Conjunctival epithelial markers and their uses

Abstract

The present invention provides: a production method for a conjunctival epithelial cell mass that is characterized by including (1) a step for inducing differentiation of conjunctival epithelial stem / precursor cells from pluripotent stem cells, (2) a step for collecting cells that express a conjunctival epithelium marker selected from the group that consists of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2 from the resulting conjunctival epithelial stem / precursor cells, and (3) a step for culturing and maturing the cells that express the conjunctival epithelial marker; a detection method for conjunctival epithelial cells that is characterized by detecting the cells of an eye surface epithelial cell mass that express a conjunctival epithelial cell marker selected from the group that consists of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2; and a method for improving the purity of conjunctival epithelial cells in an eye surface epithelial cell mass that is characterized by collecting cells that express a conjunctival epithelial marker selected from the group that consists of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. The present invention can be used to produce conjunctival epithelial cell masses and to detect and improve the purity of conjunctival epithelial cells.

Description

Technical Field

[0001] The present invention relates to a conjunctival epithelial marker and its use. Specifically, it relates to a conjunctival epithelial marker for detecting or purifying conjunctival epithelial cells, a method for producing a population of conjunctival epithelial cells using the marker, and a method for improving the purity of conjunctival epithelial cells.

Background Art

[0002] The ocular surface is covered with conjunctival epithelium and corneal epithelium, both of which play important roles indispensable for maintaining the homeostasis of the ocular surface. In particular, research on human corneal epithelium has progressed relatively far, and it has been elucidated that corneal epithelial stem / progenitor cells exist in the limbus region and function to maintain corneal epithelium. On the other hand, there are many unclear points regarding human conjunctival epithelium. Although it is said that conjunctival epithelial stem / progenitor cells are abundant in the conjunctival fornix region, their localization sites and molecular mechanisms are not yet clearly understood (Non-Patent Document 1).

[0003] In addition, although corneal epithelial stem / progenitor cell markers have already been reported, conjunctival epithelial markers that can also be used for conjunctival epithelial stem / progenitor cells have not been reported at present. If such a conjunctival epithelial marker can be discovered, it will be possible to elucidate the localization sites and molecular mechanisms of conjunctival epithelial stem / progenitor cells, and it is expected to be useful for clarifying the pathogenesis of conjunctival diseases and establishing treatment methods.

[0004] In the two-dimensional ocular tissue organoid (Self-formed Ectodermal Autonomous Multi-zone: SEAM) developed by the group of the present inventors, corneal epithelium or conjunctival epithelium can be preferentially induced by adding various growth factors to human iPS cells from a certain time, and it has been confirmed that each contains stem / progenitor cells (Non-Patent Documents 2, 3, 4). However, since the ocular surface epithelial stem / progenitor cells in SEAM include not only conjunctival epithelial stem / progenitor cells but also corneal epithelial stem / progenitor cells, the establishment of a preparation method capable of forming a more highly pure population of conjunctival epithelial cells is required.

Prior Art Documents

[0005] [Non-Patent Document 1] Gipson IK, Prog Retin Eye Res., 54, 49-63, 2016 [Non-Patent Document 2] Hayashi R, et al., Nature, 531(7594), 376-380, 2016 [Non-Patent Document 3] Hayashi R et al., Nat. Protoc., 12(4), 683-696, 2017 [Non-Patent Document 4] Nomi K et al., Cell Rep., 34(5), 108715, 2021 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to identify a conjunctival epithelial marker that can be used for the detection and purification of conjunctival epithelial cells, and to provide a method for producing a population of conjunctival epithelial cells using the marker, a method for detecting conjunctival epithelial cells, and a method for improving their purity. [Means for solving the problem]

[0007] To solve the above problems, the present invention includes the following inventions. [1] A method for producing a conjunctival epithelial cell population, comprising: (1) a step of differentiating pluripotent stem cells into conjunctival epithelial stem / progenitor cells; (2) a step of recovering cells expressing a conjunctival epithelial marker from the obtained conjunctival epithelial stem / progenitor cells; and (3) a step of culturing and maturing the cells expressing the conjunctival epithelial marker, characterized in that the conjunctival epithelial marker is selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. [2] The manufacturing method according to [1], wherein the conjunctival epithelial marker is selected from the group consisting of BST2, SLC2A3, AGR2, and TMEM54. [3] The method for producing the product according to [1], further comprising culturing in a culture medium containing an EGF (Epidermal Growth Factor) receptor agonist in step (1). [4] The method for producing the product according to [1], further comprising culturing in a culture medium containing a KGF (Keratinocyte Growth Factor) receptor agonist in step (3). [5] The method for producing a pluripotent stem cell according to [1], wherein step (1) includes inducing SEAM (Self-formed Ectodermal Autonomous Multi-zone) from pluripotent stem cells. [6] The manufacturing method according to any one of [1] to [5], wherein the conjunctival epithelial cell population has the shape of a sheet. [7] The method for manufacturing according to any one of [1] to [5] above, wherein the pluripotent stem cells are iPS cells. [8] A method for detecting conjunctival epithelial cells, characterized by detecting cells expressing a conjunctival epithelial marker selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2 in a population of ocular surface epithelial cells. [9] A method for improving the purity of conjunctival epithelial cells in an ocular surface epithelial cell population, characterized by collecting cells that express a conjunctival epithelial marker selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2.

[10] A composition for the detection or purification of conjunctival epithelial cells, characterized by comprising a substance that specifically binds to a conjunctival epithelial marker selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. [Effects of the Invention]

[0008] The present invention provides a method for producing a conjunctival epithelial cell population using a conjunctival epithelial marker that can be used for the detection and purification of conjunctival epithelial cells, a method for detecting conjunctival epithelial cells, and a method for improving their purity. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the time schedule for differentiation induction from iPS cells to corneal epithelial stem / progenitor cells or conjunctival epithelial stem / progenitor cells in Example 1. [Figure 2] Figure 2 shows the results of immunohistochemical staining observations of BST2 and SLC2A3 expression in various tissues. The first and third rows show merge images of the fluorescence signals and nuclear stains of BST2 and SLC2A3, respectively, while the second and fourth rows show the fluorescence signal images of BST2 and SLC2A3, respectively. [Figure 3] Figure 3 shows the results of immunohistochemical staining observations of AGR2 and TMEM54 expression in various tissues. The first and third rows show merge images of the fluorescence signals and nuclear stains of AGR2 and TMEM54, respectively, while the second and fourth rows show the fluorescence signal images of AGR2 and TMEM54, respectively. [Figure 4] Figure 4 shows the results of immunohistochemical staining observations of OLR1 and TRIM29 expression in various tissues. The first and third rows show merge images of the fluorescence signals and nuclear stains of OLR1 and TRIM29, respectively, while the second and fourth rows show the fluorescence signal images of OLR1 and TRIM29, respectively. [Figure 5] Figure 5 shows the results of observing CITED2 expression in various tissues by immunohistochemical staining. The first row shows a merge image of the CITED2 fluorescence signal and nuclear stain, and the second row shows the CITED2 fluorescence signal image. [Figure 6] Figure 6 summarizes the detection results from Figures 2 to 5. [Figure 7]Figure 7 shows the results of measuring the expression levels of BST2, PAX6, and p63 by RT-qPCR before and after maturation culture, after collecting CD200-negative / CD104-positive / BST2-positive cells (BST2+ in the figure) and CD200-negative / CD104-positive / BST2-negative cells (BST2- in the figure) by FACS from the cells at the 12th week after induction of conjunctival epithelial differentiation (see Figure 1). (A) shows the results of BST2, (B) shows the results of PAX6, and (C) shows the results of p63. [Figure 8] Figure 8 shows the results of measuring the expression levels of conjunctival markers by RT-qPCR before and after maturation culture, after collecting CD200-negative / CD104-positive / BST2-positive cells (BST2+ in the figure) and CD200-negative / CD104-positive / BST2-negative cells (BST2- in the figure) by FACS from the cells at the 12th week after induction of conjunctival epithelial differentiation (see Figure 1). (A) shows the results of MUC5AC, (B) shows the results of MUC4, (C) shows the results of K13, and (D) shows the results of K7. [Figure 9] Figure 9 shows the results of measuring the expression levels of corneal markers by RT-qPCR before and after maturation culture, after collecting CD200-negative / CD104-positive / BST2-positive cells (BST2+ in the figure) and CD200-negative / CD104-positive / BST2-negative cells (BST2- in the figure) by FACS from the cells at the 12th week after induction of conjunctival epithelial differentiation (see Figure 1). (A) shows the results of K12, and (B) shows the results of K3. [Figure 10] Figure 10 shows the results of observing the expression of various markers in the cell sheet obtained by mature culture of the CD200-negative / CD104-positive / BST2-positive cells in Figures 7 to 9 by immunohistochemical staining. [Figure 11] Figure 11 shows the results of observing the expression of various markers in the cell sheet obtained by mature culture of the CD200-negative / CD104-positive / BST2-negative cells in Figures 7 to 9 by immunohistochemical staining. [Figure 12] Figure 12 shows the results of PAS staining of the cell sheet obtained by mature culture of the CD200-negative / CD104-positive / BST2-positive cells in Figures 7 to 9. [Figure 13]FIG. 13 is a diagram showing the results of a colony assay using CD200-negative / CD104-positive / BST2-positive cells (BST2+ in the figure) and CD200-negative / CD104-positive / BST2-negative cells (BST2- in the figure) after maturation culture. [Figure 14] FIG. 14 is a diagram showing the results of measuring the expression levels of various genes by RT-qPCR after maturation culture of CD200-negative / CD104-positive / BST2-positive cells (BST2+ in the figure) and CD200-negative / CD104-positive / SSEA-4 weakly positive cells (SSEA4 low in the figure) from the cells at the 12th week (see FIG. 1) after conjunctival epithelial induction. (A) shows the results of MUC5AC, (B) shows the results of K13, and (C) shows the results of K12. [Figure 15] FIG. 15 is a diagram showing the results of observing the expression of various markers in the cell sheet after maturation culture of FIG. 14 by immunohistochemical staining. (A) shows the results of BST2+, and (B) shows the results of SSEA4 low.

Mode for Carrying Out the Invention

[0010] 〔Method for Producing a Conjunctival Epithelial Cell Population〕 The present invention provides a method for producing a conjunctival epithelial cell population. The method for producing a conjunctival epithelial cell population of the present invention may include the following steps (1) to (3). (1) A step of inducing differentiation of pluripotent stem cells into conjunctival epithelial stem / progenitor cells (2) A step of recovering cells expressing a conjunctival epithelial marker from the obtained conjunctival epithelial stem / progenitor cells (3) A step of culturing and maturing cells expressing a conjunctival epithelial marker

[0011] In step (1), differentiation induction from pluripotent stem cells to conjunctival epithelial stem / progenitor cells is initiated. Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatocyte stem (GS) cells, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent cells (Muse cells) derived from bone marrow stem cells. The pluripotent stem cells used in the present invention may be iPS cells, ES cells, or ntES cells, with iPS cells being preferred.

[0012] The conjunctival epithelial stem / progenitor cells that are induced to differentiate include cells that have the ability to differentiate into conjunctival epithelium, and may include conjunctival epithelial stem cells, conjunctival epithelial progenitor cells, and cells in the process of being induced to differentiate into conjunctival epithelial stem / progenitor cells.

[0013] The method for inducing differentiation from pluripotent stem cells into conjunctival epithelial stem / progenitor cells is not particularly limited and can be appropriately selected from known methods for inducing differentiation of pluripotent stem cells into conjunctival epithelial stem / progenitor cells. In one embodiment, the method for inducing differentiation from pluripotent stem cells into conjunctival epithelial stem / progenitor cells may be the method described in Non-Patent Document 2 as a method for inducing two-dimensional ocular tissue organoids (Self-formed Ectodermal Autonomous Multi-zone: SEAM) from pluripotent stem cells, or the method described in Non-Patent Document 4 as a method for culturing using a medium containing an EGF (Epidermal Growth Factor) receptor agonist, or a combination of these methods from Non-Patent Documents may be used. Examples of EGF receptor agonists include EGF.

[0014] The culture medium used to induce differentiation from pluripotent stem cells into conjunctival epithelial stem and progenitor cells can be any medium suitable for differentiation into ocular surface epithelium, and is sometimes referred to as ocular surface differentiation medium (ODM). Any medium (serum-free medium) that can be used for culturing epithelial cells, such as the medium used in autonomous differentiation, can be used as ocular surface differentiation medium.

[0015] The ocular surface differentiation medium may contain ROCK inhibitors in addition to EGF receptor agonists. "ROCK inhibitors" refer to substances that inhibit Rho kinase (ROCK: Rho-associated, coiled-coil containing protein kinase), such as N-(4-pyridyl)-4β-[(R)-1-aminoethyl]cyclohexane-1α-carboxamide (Y-27632), Fasudil (HA1077), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), and 4β-[(1R) -1-aminoethyl]-N-(4-pyridyl)benzene-1αcarboxamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4β-[(R)-1-aminoethyl]cyclohexane-1αcarboxamide (Y-30141), N-(3-{[2-(4-amino-1,2,5-oxadiazole-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridine-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A) and N-(6-fluoro-1H-indazole-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A) can be used.

[0016] The differentiation induction period in step (1) is not particularly limited and can be any period during which conjunctival epithelial markers are expressed in cells that are in the process of differentiating from pluripotent stem cells to conjunctival epithelial stem / progenitor cells or in differentiated cells. For example, when differentiating pluripotent stem cells to conjunctival epithelial stem / progenitor cells using the methods described in Non-Patent Documents 2 and 4, the differentiation induction period in step (1) can be, for example, 10 to 14 weeks. When using a differentiation induction method different from the above, the differentiation induction period in step (1) can be determined by conducting appropriate preliminary studies.

[0017] In step (2), cells expressing a conjunctival epithelial marker are collected from the conjunctival epithelial stem / progenitor cells obtained in step (1). The conjunctival epithelial marker in the present invention may be selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2, and is preferably selected from the group consisting of BST2, SLC2A3, AGR2, and TMEM54. BST2 is bone marrow stromal cell antigen 2 (also known as CD317, Tetherin, HM1.24), SLC2A3 is solute carrier family 2 member 3 (also known as GLUT3), AGR2 is anterior gradient 2 (also known as AG2, AG-2, HPC8, GOB-4, HAG-2, XAG-2, PDIA17, HEL-S-116), TMEM54 is transmembrane protein 54 (also known as BCLP, CAC1, CAC-1), OLR1 is oxidized low density lipoprotein receptor 1 (also known as LOX1, LOXIN, SLOX1, CLELC8A, SCARE1), TRIM29 is tripartite motif containing 29 (also known as ATDC), and CITED2 is a Cbp / p300 interacting transactivator with a Glu / Asp rich carboxy-terminal domain This refers to cell 2 (also known as ASD8, MRG1, VSD2, MRG-1, P35SRJ). The present invention's method for producing a conjunctival epithelial cell population reduces the proportion of cells that do not express conjunctival epithelial markers in the conjunctival epithelial stem / progenitor cells after differentiation induction, while increasing the proportion of cells that express conjunctival epithelial markers. As a result, the resulting conjunctival epithelial cell population has an improved proportion of conjunctival epithelium.

[0018] The method for recovering cells expressing conjunctival epithelial markers is not particularly limited, but examples include using a cell sorter or affinity chromatography. When using a cell sorter, for example, a cell suspension of conjunctival epithelial stem and progenitor cells can be prepared, and a substance that specifically binds to each conjunctival epithelial marker can be added to the cell suspension to recover cells expressing the conjunctival epithelial markers. Alternatively, when using affinity chromatography, for example, a cell suspension of conjunctival epithelial stem and progenitor cells can be prepared, and cells expressing conjunctival epithelial markers can be passed through a column packed with a carrier to which a substance that specifically binds to each conjunctival epithelial marker is bound, allowing the cells to bind to the substance that specifically binds to the conjunctival epithelial marker, and then the cells expressing the conjunctival epithelial markers can be recovered by dissociating the binding from the substance that specifically binds to the conjunctival epithelial marker.

[0019] The substance that specifically binds to the conjunctival epithelial marker is not particularly limited as long as it specifically binds to BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, or CITED2. Examples include small molecule compounds, peptides, antibodies, antibody fragments, glycans, etc., that have the ability to bind to BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, or CITED2. Preferably, it is an anti-BST2 antibody, anti-SLC2A3 antibody, anti-AGR2 antibody, anti-TMEM54 antibody, anti-OLR1 antibody, anti-TRIM29 antibody, or anti-CITED2 antibody.

[0020] Step (2) may further include a step of recovering the target cells using known cell surface markers used for the isolation and analysis of ocular surface epithelial cells from conjunctival epithelial stem / progenitor cells. Examples of known cell surface markers include CD104 and ITGB4 as cell surface markers expressed on conjunctival epithelial stem / progenitor cells, CD200 as a cell surface marker not expressed on conjunctival epithelial stem / progenitor cells, and SSEA-4 as a cell surface marker weakly expressed on conjunctival epithelial stem / progenitor cells. The method for recovering the target cells is not particularly limited, and a known recovery method can be used as appropriate depending on the expression status of the cell surface markers. The step of recovering the target cells may be performed simultaneously with step (2), or before or after step (2).

[0021] In step (3), cells expressing the conjunctival epithelial marker obtained in step (2) are matured. The method for maturing the cells expressing the conjunctival epithelial marker is not particularly limited and can be appropriately selected from known methods for maturing conjunctival epithelial stem / progenitor cells into conjunctival epithelium. In one embodiment, the method described in Non-Patent Literature 4 may be used as the method for culturing using a medium containing a KGF (Keratinocyte Growth Factor) receptor agonist. Examples of KGF receptor agonists include KGF. It is preferable to mature the cells expressing the conjunctival epithelial marker in a feeder cell-free environment from the viewpoint of increasing the purity of the conjunctival epithelial cell population.

[0022] The culture medium used for maturating cells expressing conjunctival epithelial markers can be any medium suitable for the maintenance culture of ocular surface epithelial cells, and may sometimes be referred to as ocular surface epithelium maintenance medium (OEM). Any medium (serum-free medium) that can be used for culturing epithelial cells can be used as ocular surface epithelium maintenance medium.

[0023] The ocular surface epithelium maintenance medium may further contain serum substitutes in addition to EGF receptor agonists or KGF receptor agonists. Examples of "serum substitutes" include albumin (e.g., lipid-rich albumin), transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc, selenium), B27® supplements, N2 supplements, etc. The concentration in the medium is 0.01 to 10% by weight, preferably 0.5 to 4% by weight, in the case of B27 supplements.

[0024] Furthermore, in addition to the above, ocular surface differentiation medium and ocular surface epithelial maintenance medium may appropriately contain various nutrients necessary for cell maintenance and proliferation, as well as various components necessary for differentiation induction. For example, nutrients may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, and dextrin; hydrocarbons such as fatty acids, oils and fats, lecithin, and alcohols; nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, and sodium nitrate; inorganic salts such as sodium chloride, potassium salts, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts (e.g., monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, manganese sulfate); various vitamins; amino acids, etc. The content of these components can be adjusted in accordance with common technical knowledge.

[0025] The duration of step (3) is not particularly limited and may be, for example, 2 to 10 weeks. The shape of the conjunctival epithelial cell population after maturation is not particularly limited and may consist of separated cell populations, or the cells may adhere to each other to form aggregates or sheets, with the sheet shape being preferable.

[0026] The conjunctival epithelial cell population produced by the present invention may be a human conjunctival epithelial cell population or a conjunctival epithelial cell population from an organism other than a human. The organism other than a human is not particularly limited and may be a mammal, for example. Examples of mammals include monkeys (crab-eating macaques, rhesus macaques, etc.), chimpanzees, dogs, cats, cattle, horses, pigs, rabbits, mice, rats, and the like.

[0027] [Method for detecting conjunctival epithelial cells] The present invention provides a method for detecting conjunctival epithelial cells (hereinafter referred to as "the detection method of the present invention"). The detection method of the present invention only needs to include a step of detecting cells expressing a conjunctival epithelial marker in a population of ocular surface epithelial cells, and the conjunctival epithelial marker may be selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. The population of ocular surface epithelial cells is a broad concept that includes cells having the ability to differentiate into ocular surface epithelium and cells that have terminally differentiated into ocular surface epithelium, and may include conjunctival epithelial stem / progenitor cells, conjunctival epithelial cells, conjunctival epithelial goblet cells, corneal epithelial stem / progenitor cells, and corneal epithelial cells. In one embodiment, the population of ocular surface epithelial cells may be obtained by differentiating pluripotent stem cells, and may be a surgical specimen containing ocular surface epithelium or a cell suspension prepared from said surgical specimen.

[0028] The method for detecting cells expressing conjunctival epithelial markers in a population of ocular surface epithelial cells is not particularly limited, and known methods can be used. For example, a method may be used to detect cells expressing conjunctival epithelial markers by labeling a substance that binds to the conjunctival epithelial marker, or a method may be used to indirectly detect cells expressing conjunctival epithelial markers by further labeling the substance that binds to the conjunctival epithelial marker with another substance. In one embodiment, the method for detecting cells expressing conjunctival epithelial markers in a population of ocular surface epithelial cells may be to detect the expression of conjunctival epithelial markers present on the surface of the cells, or to detect the expression of conjunctival epithelial markers present inside ocular surface epithelial cells that have undergone cell fixation and / or permeabilization.

[0029] [Method for improving the purity of conjunctival epithelial cells] The present invention provides a method for improving the purity of conjunctival epithelial cells (hereinafter referred to as "the purity improvement method of the present invention"). The purity improvement method of the present invention only needs to include a step of recovering cells expressing a conjunctival epithelial marker from a population of ocular surface epithelial cells, and the conjunctival epithelial marker may be selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. As the population of ocular surface epithelial cells, cells similar to those exemplified in the ocular surface epithelial cell population in the above-described method for detecting conjunctival epithelial cells can be used. The method for recovering cells expressing a conjunctival epithelial marker can be carried out in the same manner as step (2) in the above-described method for producing a population of conjunctival epithelial cells.

[0030] [Composition for the detection or purification of conjunctival epithelial cells] The present invention provides a composition for the detection or purification of conjunctival epithelial cells (hereinafter referred to as "the composition of the present invention"), comprising a substance that specifically binds to a conjunctival epithelial marker selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. The composition of the present invention may contain any substance similar to the substance exemplified as a substance that specifically binds to a conjunctival epithelial marker in the method for producing a conjunctival epithelial cell population of the present invention. The composition of the present invention may contain other components in addition to the conjunctival epithelial marker, and these other components may include, for example, solvents, additives, excipients, etc.

[0031] The present invention includes a conjunctival epithelial cell detection kit, a cell marker for the detection or purification of conjunctival epithelial cells, etc., which contains a substance that specifically binds to a conjunctival epithelial marker selected from the group consisting of BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2. [Examples]

[0032] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0033] [Example 1: Search for conjunctival epithelial markers] 1-1 Differentiation into corneal or conjunctival epithelium Based on the method described in Non-Patent Document 2, human iPS cells were cultured for 4 weeks in differentiation medium (DM) on culture dishes coated with laminin 511E8 fragment to form two-dimensional ocular tissue organoids (Self-formed Ectodermal Autonomous Multi-zone: SEAM). Subsequently, based on the differentiation induction methods described in Non-Patent Documents 3 and 4, the cells were cultured for 4 weeks in ocular surface differentiation medium (ODM) containing Y-27632 and EGF or KGF to induce differentiation into specific ocular surface epithelial cells. Then, the cells were cultured for approximately 4 weeks in ocular surface epithelium maintenance medium (OEM) containing Y-27632, EGF or KGF, and B-27 supplement. EGF was used for conjunctival epithelial differentiation induction, and KGF was used for corneal epithelial differentiation induction. The time schedule is shown in Figure 1.

[0034] Specifically, human iPS cell line [YZWJs524 strain (Kyoto University iPS Cell Research Foundation)] is cultured in laminin 511E8-coated culture dishes at a rate of 100-700 cells / cm². 2The cells were seeded at a density of [density] and maintained in StemFit® medium (Ajinomoto Co., Inc.) for 8-10 days. Then, they were cultured for 4 weeks in differentiation medium [DM; GMEM medium (Life Technologies) containing 10% knockout serum replacement (KSR, Life Technologies), 1 mM sodium pyruvate (Life Technologies), 0.1 mM non-essential amino acids (Life Technologies), 2 mM l-glutamine (Life Technologies), 1% penicillin-streptomycin solution (Life Technologies), and 55 μM 2-mercaptoethanol (Life Technologies)], and the cells from the third and fourth layers of the formed SEAM were harvested. To induce differentiation of corneal epithelium, cells from layers 3 and 4 of SEAM, collected from corneal differentiation medium, were cultured for 4 weeks in corneal differentiation medium [a 1:1 (v / v) mixture of differentiation medium containing 10 ng / mL KGF (R&D Systems) and 10 μM Y-27632 (Wako) and Cnt-20 or Cnt-PR medium (EGF and FGF2-free, CellnTEC Advanced Cell Systems)]. After that, the medium was changed to corneal epithelial maintenance medium [DMEM / F-12 (2:1 (v / v)) medium (Life Technologies) containing 2% B27 Supplement (Life Technologies), 1% penicillin-streptomycin solution, 10 ng / mL KGF, and 10 μM Y-27632], and cultured for another 4 weeks to induce differentiation. When inducing differentiation of conjunctival epithelium, conjunctival differentiation medium and conjunctival epithelial maintenance medium were used, supplemented with 10 ng / mL EGF (Wako) instead of 10 ng / mL KGF.

[0035] 1-2 Sample preparation Cells from layers 3 and 4 of SEAM at 4 weeks of differentiation induction were designated as the undifferentiated ocular surface epithelial cell group. At 12 weeks of differentiation induction into corneal epithelium, CD200-negative / SSEA-4 weakly positive / CD104-positive cells were collected by FACS and designated as Corneal Group 1, and CD200-negative / SSEA-4 strongly positive / CD104-positive cells were designated as Corneal Group 2. At 12 weeks of differentiation induction into conjunctival epithelium, CD200-negative / SSEA-4 weakly positive / CD104-positive cells were collected by FACS and designated as Conjunctival Group 1, and CD200-negative / SSEA-4 strongly positive / CD104-positive cells were designated as Conjunctival Group 2. For the undifferentiated ocular surface epithelial cell group, after dissociating the cells with Accutase at 37°C for 60 min, the necessary cell groups from layers 3 and 4 were manually obtained and suspended in PBS with 0.04% BSA at approximately 1,000 cells / μl. For corneal groups 1 and 2, and conjunctival groups 1 and 2, the cell populations isolated in each fraction by FACS were suspended in PBS with 0.04% BSA at approximately 1,000 cells / μl.

[0036] 1-3 scRNAseq analysis DNA concentration was measured using Qubit (Thermo Fisher Scientific) for the five samples prepared above (undifferentiated ocular surface epithelial cells, cornea group 1, cornea group 2, conjunctiva group 1, and conjunctiva group 2), confirming that the DNA concentration of the samples was sufficient at 100 ng / μl or higher. Libraries were prepared according to the protocol of the MGI Easy Universal Library Conversion Kit (App-A) (MGI). Base sequence data (FASTQ format) was obtained using the next-generation sequencer "DNBSEQ-G400 (MGISEQ-200RS)". Each obtained FASTQ file was analyzed using the Cell Ranger pipeline ver 3.1.0 (10X Genomics) and the reference dataset [Human reference (GRCh38) dataset required for Cell Ranger] with the [cell ranger count] command. Since a sufficient number of detectable cells and genes were obtained for each sample (data not shown), cluster analysis was performed to group each cell based on the similarity of gene expression patterns.

[0037] 1-4 Analysis of scRNAseq data Cluster analysis was performed on each sample using t-SNE dimensionality reduction on the Loupe Cell Browser (data not shown). Undifferentiated ocular surface epithelial cells could be classified into 12 clusters from 0 to 11, with cluster 1 being the most undifferentiated cell population. Conjunctival epithelium could be classified into 10 clusters from 0 to 9, with clusters 2, 3, 5, and 7 being the most undifferentiated cell populations. Corneal epithelium could also be classified into 11 clusters from 0 to 10, with clusters 0, 1, and 2 being the most undifferentiated cell populations. Since multiple clusters were candidate for both conjunctival and corneal epithelium, Velocyto analysis was performed (data not shown). Velocyto analysis revealed that cluster 2 was the most undifferentiated cell population in conjunctival epithelium, and cluster 0 was the most undifferentiated cell population in corneal epithelium. In each group, we searched for candidate genes for conjunctival epithelial markers from clusters obtained as undifferentiated cell populations, and ultimately obtained several candidate conjunctival epithelial markers (BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2).

[0038] 1-5 Immunohistochemical staining of humans and cynomolgus monkeys Human bulbar conjunctiva, limbal cornea, central cornea, and cynomolgus monkey conjunctival fornix tissues were embedded in OCT compound (4583, Tissue-Tek) for section preparation and stored at -80°C. 5 μm frozen sections were then prepared using a cryostat and dried at room temperature for at least 30 minutes. After blocking with 5% NST (room temperature, 1 hour), each section was reacted with the primary antibody shown below (4°C, overnight). After washing twice with TBS for 5 minutes, the sections were treated with Alexa Fluor 488-labeled secondary antibody (1:200) and Hoechst 33342 (1:100) (room temperature, 1 hour), and observed under a fluorescence microscope.

[0039] The following antibodies were used as primary antibodies. • Anti-BST2 antibody (1:200; HPA017060, manufactured by Atlas Antibody) • Anti-SLC2A3 antibody (1:200; HPA006539, manufactured by Atlas Antibody) ·Anti-AGR2 antibody (1:200; ab227584, manufactured by ABCAM) • Anti-TMEM54 antibody (1:200; HPA061992, manufactured by Sigma-Aldrich) ·Anti-OLR1 antibody (1:200; 11837-1-AP, manufactured by proteintech) • Anti-TRIM29 antibody (1:200; sc-376125, manufactured by Santa Cruz Biotechnology) ·Anti-CITED2 antibody (1:200; sc-21795, manufactured by Santa Cruz Biotechnology) The following antibodies were used as secondary antibodies. ·Donkey anti-rabbit IgG Alexa Flour 488 conjugate (1:200; A-21206, manufactured by Life Technologies) ·Donkey anti-mouse IgG Alexa Flour 488 conjugate (1:200; A-21202, manufactured by Life Technologies)

[0040] Figures 2-5 show the results of immunohistochemical staining using various antibodies against conjunctival epithelial markers in each tissue, and observation of the fluorescence signals. The first and third rows show merge images of the fluorescence signals and nuclear stains of candidate conjunctival epithelial markers, while the second and fourth rows show the fluorescence signal images of candidate conjunctival epithelial markers. The scale bar is 50 μm. Based on the results in Figures 2-5, Figure 6 shows the detection results of conjunctival epithelial markers in each tissue. In Figure 6, "◎" means "significant expression was detected," "〇" means "detected," and "×" means "not detected." BST2 was significantly detected in the basal layer of human conjunctival epithelium and the basal layer of the conjunctival fornix of cynomolgus monkeys, while it was not detected in the superficial layer of human conjunctival epithelium, conjunctival goblet cells, human limbus, and central cornea. SLC2A3 was detected in human conjunctival epithelium, conjunctival goblet cells, the superficial layer of the limbus, and the conjunctival fornix of cynomolgus monkeys, but was not detected in the base of the limbus or the central cornea of ​​humans. AGR2 was detected in human conjunctival goblet cells and the conjunctival fornix of cynomolgus monkeys, but was not detected in human conjunctival epithelium, the limbus, or the central cornea. TMEM54 was detected in human conjunctival goblet cells and the conjunctival fornix of cynomolgus monkeys, but was not detected in human conjunctival epithelium, the limbus, or the central cornea. OLR1 was significantly detected in the base of human conjunctival epithelium and was also detected in the superficial layer of conjunctival epithelium, conjunctival goblet cells, the base of the limbus, the central cornea, and the conjunctival fornix of cynomolgus monkeys, but was not detected in the superficial layer of the limbus of humans. TRIM29 was detected in all areas of human conjunctival epithelium, conjunctival goblet cells, limbus, central cornea, and cynomolgus monkey conjunctival fornix. CITED2 was detected in all areas of human conjunctival epithelium, conjunctival goblet cells, limbus, central cornea, and cynomolgus monkey conjunctival fornix, and the coincidence of the CITED2 signaling site and DAPI signaling site suggests that it is nuclearly localized. All candidate conjunctival epithelial markers (BST2, SLC2A3, AGR2, TMEM54, OLR1, TRIM29, and CITED2) were detected in human conjunctival epithelium, conjunctival goblet cells, and / or cynomolgus monkey conjunctival fornix, suggesting that they function as conjunctival epithelial markers.

[0041] [Example 2: Examination of effectiveness as a conjunctival epithelial marker] 2-1 FACS From the cell population at 12 weeks after differentiation induction into conjunctival epithelium in Example 1, CD200-negative cells were collected by sorting using a cell sorter SH800 (SONY). Subsequently, the cells were separated using BST2 and CD104 to collect CD200-negative / CD104-positive / BST2-positive cells and CD200-negative / CD104-positive / BST2-negative cells, respectively.

[0042] The following antibodies were used in FACS. ·Mouse monoclonal anti-CD104 Alexa Fluor 647 conjugate (Cat# 624024, manufactured by BD Biosciences) ·Mouse monoclonal anti-CD200 PE-Cy7 conjugate (Cat# 624052, manufactured by BD Biosciences) ·Mouse monoclonal anti-CD317(BST2) PE conjugate (Cat# 348406, manufactured by BioLegend)

[0043] 2-2 RT-qPCR CD200-negative / CD104-positive / BST2-positive cells and CD200-negative / CD104-positive / BST2-negative cells recovered by FACS were designated as BST2+ and BST2-, respectively, before maturation culture. Laminin 511E8 fragment (0.5 μg / cm³) 2 CD200-negative / CD104-positive / BST2-positive cells and CD200-negative / CD104-positive / BST2-negative cells were cultured for 3-4 weeks on cell culture inserts coated with ), and these were designated as BST2+ and BST2- cells after mature culture, respectively. The corneal epithelial maintenance medium used in Example 1 was used as the culture medium. Cells were treated with QIAzol, RNA was extracted, and subjected to RT-qPCR (n=7).

[0044] The results of RT-qPCR are shown in Figures 7-9. As shown in Figure 7, for BST2, gene expression was observed only in BST2+ before mature culture, and gene expression was also observed in BST2+ after mature culture, while gene expression was not observed in BST2-. For the ocular surface markers PAX6 and p63, gene expression was observed under all conditions. As shown in Figure 8, for MUC5AC, MUC4, K13, and K7, known as conjunctival markers, gene expression levels were significantly increased in BST2+ after mature culture. On the other hand, as shown in Figure 9, for K12 and K3, known as corneal markers, gene expression levels were significantly increased in BST2- after mature culture.

[0045] 2-3 Immunohistochemical staining CD200-negative / CD104-positive / BST2-positive cells (hereinafter referred to as BST2+) or CD200-negative / CD104-positive / BST2-negative cells (hereinafter referred to as BST2-) were matured and cultured in the same manner as described in 2-2, and cell sheets obtained from each cell type were collected. Frozen sections were prepared using the same procedure as in Example 1 and dried at room temperature for at least 30 minutes. After blocking with 5% NST (room temperature, 1 hour), each was reacted with the primary antibody shown below (4°C, overnight). After washing twice with TBS for 5 minutes, the cells were treated with Alexa Fluor 488-labeled secondary antibody (1:200) and Hoechst 33342 (1:100) (room temperature, 1 hour), and then observed under a fluorescence microscope.

[0046] The following antibodies were used as primary antibodies. • Anti-MUC5AC antibody (1:100; sc-33667, manufactured by Santa Cruz Biotechnology) ·Anti-K13 antibody (1:200; ab16112, manufactured by ABCAM) ·Anti-K7 antibody (1:200; ab9021, manufactured by ABCAM) • Anti-PAX6 antibody (1:300; sc-11357, manufactured by Santa Cruz Biotechnology) • Anti-BST2 antibody (1:200; HPA017060, manufactured by Atlas Antibody) • Anti-MUC4 antibody (1:200; sc-33654, manufactured by Santa Cruz Biotechnology) ·Anti-K12 antibody (1:1000; ab185627, manufactured by ABCAM) ·Anti-K3 antibody (1:200; cat#61807, manufactured by PROGEN Biotechnik) • Anti-p63 antibody (1:200; sc-8431, manufactured by Santa Cruz Biotechnology) The following antibodies were used as secondary antibodies. ·Donkey anti-rabbit IgG Alexa Flour 488 conjugate (1:200; A-21206, manufactured by Life Technologies) ·Donkey anti-mouse IgG Alexa Flour 488 conjugate (1:200; A-21202, manufactured by Life Technologies)

[0047] The results of immunohistochemical staining using various antibodies on each cell sheet and observation of fluorescence signals are shown in Figures 10-11. The scale bar is 50 μm. In cell sheets obtained by mature culture of BST2+, conjunctival markers MUC5AC, MUC4, K13, and K7, as well as ocular surface markers PAX6 and p63, were detected, while corneal markers K12 and K3 were not detected (Figure 10). On the other hand, in cell sheets obtained by mature culture of BST2-, conjunctival markers MUC5AC, MUC4, K13, and K7 were not detected, while corneal markers K12 and K3, as well as ocular surface markers PAX6 and p63, were detected (Figure 11). BST2 showed a similar trend to the results of RT-qPCR after mature culture in each cell sheet; it was detected in cell sheets obtained by mature culture of BST2+, but not in cell sheets obtained by mature culture of BST2-.

[0048] 2-4 PAS staining Cell sheets obtained by mature culture of BST2+ cells were stained using a PAS staining kit (MERCK KGaA). The slides were observed using an Axio Imager.A2 (Carl Zeiss).

[0049] Figure 12 shows the results of PAS staining performed on cell sheets obtained by mature culture of BST2+ cells. The presence of conjunctival goblet cells on the surface of the cell sheets was confirmed.

[0050] 2-5 Colony assay BST2+ or BST2- cells recovered by FACS were seeded at 2000-5000 cells / well in 12-well plates onto mitomycin C-treated NIH-3T3 feeder cells. Subsequently, the cells were incubated at 37°C for 10-14 days in KCM medium containing 20 ng / mL of KGF and 10 mM of Y-27632 [a 3:1 (v / v) mixture of glutamine-free DMEM and Nutrient Mixture F-12 Ham (Thermo Fisher Scientific), which also contained 5% FBS (Japan Bio Serum), 0.4 μg / mL hydrocortisone succinate (Wako), 2 nM 3,3',5-triiodo-l-thyronine sodium salt (MP Biomedicals), 1 nM cholera toxin (List Biological Laboratory), 2.25 μg / mL bovine transferrin HOLO form (Thermo Fisher Scientific), 2 mM l-glutamine, and 0.5% insulin transferrin selenium solution (Thermo Fisher Scientific)]. Colonies were fixed using 10% formaldehyde neutral buffer at room temperature for 1 hour, and then stained with rhodamine B (WAKO) at room temperature for 2 hours. The number of colonies formed was evaluated using a dissecting microscope, and the colony formation efficiency (CFE) was calculated by dividing the number of colonies formed after culture by the number of seeded cells (n=5).

[0051] The results of the colony assay are shown in Figure 13. Both BST2+ and BST2- were confirmed to have colony-forming ability. BST2+ showed a higher colony formation rate compared to BST2-.

[0052] [Example 3: Comparison with the conventional method] 3-1 FACS In Example 2, CD200-negative / CD104-positive / BST2-positive cells were used. In the conventional method, similar to the conjunctival group 1 in Example 1, CD200-negative / CD104-positive / SSEA-4 weakly positive cells were collected from a cell population at 12 weeks after differentiation into conjunctival epithelium. Weakly positive means cells with slightly lower expression levels compared to normal cells, and includes cells with expression levels intermediate between positive and negative.

[0053] 3-2 RT-qPCR CD200-negative / CD104-positive / BST2-positive cells and CD200-negative / CD104-positive / SSEA-4 weakly positive cells were subjected to laminin 511E8 fragment (0.5 μg / cm³) using the corneal epithelial maintenance medium used in Example 1. 2 CD200-negative / CD104-positive / BST2-positive cells and CD200-negative / CD104-positive / SSEA-4 weakly positive cells were cultured for 3-4 weeks on cell culture inserts coated with ), and these were designated as BST2+ and SSEA4 low, respectively. The cells were treated with QIAzol, RNA was extracted, and subjected to RT-qPCR. The gene expression levels of MUC5AC, K13, and K12 were evaluated by RT-qPCR (n=3).

[0054] The results of RT-qPCR are shown in Figure 14. The BST2+ group showed higher gene expression levels for the conjunctival goblet cell marker MUC5AC and the conjunctival epithelial cell marker K13 compared to the conventional SSEA4 low group. Furthermore, while significant gene expression of the corneal epithelial cell marker K12 was observed in the conventional SSEA4 low group, almost no K12 gene expression was observed in the BST2+ group.

[0055] 3-3 Immunohistochemical staining The immunohistochemical staining results of MUC5AC, K13, and K12 were compared in the same manner as in Example 2, steps 2-3, except that cell sheets obtained by mature culture of BST2+ and cell sheets obtained by mature culture of SSEA4 low were used. Figure 15 shows the results of immunohistochemical staining using various antibodies and observation of fluorescence signals in cell sheets obtained by mature culture of BST2+ or SSEA4 low. (A) shows the results for cell sheets obtained by mature culture of BST2+, and (B) shows the results for cell sheets obtained by mature culture of SSEA4 low using the conventional method. The scale bar is 50 μm. Conjunctival epithelial cell marker K13 was detected in both cell sheets obtained by mature culture of BST2+ and cell sheets obtained by mature culture of SSEA4 low. In particular, conjunctival goblet cell marker MUC5AC was detected at a higher rate in cell sheets obtained by mature culture of BST2+ compared to cell sheets obtained by mature culture of SSEA4 low. In addition, corneal epithelial cell marker K12 was detected in cell sheets obtained by mature culture of SSEA4 low using the conventional method, indicating that cells that had differentiated into corneal epithelial cells after mature culture were included. Based on the above, by collecting CD200-negative / CD104-positive / BST2-positive cells, it is possible to produce conjunctival epithelial sheets with even higher purity compared to conventional methods.

Claims

1. A method for producing a population of human conjunctival epithelial cells, (1) A process of differentiating human pluripotent stem cells into conjunctival epithelial stem cells and progenitor cells. (2) A step of collecting cells expressing conjunctival epithelial markers from the obtained conjunctival epithelial stem and progenitor cells, and (3) A step of culturing and maturing cells that express conjunctival epithelial markers, A method for producing a product comprising the above, wherein the conjunctival epithelial marker is BST2.

2. The method for producing the product according to claim 1, wherein step (1) includes culturing using a culture medium containing an EGF (Epidermal Growth Factor) receptor agonist.

3. The method for producing the product according to claim 1, wherein step (3) comprises culturing using a culture medium containing a KGF (Keratinocyte Growth Factor) receptor agonist.

4. The method for producing a human pluripotent stem cell according to claim 1, wherein step (1) includes inducing SEAM (Self-formed Ectodermal Autonomous Multi-zone) from human pluripotent stem cells.

5. The manufacturing method according to any one of claims 1 to 4, wherein the population of human conjunctival epithelial cells has the shape of a sheet.

6. The manufacturing method according to any one of claims 1 to 4, wherein the human pluripotent stem cell is a human iPS cell.

7. A method for detecting conjunctival epithelial cells, characterized by detecting cells expressing BST2 in a population of human ocular surface epithelial cells.

8. A method for improving the purity of conjunctival epithelial cells in a population of human ocular surface epithelial cells, characterized by collecting cells that express BST2.

9. A composition for the detection or purification of human conjunctival epithelial cells, characterized by comprising a substance that specifically binds to BST2.

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