy, and differentiation potential; however, the full relationship with naturally occurring pluripotent stem cells is still being evaluated. Induced pluripotent cells are typically derived from adult skin cells, blood cells, stomach cells, or liver cells, but other substitutes are possible. Those skilled in the art are well aware of the potential of iPS cells for research and therapeutic purposes.

[0024] Pluripotent stem cells are difficult to maintain in cell culture because they tend to differentiate spontaneously according to the natural fate of cells. To prevent undesirable differentiation, pluripotent stem cells are usually cultured on feeder cells. Even as feeder cells for human pluripotent cells, animal-derived feeder cells such as mouse embryonic fibroblasts (mouse fetal fibroblasts) (MEFs) are widely used. In addition, as an alternative to animal-derived materials, human-derived feeder cells such as human foreskin feeder cells are also used in the culture of human pluripotent stem cells. Therefore, in some embodiments, pluripotent stem cells cultured on feeder cells, preferably human feeder cells such as human foreskin feeder cells, may be used in the method for producing ABCG2-positive limbal stem cells.

[0025] To overcome the drawbacks associated with the use of feeder cells, new feeder-free cell culture methods have been developed. Therefore, the term "feeder-free culture," or its linguistic variations, refers to the cultivation of pluripotent stem cells without the use of any feeder cells.

[0026] The use of feeder cells can be omitted, for example, by replacing them with a suitable substrate coating, as is well known in the art. Suitable coating materials include, but are not limited to, human or animal extracellular matrix (ECM) proteins, such as laminin, collagen, vitronectin, fibronectin, nidogen, proteoglycans, and E-cadherins, as well as their isoforms, fragments, and peptide sequences. Non-limiting examples of ECM protein isoforms include different isoforms of laminin, such as laminin-511, laminin-521, laminin-322, and laminin-411. Non-limiting examples of fragments include the E8 fragment of the human laminin isoform, while non-limiting examples of peptide sequences include the Arg-Gly-Asp (RGD) sequence of vitronectin. The above-mentioned ECM proteins, isoforms, fragments, or peptide sequences may be fused with other proteins, and an N-cadherin domain fused with an IgG-Fc domain is a non-limiting example of such a fusion protein. Suitable alternative or additional coating materials include, but are not limited to, ECM or basement membrane extracts from various tissue or cell types of human or animal origin, such as mouse embryonic fibroblasts, human fibroblasts, mesenchymal stem cells, and tumors. Further suitable coating materials include natural or synthetic biomaterials and hybrids thereof, either alone or functionalized with ECM proteins or their sequences, or with other chemical or physical surface modifications. Non-limiting examples of such biomaterials include PMEDSAH (poly[2-(methacryloyloxy)ethyl-dimethyl-(3-sulfopropyl)ammonium hydroxide]) and collagen graft mixed cellulose ester membranes (MCE-COL).Further, non-limiting examples of suitable coating materials include commercial products such as Corning® Synthemax® surfaces, Corning® PureCoat®, CELLstart®, Matrigel®, or Geltrex®. Any of the aforementioned proteins, isoforms, fragments, peptide sequences, fusion proteins, extracts, biomaterials, or commercial products may be used alone or in any suitable combination or mixture to replace feeder cells, as is well known in the art. Means and methods for obtaining, selecting, and using suitable coating materials to replace feeder cells are readily available in the art.

[0027] Accordingly, pluripotent stem cells cultured under feeder-free conditions may be used in some embodiments of the method for producing ABCG2-positive limbal stem cells.

[0028] Preferred extracellular matrix proteins include laminins, which are heterotrimeric glycoproteins containing α, β, and γ chains, with five, four, and three gene variants, respectively. Laminin molecules are named according to their chain composition. Thus, as used herein, the term "laminin-521" refers to laminin containing α5, β2, and γ1 chains, while the term "laminin-511" refers to laminin containing α5, β1, and γ1 chains. Preferred laminins include recombinant human laminin-521, recombinant human laminin-511, and recombinant human laminin-511 fragment E8.

[0029] Human pluripotent stem cells (PPTs) grow as colonies on feeder cells, but in feeder-free culture on laminin 521, they grow as monolayers, allowing for more efficient proliferation. Furthermore, flow cytometry analysis shows that PPTs cultured in a feeder-free culture system exhibit a higher positive rate for pluripotency markers compared to cells cultured on feeder cells.

[0030] In fact, human pluripotent stem cells obtained from cultures containing feeder cells appear to be different from those obtained from feeder-free cultures. As demonstrated in the experimental section of International Publication No. 2018 / 037161, the corneal differentiation method disclosed in European Patent Application Publication No. 2828380 yields good results for human pluripotent stem cells obtained from cultures containing feeder cells, but does not yield similarly good results for pluripotent stem cells obtained from feeder-free cultures. On the other hand, the method in International Publication No. 2018 / 037161 allows for the excellent differentiation of human pluripotent stem cells obtained from feeder-free cultures, first into ophthalmic progenitor cells, and then into p63-positive corneal epithelial progenitor cells (i.e., limbal stem cells). Hereinafter, the present invention provides excellent maintenance of ABCG2-positive limbal stem cells. Thus, in some embodiments of the present invention, undifferentiated pluripotent stem cells obtained from feeder-free cultures are used to produce ABCG2-positive limbal stem cells. However, the present invention is not limited to the differentiation of human pluripotent stem cells obtained from feeder-free culture, and may also be used to differentiate pluripotent stem cells cultured on feeder cells into ABCG2-positive limbal stem cells.

[0031] As used herein, the term “ocular progenitor cells” broadly refers to any cell lineage of the eye that is derived from pluripotent stem cells and is characterized by downregulation of the pluripotency marker OCT-4 (also known as POU5F1) and upregulation of PAX6, a gene that exhibits differentiation into eye-specific cell lineages. Means and methods for quantifying pluripotency markers are readily available in the art.

[0032] As used herein, the term “p63-positive limbal stem cells” refers to a population of limbal stem cells that express p63 on their cell surface.

[0033] As used herein, the term “ABCG2-positive limbal stem cells” refers to a population of limbal stem cells that express ATP-binding cassette subfamily G member 2 (ABCG2) on their cell surface.

[0034] Cell surface markers such as ABCG2 and p63 may be quantified by any available technique suitable for this purpose, including but not limited to immunofluorescence and flow cytometry, such as fluorescence-activated cell sorting (FACS).

[0035] Method for producing ABCG2-positive limbal stem cells International Publication No. 2018 / 037161 discloses a method for producing differentiated ophthalmic cells by first inducing pluripotent stem cells obtained from feeder-free culture into ophthalmic progenitor cells in the presence of a TGF-β inhibitor and fibroblast growth factor (FGF), and then in the presence of bone morphogenetic protein 4 (BMP-4). The ophthalmic progenitor cells thus obtained are then differentiated into p63-positive corneal epithelial progenitor cells (p63-positive limbal stem cells) by removing the TGF-β inhibitor, FGF, and BMP-4, and culturing the ophthalmic progenitor cells in the presence of one or more supplements selected from the group consisting of epidermal growth factor (EGF), hydrocortisone, insulin, isoproterenol, and triiodothyronine. Optionally, these p63-positive corneal epithelial progenitor cells can then be matured into mature corneal epithelial cells or corneal stratified epithelium.

[0036] Surprisingly, careful analysis of the expression patterns of several pluripotency markers, limbal stem cell markers, and corneal epithelial markers at different time points using the methodology described in International Publication No. 2018 / 037161 revealed that ABCG2 was expressed only transiently, peaking at days 10-11, and then gradually decreasing to very low levels by day 21 of the differentiation protocol (including a 4-day induction period). Simultaneously, the expression of the p63 ΔNp63α isoform steadily increased. In addition, it was unexpectedly found that strong ABCG2 expression could be maintained for at least 35 days without passaging, and for at least 50 days with passaging of subconfluent cultures, by supplementing the culture medium with at least epidermal growth factor (EGF) and at least one Wnt activator.

[0037] Thus, the present invention provides a method for producing and maintaining ABCG2-positive limbal stem cells, the method comprising an induction phase, a differentiation phase, and a maintenance phase, as will be described in detail below and summarized in Table 1.

[0038] [Table 1]

[0039] lag period During the induction phase, the obtained pluripotent stem cells are induced into surface ectoderm and ocular progenitor cells.

[0040] The induction phase may be carried out in either suspension culture or adherent culture. When the induction phase is carried out in adherent culture, it may be advantageous to use a substrate coated with materials such as extracellular matrix (ECM) proteins or combinations thereof, as is generally known in the art. Preferred ECM proteins include, but are not limited to, laminin, collagen, vitronectin, fibronectin, idogen, and proteoglycans or their peptide sequences. Furthermore, the induction phase may be carried out in adherent culture using any coating suitable for replacing feeder cells.

[0041] In some embodiments, it is preferable to carry out the induction phase in suspension culture. In such embodiments, the first step of the induction phase includes forming embryoid bodies from pluripotent stem cells obtained from feeder-free culture. As used herein, the term “embryoid body” (EB) refers to a three-dimensional cell aggregate. Embryoid body formation can be achieved by various aggregation-promoting methods known in the art.

[0042] As used herein, the term “aggregation-promoting method” refers to any method that can promote the formation of embryoid bodies from pluripotent stem cells by physical or chemical means.

[0043] Embryoid body formation can be achieved, for example, by employing a physical aggregation-promoting method in which pluripotent stem cells are cultured in suspension culture in the presence of a non-adhesion-promoting cell culture surface such as Corning Corning® Costar® ultra-low adhesion surface, or in the presence of one or more agents that prevent cell adhesion.

[0044] A further non-limiting example of a suitable physical method to promote aggregation in order to achieve EB formation is suspension culture. In this method, a suspension containing pluripotent stem cells is plated in a regular arrangement on the lid of a petri dish, and then the inverted lid is placed on the bottom of a petri dish filled with a suitable liquid such as PBS to prevent the droplets from drying out. Ultimately, the stem cells fall to the bottom of the hanging droplets and aggregate into one EB per drop. Commercial products based on the suspension method, such as Perfecta3D® Hanging Drop Plates (3D Biomatrix), may also be used.

[0045] Appropriate physical aggregation-promoting methods include using multiwell and microfabrication techniques to induce the formation of EBs of controlled size, for example, using low-adhesion 96-well plates or microwell arrays. Non-limiting examples of such plates or arrays include microwell-equipped poly(dimethylsiloxane) (PDMS) molds with patterned microwells, agarose hydrogel microwell arrays, and commercially available AggreWell® (STEMCELL Technologies) microarray plates.

[0046] Furthermore, as a physical method to promote aggregation in order to obtain EB formation, forced aggregation by centrifugation, rotation, or other physical environments such as a microgravity environment may be employed.

[0047] A further preferred method for promoting aggregation involves culturing cells in the presence of a drug that aids in cell aggregation. Non-exclusive examples of such drugs include high molecular weight crowders (high molecular weight intermingling molecules) that bring cells into closer contact, such as galactose derivatives (e.g., carrageenan), glucose derivatives (e.g., dextran sulfate), polyethylene glycol, and Ficoll®.

[0048] Alternatively, a physical aggregation-promoting method may be used, in which pluripotent stem cells are encapsulated or captured in a hydrogel such as methylcellulose, fibrin, hyaluronic acid, dextran, alginic acid, or agarose, thereby generating individually separated EBs in a semi-solid suspension medium.

[0049] Any suitable physical aggregation-promoting method may be used in combination with one or more aggregation-promoting chemical agents, such as brevistatin or Rho-related kinase (ROCK) inhibitors, or other apoptosis inhibitors that increase single-cell viability and / or promote stem cell aggregation. Alternatively, cell aggregation may be promoted solely by chemical means, for example, by using the above-mentioned chemical agents.

[0050] Conventionally, embryoid bodies may also be formed from pluripotent stem cells by manually separating attached colonies or regions of attached colonies. However, in some embodiments of the method of the present invention, manual separation is not a viable option for obtaining embryoid bodies because pluripotent stem cells cultured under feeder-free conditions do not spontaneously re-aggregate after being manually dissociated into small aggregates or single cells. Adding one or more aggregation promoters, such as ROCK inhibitors or brevistatin, significantly reduces apoptosis induced by dissociation, allowing embryoid body formation after dissociation.

[0051] The time required for embryoid body formation can vary depending on various factors, such as the method used. Typically, the duration of this process is between approximately 1 hour and 48 hours, more specifically between approximately 5 hours and 24 hours, or overnight, i.e., approximately 18 hours. However, in some cases, the duration of this process may be longer than 48 hours. In some embodiments, pluripotent stem cells are cultured in the presence of brevistatin for approximately 1 day for embryoid body formation.

[0052] In the second step of the induction phase, embryoid bodies obtained from the first step of the induction phase are subjected to induction medium containing an active "induction supplement," namely a TGF-β inhibitor and FGF. If adherent cells are used (i.e., the step of forming embryoid bodies is omitted), the subjecting of the cells to the induction medium may be considered the first step of the induction phase. The above induction supplement has been shown to enhance the induction of pluripotent stem cells into ophthalmic progenitor cells and improve the efficiency of further differentiation into clinically valuable limbal stem cells.

[0053] In some preferred embodiments, the amount of TGF-β inhibitor in the induction medium is about 1 μM to about 100 μM, preferably about 1 to about 30 μM, and / or the amount of fibroblast growth factor is about 1 ng / ml to about 1000 ng / ml, preferably about 2 ng / ml to about 100 ng / ml, more preferably about 30 ng / ml to about 80 ng / ml.

[0054] The induction medium of the present invention may be considered to consist of a basal medium and the induction supplement of the present invention, or to include a basal medium and the induction supplement of the present invention. However, further supplements common in the art may be applied. As used herein, the term “common cell culture supplements” refers to materials used in substantially all cell culture media, including antibiotics, L-glutamine, and serum, serum albumin, or serum substitutes, preferably specified (limited) serum substitutes.

[0055] On the other hand, in some embodiments, the induction medium does not contain any materials other than the induction supplement, basal medium, antibiotic, L-glutamine, and specified serum substitute.

[0056] In some more specific embodiments, a TGF-β inhibitor of formula I or formula II and bFGF are used as the inducing supplement. In some even more specific embodiments, the inducing supplement is SB-505124 and bFGF. In some even more specific embodiments, SB-505124 is used at a concentration of about 10 μM and bFGF is used at a concentration of about 50 ng / ml.

[0057] Any of the embodiments described above may form the basis for additional or alternative embodiments in which the induction medium does not include any supplements that are generally known to induce differentiation into lineages other than the oculobular lineage, including neural differentiation. Such generally known supplements include, but are not limited to, retinoic acid, ascorbic acid, brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF). In some preferred embodiments, the induction medium does not contain a Wnt inhibitor.

[0058] The time used to induce adherent cells or embryoid bodies with induction supplements can vary depending on various factors such as the cell line, the initial differentiation state of the cells, and the specific supplement used and its concentration. Typically, the duration of this process ranges from about 1 to 7 days (i.e., about 22 to 185 hours), more specifically, from about 1 to 5 days (i.e., about 22 to 132 hours). In some embodiments, embryoid bodies are cultured in the presence of induction supplements for about 1 day (i.e., about 22 to 26 hours).

[0059] In the next step after the induction phase, the first induction supplement (TGF-β inhibitor and FGF) is removed, and the adherent cells or embryoid bodies obtained from the previous step are cultured in the presence of bone morphogenetic protein 4 (BMP-4) to guide the cells to the surface ectoderm and simultaneously prevent differentiation into the nervous system. Typical concentrations range from approximately 1 ng / ml to approximately 1000 ng / ml, preferably approximately 10 ng / ml to approximately 50 ng / ml, and more preferably approximately 25 ng / ml.

[0060] The time used to induce adherent cells or embryoid bodies using BMP-4 may vary depending on various factors such as the concentration of BMP-4. Typically, the duration of this process varies from about 12 hours to about 5 days (i.e., about 12 hours to about 132 hours), preferably from about 24 hours to about 4 days (i.e., about 24 hours to about 105 hours), and more specifically from 2 days (i.e., about 43 hours to about 53 hours), and this time may or may not include changing the culture medium with fresh medium. In some embodiments, this process is carried out for about 2 days, preferably first for about 1 day in medium supplemented with a preferably about 25 ng / ml amount of BMP-4, and then for a further 1 day in fresh medium also preferably supplemented with about 25 ng / ml amount of BMP-4.

[0061] The overall duration of the induction phase may vary depending on various factors. Typically, the duration of the induction phase can range from about two days to several days. A preferred time range is about 2.5 days to about 18 days, i.e., about 54 hours to about 475 hours. In some embodiments, the preferred overall duration of the induction phase is about 3 days to about 7 days (i.e., about 65 hours to about 185 hours), or about 3 days to about 5 days (i.e., about 65 hours to about 132 hours). More specifically, the preferred overall duration of the induction phase is four days, including any one day of embryoid body formation. As used herein, the term "about" means a variation of about 10% of the specified value. Thus, for example, the term "about 3 days" has a variation of 65 hours to 79 hours, while the term "about 7 days" has a variation of 152 hours to 186 hours. Short induction times can lead to weakened downregulation of OCT4 and upregulation of PAX6, potentially resulting in less efficient differentiation, as evidenced by the reduced expression of precursor markers such as PAX6 and p63. Furthermore, since human pluripotent stem cells are known to differentiate into the nervous system, particularly in the presence of basic fibroblast growth factor, longer induction times can be expected to result in more neural differentiation.

[0062] Coagulation promoter As used herein, the functional term “aggregation promoter” refers to a drug that can promote the formation of embryoid bodies. Non-limiting examples of aggregation promoters include brevistatin and Rho-related kinase (ROCK) inhibitors, which are disclosed in detail below.

[0063] Brevistatin is a selective and reversible inhibitor of the cell permeability of non-muscular myosin II. Its name derives from its ability to inhibit cellular blebing. The actin-myosin cytoskeleton is a dynamic system essential for cell contraction, motility, and tissue construction. The actin-myosin motor consists of actin filaments and non-muscular myosin II heavy chains that slide along the actin filaments, causing contraction. This process is initiated (triggered) by the binding of myosin light chains, which are activated by kinase-mediated phosphorylation, such as Rho-associated kinase (ROCK). When contact with the ECM and epithelial cells is lost, actin-myosin can contract freely, leading to phenotypic changes such as excessive blebing of the cell membrane and ultimately cell death. Inhibiting actin-myosin contraction in human ES cells, which exhibit individual variability, dramatically improves cell viability and cloning efficiency. Actin-myosin contraction is a downstream target of ROCK regulation. Brevistatin is readily available in this field.

[0064] ROCK inhibitors are highly potent and selective, cell-permeable inhibitors of protein kinases belonging to the Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) family. Non-exclusive examples of ROCK inhibitors include chroman 1, fasudil, fasudil hydrochloride, GSK269962A, GSK429286A, H-1152, H-1152 dihydrochloride, hydroxyfasudil, hydroxyfasudil hydrochloride, K-115, K-115 free base, LX7101, RKI-1447, ROCK inhibitor (azaindole 1), SAR407899, SAR407899 hydrochloride, SLx-2119, SR-3677, thiazovibin, and Y-27632, all of which are available, for example, from MedChem Express.

[0065] A preferred ROCK inhibitor is Y-27632 (dihydrochloride trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), which inhibits both ROCK1 and ROCK2 by competing with ATP for binding to the catalytic site. Furthermore, Y-27632 is a potent inhibitor of apoptosis (anoikis) in pluripotent stem cells, allowing the survival of dissociated human pluripotent stem cells, improving embryoid body formation in forced aggregation protocols, and enhancing the post-thaw viability of cryopreserved single human ES cells.

[0066] Those skilled in the art can easily determine, using various readily available methods in the art, whether a given agent has aggregation-promoting activity and whether it is suitable for use in such methods. Non-limiting examples of such methods include visual evaluation of aggregate formation and cell viability assays.

[0067] TGF-beta (TGF-β) inhibitor As used herein, "TGF-β inhibitor" refers to a substance that can functionally inhibit transforming growth factor β1.

[0068] Transforming growth factor β1 (TGF-β1) is a member of a large superfamily of multifaceted cytokines involved in many biological activities, including growth, differentiation, migration, cell survival, and adhesion, in both diseased and normal states. Nearly 30 members have been identified within this superfamily. These are thought to be classified into two main branches: TGFβ / Activin / Nodal and BMP / GDF (Bone Morphogenetic Protein / Growth and Differentiation Factor). These are highly diverse and often have complementary functions. Some are expressed only for short periods during embryonic development or in only limited cell types (e.g., anti-Müllerian hormone, AMH, inhibin), while others are widely expressed during embryogenesis and in adult tissues (e.g., TGFβ1, BMP4). TGF-β1 is a potent regulator of extracellular matrix (fibrosis factor) synthesis and is also involved in wound healing.

[0069] Appropriate TGF-β inhibitory functions may be found in proteins and small organic compounds, both chemically and structurally. Those skilled in the art know means of isolating proteins from biological matrices or of producing proteins by recombinant techniques.

[0070] Compounds exhibiting TGF-β inhibitory activity may be found by screening. Preferably, TGF-β inhibitors are organic molecules with relatively low molar mass, for example, small molecules with a molar mass of less than 800 g / mol, preferably less than 500 g / mol. A suitable low-molar TGF-β inhibitor with a general structure, formula I, may be described as follows: [ka] In the above formula I, R1 represents a C1-C5 aliphatic alkyl group, a carboxylic acid, or an amide; R2 represents a C1-C5 aliphatic alkyl group; and R3 and R4 represent a heteroatom, an aliphatic alkyl group containing O or N. These may be linked together to form a 5-membered or 6-membered heterocycle.

[0071] A typical structure is one that contains a heterocycle with two oxygen atoms and can be called a low-molecular-weight molecule of general formula II. [ka] In the above formula II, R1 represents a C1-C5 aliphatic alkyl group, an aromatic carboxylic acid, or an amide, and R2 represents a C1-C5 aliphatic alkyl group.

[0072] One non-limiting example of such a TGF-β inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide, also known as SB 431542, which is marketed by multiple suppliers as a selective inhibitor of transforming growth factor-β1 receptor (ALK5), ALK4, and ALK7. Another non-limiting example of a specific TGF inhibitor is 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazole-4-yl)-6-methylpyridine hydrochloride hydrate, also known as SB 505124.

[0073] However, other small molecules that exhibit TGF-β inhibitory activity or are commercially available as TGF inhibitors may also be equally suitable in the context of the present invention. When selecting the above TGF-β inhibitors from substances obtained by chemical synthesis or recombinant production, a standard culture medium can be provided. This also satisfies the requirements of xeno-free and serum-free conditions.

[0074] A person skilled in the art can easily determine, using various readily available methods in the art, whether a given drug has TGF-β inhibitory activity and whether it is suitable for use in such methods.

[0075] Fibroblast growth factor The induction medium of the present invention requires fibroblast growth factor to contribute to differentiation. Fibroblast growth factor, or FGF, is a group of growth factors generally involved in angiogenesis, wound healing, and embryonic development. FGF is a heparin-binding protein, and its interaction with heparan sulfate proteoglycans bound to the cell surface has been shown to be essential for FGF signaling. FGF plays an important role in the proliferation and differentiation processes of various cells and tissues.

[0076] A preferred fibroblast growth factor suitable for use in the present invention is basic FGF (bFGF or FGF-2). However, in some embodiments, other materials may be used instead of FGF, such as specific synthetic small peptides designed to activate the fibroblast growth factor receptor (e.g., produced by recombinant DNA variants or mutants). The fibroblast growth factor may be included in the serum substitute used as the basal medium, or it may be added separately to the final cell culture medium according to the present invention.

[0077] differentiation stage Following the induction phase, which is preferably carried out in suspension culture, a differentiation phase takes place in adherent culture. In the latter stage, the ophthalmic progenitor cells produced in the induction phase are differentiated into ABCG2-positive limbal stem cells.

[0078] The differentiation phase should be performed in adherent culture, and since the ability to adhere to the extracellular matrix (ECM) is considered important for epithelial cells, it is advantageous to use substrates such as cell culture plates or bottles coated with ECM proteins that are generally known in the art. Preferred ECM proteins include collagen IV and laminin, preferably laminin-521 and / or laminin-511. More preferably, the cell culture substrate is coated with a mixture of collagen IV and laminin 521 and / or laminin 511, even more preferably 5 μg / cm³. 2Collagen IV and 0.75 μg / cm³ 2 The substrate is coated with laminin 521. Other suitable coating materials, though not limited to these, include collagen I, vitronectin, fibronectin, nidogen, proteoglycans, or their peptide sequences, commercially available adherent culture substrates containing them, e.g., CELLstart®, and basement membrane extracts such as Matrigel® or Geltrex®. Furthermore, any coating suitable for replacing feeder cells may be used during differentiation. If xeno-free conditions are desired for use in humans, the substrate should be coated with one or more ECM proteins of human or recombinant human origin. Means and methods for coating cell culture substrates are generally available in the art.

[0079] Typically, to obtain ABCG2-positive limbal stem cells, ocular progenitor cells need to be cultured for about 3 to 14 days, preferably about 7 days, under the corneal differentiation conditions of the present invention. In some preferred embodiments, ABCG2-positive limbal stem cells are obtained by performing an induction phase of about 2.5 to 18 days, followed by a corneal differentiation phase of about 3 to 14 days. In some even more preferred embodiments, ABCG2-positive limbal stem cells are obtained by performing an induction phase of about 4 days, followed by a corneal differentiation phase of about 6 to 7 days. After differentiation of this length, the purest ABCG2-positive cell population can be obtained. Shorter differentiation times result in a more heterogeneous cell population, while longer differentiation times result in the simultaneous disappearance of ABCG2 expression and the emergence of p63 expression, followed by final maturation into corneal epithelial cells. Non-limiting examples of markers of mature corneal epithelium include CK12 and CK3.

[0080] As used herein, the term “corneal differentiation medium” refers to a cell culture medium that supports the differentiation of cells into corneal lineages, preferably into ABCG2-positive limbal stem cells. Those skilled in the art can easily determine, using various methods readily available in the art, whether a given cell medium is considered a corneal differentiation medium and, consequently, whether it is suitable for use in this differentiation stage.

[0081] Accordingly, a culture medium suitable for use during the differentiation phase may be any corneal medium, such as CnT-30, commercially available from CELLnTECH, or any supplemental hormonal epithelial medium (SHEM) suitable for culturing corneal epithelial cells. In some other embodiments, the differentiation medium may be composed of adding one or more differentiation supplements selected from the group consisting of epidermal growth factor (EGF), hydrocortisone, insulin, isoproterenol, and triiodothyronine to any suitable basal medium. In some embodiments, the corneal differentiation medium does not contain any materials other than the one or more differentiation and maturation supplements, basal medium, antibiotics, L-glutamine, and specified serum substitutes. That is, in some embodiments, the corneal differentiation medium does not contain any materials such as TGF-β inhibitors, fibroblast growth factor, and BMP-4, or functionally equivalent drugs. In some further embodiments, the corneal differentiation medium does not contain Wnt inhibitors either.

[0082] Any of the embodiments described above may form the basis for additional or alternative embodiments in which the differentiation medium does not include any supplements that are generally known to induce differentiation into cell lineages other than the ocular lineage, including neural differentiation. Such commonly known supplements include, but are not limited to, retinoic acid, ascorbic acid, BDNF, and GDNF.

[0083] maintenance period Following the differentiation phase described above, a maintenance phase is performed using adherent culture. During this stage, the ABCG2-positive phenotype of the limbal stem cells obtained during the differentiation phase is maintained.

[0084] Suitable culture media for use in the maintenance phase may be, for example, any corneal medium or any supplement hormone epithelial medium (SHEM) available in the art and suitable for culturing corneal epithelial cells. Non-limiting examples include CnT-07 and CnT-30, commercially available from CELLnTECH, and XFDM-, disclosed in Hongisto et al., 2017, Stem Cells. In some other embodiments, the maintenance medium may consist of any suitable basal medium to which at least epidermal growth factor (EGF) and at least one Wnt activator are added as differentiation supplements. Preferred Wnt activators include glycogen synthase kinase 3 (GSK3) inhibitors such as CHIR99021, and their substitutes such as R-spongin or RS-246204. A further preferred maintenance supplement is noggin. In some embodiments, the maintenance medium may further contain hydrocortisone, insulin, isoproterenol, and triiodothyronine. Alternatively or additionally, in some embodiments, the maintenance medium does not contain any materials other than the maintenance supplements, basal medium, antibiotics, L-glutamine, and specified serum substitutes. Accordingly, in some embodiments, the medium does not contain any materials such as TGF-β inhibitors, fibroblast growth factor, and BMP-4, or functionally equivalent agents.

[0085] In some embodiments, the ABCG2-positive phenotype may be maintained for at least 50 days across six passages and through cryopreservation.

[0086] epidermal growth factor In the differentiation medium of the present invention, epidermal growth factor is required to maintain ABCG2 expression in limbal stem cells.

[0087] In some preferred embodiments, the amount of EGF in the differentiation medium is about 1 ng / ml to about 150 ng / ml, preferably about 1 ng / ml to about 100 ng / ml, and more preferably about 5 ng / ml to about 50 ng / ml.

[0088] In some embodiments, other materials may be used instead of EGF, such as specific synthetic small peptides designed to activate the epidermal growth factor receptor (EGFR) (e.g., produced by recombinant DNA variants or mutants).

[0089] EGF may be provided as a serum substitute or in a basal medium, or EGF may be added separately to the final cell culture medium according to the present invention.

[0090] Wnt activator The Wnt family of oncogenes consists of at least 16 known members, which encode secretory signaling proteins involved in carcinogenesis, as well as several other developmental processes, including the regulation of cell fate and embryonic development. As used herein, the term “Wnt activator” refers to a substance that can activate the Wnt signaling pathway. Both protein and small molecule Wnt activators are known in the art.

[0091] Glycogen synthase kinase 3 (GSK3) inhibitors are an exemplary class of preferred Wnt activators for use in the present invention. CHIR99021, which is commercially available from multiple suppliers, is the most selective inhibitor of GSK3 and is therefore a particularly preferred Wnt activator for use in the present invention. Further GSK3 inhibitors include, but are not limited to, SB-216763, BIO(6-bromoindilbine-3'-oxime), LY2090314, and lithium chloride, all of which are commercially available.

[0092] In some preferred embodiments, the amount of GSK3 inhibitor such as CHIR99021 in the differentiation medium is about 1 μM to about 15 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, preferably about 3 μM.

[0093] Another class of Wnt activators suitable for use in the present invention is the R-spongin protein family, whose four members are designated as R-spongin-1, R-spongin-2, R-spongin-3, and R-spongin-4, and which are secreted agonists of the orthodox Wnt / β-catenin signaling pathway.

[0094] In some embodiments, the Wnt activator is R-spondin-1. Preferred concentration ranges include about 100 ng / ml to about 2 μg / ml, about 500 ng / ml to about 2 μg / ml, and preferably about 1 μg / ml.

[0095] RS-246204 is a low-molecular-weight R-spondin-1 substitute, which is also suitable for use in this invention. Preferred concentration ranges include approximately 6.25 μM to approximately 200 μM and approximately 25 μM to approximately 50 μM.

[0096] In some embodiments, the differentiation supplement may include at least one GSK3 inhibitor, such as CHIR99021, and at least one R-spongin, such as R-spongin-1. In some other embodiments, increasing the amount of the at least one GSK3 inhibitor may be used to replace the presence of the at least one R-spongin, and vice versa.

[0097] Those skilled in the art can easily determine, using various readily available methods in the art, whether a given drug has Wnt-activating properties and whether it is suitable for use in such methods. A compound can be tested for its ability to act as a Wnt activator, for example, using a commercially available test kit, such as the LEADING LIGHT® Wnt Reporter Assay Starter Kit from Enzo.

[0098] Nogin Noggin, also known as NOG, is a protein involved in the development of many body tissues, including nerve tissue, muscles, and bones.

[0099] In some embodiments of the present invention, the differentiation medium may further contain nogging or be supplemented with nogging to support ABCG2 expression in limbal stem cells.

[0100] Typically, the amount of noggin in the differentiation medium may vary from about 10 ng / ml to about 500 ng / ml, or from about 10 ng / ml to about 100 ng / ml, for example, it may be about 100 ng / ml.

[0101] LDN-193189 is a low-molecular-weight noggin substitute and is suitable for use in the present invention. A preferred concentration range is, for example, about 0.01 μM to about 1 μM. Further noggin substitutes include, but are not limited to, dorsomorphine analogs such as DMH1.

[0102] Method for maintaining the ABCG2-positive phenotype of limbal stem cells In addition to the method for producing ABCG2-positive limbal stem cells described above, the present invention also provides a method for maintaining the ABCG2-positive phenotype of limbal stem cells.

[0103] In this method, limbal stem cells that attempt to maintain the ABCG2-positive phenotype include, but are not limited to, primary limbal stem cells or limbal stem cells generated by the differentiation method described above, and may be any ABCG2-positive limbal stem cells.

[0104] A method for maintaining the ABCG2-positive phenotype of limbal stem cells comprises the step of culturing ABCG2-positive limbal stem cells in a culture medium containing EGF and at least one Wnt activator. In some embodiments, the medium comprises EGF and at least one GSK3 inhibitor (e.g., CHIR99021). In some other embodiments, the medium comprises EGF and R-spongin (e.g., R-spongin-1) or a substitute thereof (e.g., RS-246204). In some further embodiments, the medium comprises EGF, at least one GSK3 inhibitor (e.g., CHIR99021) and R-spongin (e.g., R-spongin-1) or a substitute thereof (e.g., RS-246204). In each of these embodiments, the medium may further contain nogging. The preferred concentration ranges of these supplements described above in relation to the method for producing ABCG2-positive limbal stem cells also apply to the method for maintaining the ABCG2-positive phenotype of limbal stem cells.

[0105] In some embodiments, the ABCG2-positive phenotype may be maintained for at least 50 days across six passages and through cryopreservation.

[0106] Any embodiment described in relation to the maintenance phase of the method for producing ABCG2-positive limbal stem cells applies to the method for maintaining the ABCG2-positive phenotype of limbal stem cells, unless otherwise specified.

[0107] General characteristics of culture conditions and culture media Any culture medium can be considered to consist of a basal medium and supplements. In the induction medium of the present invention, the essential supplements are, firstly, a TGF-β inhibitor and FGF, and secondly, BMP-4. In the maintenance medium of the present invention, the necessary supplements include EGF and at least one Wnt activator. However, in the context of culture media, supplements common in the art may be further applied, as long as they are not known to induce differentiation into tissues other than ocular tissue. When referring to the components of a culture medium, this term includes both supplements and materials to the basal medium.

[0108] When in use or ready for use, the culture medium of the present invention contains the appropriate essential supplements described above. However, according to common practice in the art, the materials for the medium may be provided as a concentrate containing the above components, or as a set of vials for preparing the appropriate combination before laboratory use according to the provided instructions. Often, the culture medium is diluted immediately before use to prepare the final composition. Therefore, it is understood that any stock solution or preparation kit suitable for such immediate preparation may be used to obtain the cell culture medium used in the method. For example, for the preparation of the above-described induction medium, a cell culture kit may be employed that contains TGF-β inhibitors and fibroblast growth factors as supplements, each in separate containers, or optionally in combination thereof, and optionally includes other components, such as basal media or consumables for their preparation.

[0109] In this specification, “(culture) medium” or “culture solution” broadly refers to any liquid or gel-like formulation designed to support the growth of microorganisms, cells, or small plants. When referring to formulations intended for the maintenance and growth of cells, the term “cell culture medium” is used. In the art, expressions such as induction medium, growth medium, differentiation medium, and maturation medium can be considered variations of the general expression “culture medium.” Those skilled in the art are familiar with the basic components necessary to maintain and nourish living cells in or on a culture medium, and commercially available basic media are widely available. Such basic components are usually called “basal media” and contain the necessary amino acids, minerals, vitamins, and organic compounds. Generally, basal media may be assembled from isolated pure components. If necessary, basal media may be supplemented with substances that contribute to the specific characteristics or functions of the culture medium. Very common supplements include antibiotics used to inhibit the growth of contaminants, L-glutamic acid, serum, serum albumin, and serum substitutes. Those skilled in the art are familiar with both the necessary or optional culture medium components and their concentrations.

[0110] For use in the methods and various embodiments thereof of the present invention, the basal medium may be any stem cell culture medium on which stem cells can effectively differentiate. Non-limiting examples of suitable basal media include knockout Dulbecco's modified Eagle medium (KO-DMEM), Dulbecco's modified Eagle medium (DMEM), Minimum Essential Medium (MEM), Eagle basal medium (BME), RPMI1640, F-10, F-12, Glasgow Minimum Essential Medium (G-MEM), Iskov modified Dulbecco's medium, and any combination thereof. In some preferred embodiments, a modified RegES medium (referred to herein as RegESbasic, disclosed in Vaajasaari et al., Mol Vis. 2011;17:558-75) that omits retinol, bFGF, and activin A is used as the basal medium. In some more preferred embodiments, RegESbasic is used as the basal medium for the induction medium of the present invention.

[0111] For better clinical acceptance, all media used in the method and its various embodiments are preferably substantially xenofree, substantially serum-free, or substantially defined, more preferably a combination thereof, and most preferably substantially xenofree, substantially serum-free, and substantially defined simultaneously. In this specification, “substantially” means that unintended traces are irrelevant, and what is considered and accepted as xenofree, serum-free, or defined under clinical or laboratory rules also applies herein.

[0112] As used herein, the term "xeno-free" means free from foreign substances or exotic components. Therefore, in the case of human cell cultures, the term "xeno-free" refers to a state free from non-human animal components. In other words, if xeno-free conditions are desired for producing corneal cells for human use, all components of any cell culture medium must be of human or recombinant origin.

[0113] Traditionally, serum, particularly fetal bovine serum (FBS), has been valued in cell culture for providing essential growth and survival components for in vitro cell culture of eukaryotic cells. FBS is produced from blood collected at slaughterhouses of cattle raised for meat. "Serum-free" indicates that the culture medium does not contain animal or human serum. Standardized media (limited media) are valued when there is a contradiction in using "conditioned media," which refers to unspecified (unclear) media, such as used media recovered from cultured cells that contain metabolites, growth factors, and extracellular matrix proteins secreted into the medium by the cultured cells. Unspecified media can exhibit considerable differences due to natural biological variability. Unspecified components in cell culture impair the reproducibility of cell model experiments, for example, in drug discovery and toxicity studies. Therefore, "standardized media" or "standardized culture medium" refers to a composition in which all materials are known. Typically, serum added to culture media for cell culture is replaced with known amounts of serum components, such as albumin, insulin, transferrin, and possibly specific growth factors (e.g., basic fibroblast growth factor, transforming growth factor, or platelet-derived growth factor).

[0114] Synthetic culture media (known-composition media) are growth media suitable for in vitro cell culture of human or animal cells, and are media in which all chemical components are known. Synthetic media contain no animal-derived components whatsoever and represent the purest and most consistent cell culture environment. Naturally, synthetic media cannot contain fetal bovine serum, bovine serum albumin, or human serum albumin, because these products originate from bovine or human sources and contain complex mixtures of albumin and lipids.

[0115] Synthetic media differ from serum-free media in that bovine serum albumin or human serum albumin is replaced with either a chemically distinct recombinant (lacking albumin-related lipids) or a synthetic chemical such as polyvinyl alcohol, which is a polymer capable of replicating some of the functions of BSA / HSA. The next level of standardized media after synthetic media are protein-free media. These media contain hydrolysates of animal proteins and are commonly used for culturing insects and CHO cells, but their formulations are complex.

[0116] According to several embodiments, the culture medium of the present invention comprises a xenofree serum substitute formulation. A specified xenofree serum substitute formulation or composition may be used to supplement any suitable basal medium for use in vitro derivation, maintenance, proliferation, or differentiation of stem cells. The serum substitute may be used to supplement either a serum-free basal medium or a serum-containing basal medium, or any combination thereof. Supplementing a xenofree basal medium with a xenofree serum substitute results in a final culture medium that is also xenofree. One example is described in Rajala et al., 2010, incorporated herein by reference, which describes a xenofree serum substitute applicable in the context of the present invention. Another non-limiting example of a serum substitute is KnockOut® Serum Replacement (Ko-SR) and its xenofree version, KnockOut® SR XenoFree CTS®, both commercially available from Life Technologies.

[0117] Therapeutic use The present invention also provides a method for treating eye diseases such as corneal epithelial stem cell deficiency (LSCD) in subjects where it is needed. The method includes administering an efficient amount of ABCG-positive limbal stem cells, manufactured or maintained according to the present invention, to the subject.

[0118] Accordingly, the present invention also provides ABCG2-positive limbal stem cells manufactured or maintained in accordance with the present invention for use in the treatment of ocular diseases such as LSCD.

[0119] As used herein, the term “subject” refers to any mammal, preferably human.

[0120] ABCG2-positive limbal stem cells for use in treatment may be allogeneic or autologous.

[0121] As used herein, the term “efficient quantity” refers to the quantity of ABCG2-positive limbal stem cells that at least mitigate the adverse effects of an eye disease. Since it has been reported that cultures of limbal stem cells in which p63-positive cells constitute only 3% of the cell population yield a 78% therapeutic success rate (Rama et al., 2010, N Engl J Med, 363:147-55), populations of limbal stem cells containing more than 3% ABCG2-positive cells may also be considered clinically useful and therefore may be included in the term “efficient quantity.” However, preferably, an efficient quantity of ABCG2-positive limbal stem cells refers to a cell population in which ABCG2-positive cells constitute at least 65%, preferably at least 75%, and most preferably at least 90% of the total cell population.

[0122] As used herein, the terms “treatment” or “to treat” are intended to include administering the ABCG2-positive limbal stem cells of the present invention to subjects, for purposes that may include improving, alleviating, suppressing, or curing a disease.

[0123] Numbered exemplary embodiments of the present invention 1. A method for maintaining the ABCG2-positive phenotype of limbal stem cells, comprising the step of culturing ABCG2-positive limbal stem cells in a culture medium containing EGF and at least one Wnt activator.

[0124] 2. The method according to Embodiment 1, wherein the amount of EGF is about 1 ng / ml to about 150 ng / ml, preferably about 1 ng / ml to about 100 ng / ml, and more preferably about 15 ng / ml to about 50 ng / ml.

[0125] 3. The method according to Embodiment 1 or Embodiment 2, wherein the above-mentioned Wnt activator is selected from the group consisting of GSK3 inhibitors and proteins of the R-spondin family.

[0126] 4. The method according to Embodiment 3, wherein the above-mentioned GSK3 inhibitor is selected from the group consisting of CHIR99021, SB-216763, BIO(6-bromoindilbine-3'-oxime), LY2090314, and lithium chloride.

[0127] 5. The method according to Embodiment 4, wherein the GSK3 inhibitor is CHIR99021.

[0128] 6. The method according to Embodiment 5, wherein the amount of CHIR99021 is approximately 1 μM to approximately 15 μM, approximately 1 μM to approximately 10 μM, approximately 1 μM to approximately 5 μM, or approximately 3 μM.

[0129] 7. The method according to Embodiment 3, wherein the protein of the above-mentioned R-spongin family is selected from the group consisting of R-spongin-1 and its supplement RS-246204, R-spongin-2, R-spongin-3, and R-spongin-4.

[0130] 8. The method according to Embodiment 7, wherein the protein of the R-spongin family is R-spongin-1.

[0131] 9. The method according to Embodiment 8, wherein the amount of R-spongin-1 is approximately 100 ng / ml to approximately 2 μg / ml, approximately 500 ng / ml to approximately 2 μg / ml, or approximately 1 μg / ml.

[0132] 10. The method according to Embodiment 7, wherein the amount of RS-246204 is approximately 6.25 μM to approximately 200 μM, or approximately 25 μM to approximately 50 μM.

[0133] 11. The method according to any one of Embodiments 1 to 10, wherein the culture medium further comprises noggin or a supplement thereof selected from the group consisting of LDN-193189 and dorsomorphin analogs such as DMH1.

[0134] 12. The method according to Embodiment 11, wherein the amount of noggin is approximately 10 ng / ml to approximately 500 ng / ml, approximately 10 ng / ml to approximately 100 ng / ml, or approximately 100 ng / ml.

[0135] 13. The method according to Embodiment 11, wherein the amount of LDN-193189 is approximately 0.01 μM to approximately 1 μM.

[0136] 14. The method according to any one of Embodiments 1 to 13, wherein the ABCG2-positive limbal stem cells are ABCG2-positive primary limbal stem cells or ABCG2-positive limbal stem cells derived from pluripotent stem cells.

[0137] 15. The method according to Embodiment 14, wherein the cells are human cells.

[0138] 16. A method for producing ABCG2-positive limbal corneal stem cells. a) A process for providing pluripotent stem cells, b) A step of culturing the above cells in a cell culture medium containing a TGF-β inhibitor and fibroblast growth factor (FGF), preferably basic FGF, c) The TGF-β inhibitor and FGF are removed, and the cells obtained in step b) are cultured in a cell culture medium containing bone morphogenetic protein 4 (BMP-4) to generate ophthalmic progenitor cells; d) A step of culturing the above-mentioned ocular progenitor cells in corneal differentiation medium to generate ABCG2-positive limbal stem cells, e) The ABCG2-positive limbal stem cells are cultured in a maintenance cell culture medium containing EGF and at least one Wnt activator, thereby maintaining the phenotype of the ABCG2-positive limbal stem cells obtained through steps a to d). A method that includes this.

[0139] 17. A method for producing ABCG2-positive limbal corneal stem cells, a) A step of culturing pluripotent stem cells in a cell culture medium containing a TGF-β inhibitor and fibroblast growth factor (FGF), preferably basic FGF, b) The process of removing the TGF-β inhibitor and FGF, culturing the cells obtained in step a) in a cell culture medium containing bone morphogenetic protein 4 (BMP-4), thereby generating ophthalmoprogenitor cells, c) A step of culturing the above-mentioned ocular progenitor cells in corneal differentiation medium to generate ABCG2-positive limbal stem cells, d) The above ABCG2-positive limbal stem cells are cultured in a maintenance cell culture medium containing EGF and at least one Wnt activator, thereby maintaining the phenotype of the ABCG2-positive limbal stem cells obtained through steps a to e). A method that includes this.

[0140] 18. The method according to Embodiment 16 or Embodiment 17, wherein the TGF-β inhibitor is selected from TGF-β inhibitors having a molar mass of less than 800 g / mol, preferably less than 500 g / mol.

[0141] 19. The above TGF-β inhibitor is selected from the organic molecules related to formula I, [ka] The method according to Embodiment 18, wherein in the above formula, R1 represents a C1-C5 aliphatic alkyl group, a carboxylic acid, or an amide; R2 represents a C1-C5 aliphatic alkyl group; and R3 and R4 represent a heteroatom, an aliphatic alkyl group containing O or N, and these may be linked to each other to form a 5-membered or 6-membered heterocycle.

[0142] 20. The method according to any one of Embodiments 16 to 18, wherein the fibroblast growth factor is selected from basic FGF and synthetic small peptides exhibiting fibroblast growth factor-like activity.

[0143] 21. The method according to any one of Embodiments 16 to 20, wherein the amount of TGF-β inhibitor is 1 μM to 100 μM, preferably 1 to 30 μM.

[0144] 22. The method according to any one of Embodiments 16 to 21, wherein the amount of fibroblast growth factor is 1 ng / ml to about 1000 ng / ml, preferably about 2 ng / ml to about 100 ng / ml, more preferably about 30 ng / ml to about 80 ng / ml.

[0145] 23. The method according to any one of Embodiments 16 to 22, wherein the amount of BMP-4 is 1 ng / ml to 1000 ng / ml, preferably about 10 ng / ml to 50 ng / ml, more preferably 25 ng / ml.

[0146] 24. The method according to any one of Embodiments 16 to 23, wherein the amount of EGF is about 1 ng / ml to about 150 ng / ml, preferably about 1 ng / ml to about 100 ng / ml, and more preferably about 15 ng / ml to about 50 ng / ml.

[0147] 25. The method according to any one of Embodiments 16 to 24, wherein the above-mentioned Wnt activator is selected from the group consisting of GSK3 inhibitors and proteins of the R-spondin family.

[0148] 26. The method according to Embodiment 25, wherein the above GSK3 inhibitor is selected from the group consisting of CHIR99021, SB-216763, BIO(6-bromoindilbine-3'-oxime), LY2090314, and lithium chloride.

[0149] 27. The method according to embodiment 26, wherein the GSK3 inhibitor is CHIR99021.

[0150] 28. The method according to Embodiment 27, wherein the amount of CHIR99021 is approximately 1 μM to approximately 15 μM, approximately 1 μM to approximately 10 μM, approximately 1 μM to approximately 5 μM, or approximately 3 μM.

[0151] 29. The method according to Embodiment 25, wherein the protein of the above-mentioned R-spongin family is selected from the group consisting of R-spongin-1 and its supplement RS-246204, R-spongin-2, R-spongin-3, and R-spongin-4.

[0152] 30. The method according to Embodiment 29, wherein the protein of the R-spongin family is R-spongin-1.

[0153] 31. The method according to Embodiment 30, wherein the amount of R-spongin-1 is approximately 100 ng / ml to approximately 2 μg / ml, approximately 500 ng / ml to approximately 2 μg / ml, or approximately 1 μg / ml.

[0154] 32. The method according to Embodiment 29, wherein the amount of RS-246204 is approximately 6.25 μM to approximately 200 μM, or approximately 25 μM to approximately 50 μM.

[0155] 33. The method according to any one of Embodiments 16 to 32, wherein the maintenance culture medium further comprises noggin or a supplement thereof selected from the group consisting of LDN-193189 and dorsomorphin analogs such as DMH1.

[0156] 34. The method according to Embodiment 33, wherein the amount of noggin is approximately 10 ng / ml to approximately 500 ng / ml, approximately 10 ng / ml to approximately 100 ng / ml, or approximately 100 ng / ml.

[0157] 35. The method according to Embodiment 33, wherein the amount of LDN-193189 is approximately 0.01 μM to approximately 1 μM.

[0158] 36. The method is the method according to any one of Embodiments 16 to 35, wherein the pluripotent stem cells are selected from induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, but human embryonic stem (hES) cells are used, and the method does not involve the destruction of a human embryo.

[0159] 37. The method according to any one of Embodiments 16 to 36, wherein the cells are human cells.

[0160] 38. The method according to any one of Embodiments 16 to 37, wherein step a) is to form an embryoid body from the pluripotent stem cells.

[0161] 39. The method of any one of Embodiments 17 to 37, which includes the step of forming an embryoid body from the above-mentioned pluripotent stem cells, preceding step a).

[0162] 40. The method according to Embodiment 38 or Embodiment 39, wherein the formation of the embryoid body is carried out by physical or chemical means, preferably by a method selected from the group consisting of culturing cells in the presence of an anti-adhesion agent, culturing cells in a suspension, forced aggregation by microfabrication techniques, centrifugation, etc., and culturing cells in the presence of one or more aggregation promoters such as polymer crowder, brevistatin, and ROCK inhibitor.

[0163] 41. The method according to any one of Embodiments 16 and any of Embodiments 18 to 40, referencing Embodiment 16, wherein the culture in step d) and step e) is carried out on a substrate coated with at least collagen IV and laminin, preferably laminin 521 and / or laminin 511.

[0164] 42. The method according to any one of Embodiments 17 and any of Embodiments 18 to 40, referencing Embodiment 17, wherein the culture in step c) and step d) is carried out on a substrate coated with at least collagen IV and laminin, preferably laminin 521 and / or laminin 511.

[0165] 43. The method according to any one of Embodiments 1 to 42, which is substantially xenofree, substantially serum-free, and / or carried out under specified conditions.

[0166] 44. The method according to any one of Embodiments 16 and Embodiments 18 to 43, referencing Embodiment 16, wherein the duration of step b) is 1 to 7 days, preferably 1 day.

[0167] 45. The method according to Embodiment 17 and any of Embodiments 18 to 43, which reference Embodiment 17, wherein the duration of step a) is 1 to 7 days, preferably 1 day.

[0168] 46. ​​The method according to Embodiment 16 and any of Embodiments 18 to 43, which reference Embodiment 16, wherein the duration of step c) is 0.5 to 5 days, preferably 2 days.

[0169] 47. The method according to Embodiment 17 and any of Embodiments 18 to 43, which reference Embodiment 17, wherein the duration of step b) is 0.5 to 5 days, preferably 2 days.

[0170] 48. The method of any one of Embodiments 16 and Embodiments 18 to 43, which reference Embodiment 16, wherein the duration of step d) is carried out for 3 to 14 days, preferably 6 to 7 days, more preferably 7 days.

[0171] 49. The method according to any one of Embodiments 17 and Embodiments 18 to 43, referencing Embodiment 17, wherein the duration of step c) is 3 to 14, preferably 6 to 7 days, more preferably 7 days.

[0172] 50. The method of any one of Embodiment 16 and any of Embodiments 18 to 43, when referring to Embodiment 16, wherein the duration of step e) is carried out for any desired number of days, such as less than 50 days or at least 50 days.

[0173] 51. The method of any one of Embodiment 17 and any of Embodiments 18 to 43, when referring to Embodiment 17, wherein the duration of step d) is carried out for any desired number of days, such as less than 50 days or at least 50 days.

[0174] 52. ABCG2-positive limbal stem cells for use in the treatment of eye diseases, preferably corneal epithelial stem cell deficiency (LSCD), wherein the ABCG2-positive phenotype is maintained by the method of any one of Embodiments 1 to 15, or produced by the method of any one of Claims 16 to 51.

[0175] As technology advances, it will be apparent to those skilled in the art that the concept of the present invention can be implemented in various ways. The present invention and its embodiments are not limited to the examples described below and may be modified within the scope of the claims. [Examples]

[0176] Example 1. Examination of the differentiation of human pluripotent stem cells into limbal epithelial stem cells revealed that two distinct cell populations expressing ABCG2 or ΔNp63α subsequently emerged. These experiments were conducted to analyze the differentiation hierarchy along the differentiation process from human pluripotent stem cells to limbal epithelial stem cells, using the differentiation method disclosed in International Publication No. 2018 / 037161.

[0177] Materials and methods Differentiation Protocol for International Publication No. 2018 / 037161 This study used three genetically distinct tissue-derived hPSC strains, hESC strains Regea08 / 017 and Regea11 / 013 (Skottman et al., 2010), and hiPSC strain UTA.04607.WT. Human PSC cultures were routinely maintained under serum and feeder cell-free conditions and differentiated towards the corneal epithelial lineage as previously described by Hongisto et al. 2017 Stem Cell Res and Hongisto et al. 2018 JoVE. Briefly, undifferentiated hPSC colonies were enzymatically eluted into single-cell suspensions and transferred to standard XF-Ko-SR medium on low-adhesion well plates for induction in suspension culture. On day one, 5 μM brevistatin (Sigma-Aldrich) was added to XF-Ko-SR to support embryoid body (EB) formation. During the subsequent 3-day induction period for EB cells, 10 μM SB-505124 and 50 ng / ml human basic fibroblast growth factor (bFGF; PeproTech Inc., Rocky Hill, New Jersey) were added to XF-Ko-SR medium for 1 day, followed by 25 ng / ml bone morphogenetic protein (BMP)-4 (PeproTech Inc.) for 2 days. After induction, 0.5 μg / cm³ was added to further differentiate the EB cells into epithelial cells. 2 Recombinant laminin-521 (LN-521, Biolamina, Sweden) and 5 μg / cm³ 2 Wells coated with human placental type IV collagen (Col IV, Sigma-Aldrich) were transferred to commercially available standard CnT-30 corneal differentiation medium (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland). In standard differentiation, the adhered cultures were then maintained in CnT-30, with the medium changed three times a week until analysis or cryopreservation. Representative images of cell morphology were taken with a Nikon Eclipse TE2000-S phase-contrast microscope (Nikon Instruments Europe).

[0178] Protein expression profiling in differentiation protocols using immunofluorescence Undifferentiated (UD) human pluripotent stem cells and hPSC-derived LSCs from Regea08 / 017 and UTA.04607 were used to fully characterize the expression of OCT-3 / 4, PAX-6, ABCG2, p63α, ΔNp63, CK-15, CK-14, and CK-12 using immunofluorescence (IF) labeling at time points 7, 9, 11, 14, 17, 21, and 24 (including a 4-day induction period). Cytospin samples for quantifying the expression of OCT-3 / 4, ABCG2, p63α, ΔNp63, CK-15, and CK-14 were prepared at time points 10 and 24. Before staining, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 15–20 minutes at room temperature (RT). The basic IF protocol using primary and secondary antibodies (Table 1) was performed essentially as previously described by Mikhailova et al., 2014. Raw images of stained cells were acquired using an Olympus IX51 fluorescence microscope, and ImageJ image processing and analysis software (https: / / imagej.nih.gov / ij / ) was used for cell counting analysis. IF characterization and cytospin quantification were repeated 2-3 times for both strains using cells from individual differentiation batches. Subsequently, IF analysis at days 11 and 24 confirmed similar protein expression profiles for the hESC strain Regea11 / 013.

[0179] [Table 2]

[0180] Fluorescence-activated cell sorting (FACS) analysis of ABCG2 To label the cells with FACS antibodies, they were first enzymatically dissociated and washed with pre-cooled FACS washing buffer containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich) and 2nM EDTA (Gibco, Thermo Fisher Scientific) in DPBS (Lonza). The counted cells were 2–10 × 10⁶ per sample. 5 Cells were divided into 5 ml tubes. An optimized amount of the appropriate antibody was added to 100 μl of sample volume and incubated on ice for 20 minutes in the dark. Finally, the samples were washed twice, resuspended in the washing buffer described above, and stored on ice until analysis. Cells were labeled with APC-conjugated monoclonal mouse anti-human CD338 (ABCG2) antibody, clone 5D3 (BD ​​Pharmingen, #561451) (3 μl per sample). In addition, an APC-conjugated mouse IgG2 bκ antibody (BD Pharmingen, #555745) was used as an appropriate isotype control, and unstained negative samples were prepared to gate the population. FACS analysis was performed using a BD FACSAria® Fusion cell sorter (BD Biosciences, San Jose, California, USA). Analysis from individual differentiation batches was repeated at least three times for both the Regea08 / 017 and UTA.04607.WT strains. 10,000 events were recorded for each sample.

[0181] Quantitative RT-PCR of ABCG2 Total RNA was extracted from pelletized cell samples using the RNeasy Mini Kit (Qiagen, Thermo Fisher Scientific), and the RNA concentration of each sample was measured using a NanoDrop-1000 spectrophotometer (NanoDrop Technologies). 400 ng of total RNA from each sample was used for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific). The resulting cDNA samples were analyzed for ABCG2 mRNA using qPCR with a sequence-specific TaqMan Gene Expression Assay (#HS01053790_m1, Applied Biosystems). GAPDH (Hs99999905_m1) was used as a housekeeping gene. All samples and controls were subjected to three reactions using a 7300 Real-Time PCR system (Applied Biosystems). The results were analyzed using the -2ΔΔCt method (Livak and Schmittgen, 2001) and are shown as the relative multiplier change in gene expression compared to undifferentiated control (UD-hPSC), with GAPHD as the baseline.

[0182] result To investigate protein expression at different stages of the differentiation process from hPSCs to LSCs, two genetically distinct feeder-free cultured hPSC strains, hESC strain Regea08 / 017 and hiPSC strain UTA.04607.WT, were guided to corneal lines using an established two-step protocol (Hongisto et al., 2017 Stem Cell Res Ther). Along the differentiation process, changes in the expression patterns of OCT3 / 4 and several recognized LSC and mature corneal epithelium-related marker proteins were extensively characterized using immunofluorescence (IF). Eight IF analysis time points included day 0 (UD-hPSC), day 7, day 9, day 11, day 14, day 17, day 21, and day 24 (including a 4-day induction period). During this timeframe, the expression of the pluripotency-related factor OCT-3 / 4 was significantly downregulated, while the expression of PAX-6, ΔNp63α, CK-15, and CK-14 was remarkably increased, indicating the emergence of limbal epithelial-like cell populations (Figure 2A). Interestingly, ABCG2 was expressed only transiently, peaking at days 9–11, then gradually decreasing, reaching very low levels by day 24 of differentiation (Figure 2A). Similar to previous results using this protocol (Hongisto et al., 2017), the mature corneal epithelial marker CK-12 remained undetectable throughout this characterization window (data not shown), further confirming the undifferentiated progenitor cell phenotype of the cells. Quantification of protein expression from cytospin samples revealed significant differences between the cell populations at the time points of day 10 and day 24 (Figure 2B). In a representative hESC strain (Regea08 / 017), ABCG2 expression decreased from 62.3% (SD 6.7) to 1.8% (SD 0.9), while ΔNp63α expression (as shown by double staining with ΔNp63 and p63α antibodies; Figure 2C) increased from 23.2% (SD 14.1) to 54.3% (SD 6.2). CK15 and CK14 expression increased from undetectable levels on day 10 to 37.0% (SD 12.4) and 56.2% (SD 14.3), respectively, by day 24. On the other hand, OCT3 / 4 was already expressed in less than 1.5% of cells on day 10, and further decreased to less than 1% by day 24.Given the differing behaviors of ABCG2 and ΔNp63α during differentiation, co-localization of ABCG2 and p63α was investigated using cytospin samples from day 10 and day 24. Interestingly, as shown in Figure 2D, the strongest staining of both markers was typically observed in different cells. Furthermore, ABCG2 and p63α co-localized in 31.6% of cells on day 10, compared to only 1% of cells on day 24 (Figure 2E). However, the difference observed between the two time points was not statistically significant (Mann-Whitney U test).

[0183] To confirm the expression pattern of ABCG2, fluorescence-activated cell sorting (FACS) analysis was performed not only on UD-hPSCs but also on hPSC-LSCs from days 10-11 and 24-26. As a result, ABCG2 expression was low in both UD-hPSCs and more differentiated hPSC-LSCs from days 24-25 (0.8%, SD 1.3 and 1.5%, SD 2.0, respectively, for the representative hESC strain Regea08 / 017), but the expression level of ABCG2 was significantly higher in hPSC-LSCs from days 10-11 (21.6%, SD 8.2, Mann-Whitney U test) (Figure 2F). In addition, analysis of changes in ABCG2 expression at the transcriptional level using qRT-PCR revealed that the expression level of ABCG2 mRNA was significantly higher in the hPSC-LSC population at day 10 compared to the UD-hPSC and hPSC-LSC populations at day 24 (Figure 2G). Combining these findings, the above characterization analysis revealed very different expression profiles for the proposed limbal epithelial stem cell / progenitor cell markers ABCG2, ΔNp63α, CK-15, and CK-14 during the differentiation process of hPSC-LSCs between the time points of day 10-11 and day 24.

[0184] Example 2. Maintenance of ABCG2 expression This experiment was conducted to establish the culture conditions necessary to maintain ABCG2 expression over a long period.

[0185] Materials and methods Culture conditions for maintaining the ABCG2-positive hPSC-LSC phenotype To maintain the ABCG2-positive population, the culture medium for CnT-30 was replaced with CnT-07 (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland), and on days 10-11 (including a 4-day induction period), ENRC (50 ng / ml mouse recombinant epidermal growth factor (EGF, Invitrogen), 100 ng / ml mouse recombinant noggin, 1 μg / ml human recombinant R-spongin-1 (both Peprotech), and 3 μM CHIR-99021 (Stemgent)) was supplemented. The CnT-07+ENRC medium was either directly introduced into adherent cultures, or hPSC-LSCs were simultaneously enzymatically dissolved to form a single-cell suspension, and then cultured in the new medium at a rate of 1000-5000 cells / cm². 2 The cells were subcultured at a density into new wells coated with new LN-521 / Col IV. In all cases, the hPSC-LSCs were then cultured according to standard feeding procedures. After the appearance of ABCG2-positive colonies, further proliferation and / or continued culture of the hPSC-LSCs in CnT-07+ENRC was performed, with subconfluent cultures at 1000 cells / cm³. 2 This density was achieved by subculturing in a new LN-521 / Col IV coated matrix. Cryopreservation of these cells was performed as described by Hongisto et al., 2017 Stem Cell Res Ther.

[0186] The preservation of high ABCG2 expression in hPSC-LSC colonies cultured with Cnt-07+ENRC was confirmed by IF in all three hPSC strains studied. Quantitative RT-PCR analysis was performed on Regea08 / 017 and UTA.04607.WT. The above characterization methods were basically carried out in accordance with those described in Example 1.

[0187] In addition to the above, the following modified culture conditions to maintain the ABCG2-positive hPSC-LSC phenotype were tested using the hESC strain Regea08 / 017.

[0188] In the first related experiment, different cell culture media were tested as basal media with ENRC supplementation. The basal media tested included commercially available pluripotent stem cell culture medium E8 Flex (Thermo Fisher Scientific), commercially available corneal differentiation medium CnT-30 (CELLnTEC), and a self-formulated xenofree basal medium XF-Ko-SR described by Hongisto et al., 2017 Stem Cell Res & Ther.

[0189] In the second related experiment, CnT-07 was used as a basal cell culture medium supplemented with a modified ENRC formulation. One component of ENRC was not provided under each experimental condition. Therefore, the conditions tested were CnT-07 + ENR (without CHIR-99021), CnT-07 + ENC (without R-spongin-1), CnT-07 + ERC (without Noggin), and CnT-07 + NRC (without EGF).

[0190] In both experiments described above, the tested cell culture conditions were directly introduced into hPSC-LSC cultures attached on day 11 (including a 4-day differentiation period), and the cells were cultured for 10 days according to a standard feeding regimen three times a week. At the end of the experiment, cell morphology was evaluated and representative images were taken using a Nikon Eclipse TE2000-S phase-contrast microscope (Nikon Instruments Europe). In the second experiment, the cells were fixed and subjected to IF staining using ABCG2 antibody and p63α antibody to confirm the maintenance of the ABCG2-positive hPSC-LSC phenotype.

[0191] Growth analysis For Regea11 / 013, the doubling of the cell population at the end of each subculturing was calculated using the following formula: log(N / N0) / log2 (where N0 is the number of plated cells and N is the number of cells at the end of the culture period). Similarly, the doubling time for each subculturing was calculated using the following formula: T×log2 / log(N-N0) (where T is the duration of culture in hours).

[0192] result Importantly, replacing corneal differentiation medium CnT-30 with epithelial maintenance medium CnT-07 supplemented with a specific combination of EGF, noggin, R-spondin-1, and CHIR-99201 (ENRC) maintained colony morphology and strongly expressed ABCG2. On the other hand, other tested cell culture media supplemented with the complete ENRC, namely E8 Flex, CnT-30, and XF-Ko-SR, were not effective in maintaining and expanding the desired cell morphology, in relation to unwanted cell types (data not shown). Despite their poor performance, these media are also suitable for use in the present invention, at least in some of their embodiments. Interestingly, in addition to CnT-07 supplemented with the complete combination of ENRC, the ABCG2-positive hPSC-LSC phenotype was maintained in all tested ENRC modification conditions, including CHIR-99021, namely CnT-07+ENC, CnT-07+ERC, and CnT-07+NRC.

[0193] Notably, ABCG2-positive colonies expressed only low levels of p63α, while ABCG2-negative cells under CnT-30 conditions expressed p63α (Figure 3A). Furthermore, under CnT-07+ENRC conditions, ABCG2 expression was maintained even after cell passage (Figure 3B). However, when cultured and passaged in CnT-30 differentiation medium, these cells lost robust ABCG2 expression and further differentiated into a p63α-expressing phenotype, as described in the differentiation process of hPSC-LSCs in Example 1. These IF results were confirmed in three genetically distinct cell lines: two hESC lines (Regea08 / 017 and Regea11 / 013) and one hiPSC line (UTA.04607.WT). The effect of the CnT-07+ENRC condition was consistent across all three cases, but the growth effect during further passage and the tendency to generate other cell types differed among the cell lines. Without subculturing, ABCG2-positive colonies persisted in CnT-07+ENRC for at least 24 days (35 days total culture time for hPSC-LESC), which was the longest time point analyzed for a single strain, Regea08 / 017 (data not shown).

[0194] To determine the optimal time point for passage of ABCG2-positive hPSC-LSCs, two different time points were tested. First, cells were cultured in CnT-07+ENRC for 10 days, and passage was performed on day 21 when ABCG2 expression stabilized. In the alternative approach, cells were passaged on day 11 (i.e., at the peak of ABCG2 expression during the differentiation process of hPSC-LESCs), re-seeded in CnT-07+ENRC, and further cultured. Maintenance of ABCG2-positive colonies was achieved with both methods and confirmed in all three cell lines, but there were significant cell line-specific differences in proliferation. In Regea11 / 013, the latter approach resulted in highly proliferative colonies with minimal contamination from other cell types. Colony morphology and ABCG2 / p63α expression remained unaffected by further passage (Figure 4A). In Regea11 / 013, more than 20 population doublings (PD) were observed over 5 passages, with an average population doubling time (PDT) of 50.9 hours (SD 16.4) (Figure 4B-C, black dotted line). In addition, cryopreservation between passages 2 and 3 (p2-p3) did not significantly affect the cell's proliferative capacity, as shown by the white box in Figures 4B-4C.

Claims

1. A method for maintaining the ABCG2-positive phenotype of limbal stem cells, comprising the step of culturing ABCG2-positive limbal stem cells in a culture medium containing EGF and at least one Wnt activator.

2. The method according to claim 1, wherein the Wnt activator is selected from the group consisting of a GSK3 inhibitor, preferably CHIR99021, and a protein of the R-spongin family, preferably R-spongin-1 or its supplement RS-246204.

3. The method according to claim 1 or claim 2, wherein the culture medium further comprises noggin or its supplement LDN-193189.

4. The method according to any one of claims 1 to 3, wherein the ABCG2-positive limbal stem cells are ABCG2-positive primary limbal stem cells or ABCG2-positive limbal stem cells derived from pluripotent stem cells.

5. A method for producing ABCG2-positive limbal stem cells, a) A process for providing pluripotent stem cells, b) A step of culturing the cells in a cell culture medium containing a TGF-β inhibitor and fibroblast growth factor (FGF), preferably basic FGF, c) The step of removing the TGF-β inhibitor and the FGF, culturing the cells obtained in step b) in a cell culture medium containing bone morphogenetic protein 4 (BMP-4), thereby generating ophthalmoprogenitor cells, d) A step of culturing the ocular progenitor cells in a corneal differentiation medium to generate ABCG2-positive limbal stem cells, e) A step of culturing the ABCG2-positive limbal stem cells in a maintenance cell culture medium containing EGF and at least one Wnt activator, thereby maintaining the phenotype of the ABCG2-positive limbal stem cells obtained through steps a to d) A method that includes this.

6. The method according to claim 5, wherein the Wnt activator is selected from the group consisting of a GSK3 inhibitor, preferably CHIR99021, and a protein of the R-spongin family, preferably R-spongin g-1 or its supplement RS-246204.

7. The method according to claim 5 or claim 6, wherein the maintenance culture medium in step e) further comprises noggin or its supplement LDN-193189.

8. The method according to any one of claims 5 to 7, wherein the pluripotent stem cells are selected from induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, provided that human embryonic stem (hES) cells are used, the method does not involve the destruction of a human embryo.

9. The method according to any one of claims 5 to 8, wherein step a) is to form an embryoid body from the pluripotent stem cells.

10. The method according to claim 9, wherein the formation of the embryoid body in step a) is carried out by physical or chemical means, preferably by a method selected from the group consisting of culturing cells in the presence of an anti-adhesion agent, culturing cells in a suspension, forced aggregation by microfabrication techniques, centrifugation, etc., and culturing cells in the presence of one or more aggregation promoters such as polymer crowder, brevistatin, and ROCK inhibitor.

11. The method according to any one of claims 6 to 10, wherein the culture in step d) and step e) is carried out on a substrate coated with at least collagen IV and laminin, preferably laminin 521 and / or laminin 511.

12. The method according to any one of claims 1 to 11, which is substantially xenofree, substantially serum-free, and / or carried out under specified conditions.

13. ABCG2-positive limbal stem cells, for use in the treatment of eye diseases, preferably corneal epithelial stem cell deficiency (LSCD), wherein the ABCG2-positive phenotype is maintained by the method of any one of claims 1 to 4, or produced by the method of any one of claims 5 to 12.

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