Method for preparing lacrimal gland tissue from stem cells
阅读说明：本技术 源自干细胞的泪腺组织的制作方法 (Method for preparing lacrimal gland tissue from stem cells ) 是由 西田幸二 林龙平 大久保徹 本间阳一 于 2019-03-28 设计创作，主要内容包括：一种源自干细胞的泪腺组织的制作方法,其特征在于,从由多能干细胞得到的SEAM细胞群(自我形成的外胚层自主性多区细胞群)中分离出SSEA4和CD104双阳性细胞,将所得到的细胞在含有EGF(表皮生长因子)和ROCK抑制剂的培养基中进行三维培养,由此获得表达泪腺的相关蛋白的细胞群。通过本发明,能够由包括iPS细胞的多能干细胞制造泪腺类器官,因此对于利用细胞医疗进行的泪腺相关疾病的再生治疗和与上述疾病相关的研究极其有用。(A method for producing a lacrimal gland tissue derived from stem cells, characterized by isolating cells that are both SSEA4 and CD104 positive from a SEAM cell population (self-forming ectoderm autonomous multizone cell population) obtained from pluripotent stem cells, and three-dimensionally culturing the obtained cells in a medium containing EGF (epidermal growth factor) and a ROCK inhibitor, thereby obtaining a cell population expressing a protein related to lacrimal gland. The present invention enables the production of lacrimal gland-like organs from pluripotent stem cells including iPS cells, and is therefore extremely useful for regenerative therapy of lacrimal gland-related diseases by cellular medical therapy and for research relating to the above-mentioned diseases.)
1. A method for producing a lacrimal gland tissue derived from stem cells, characterized by isolating cells that are both SSEA4 and CD104 positive from a SEAM cell population (self-forming ectoderm autonomous multizone cell population) obtained from pluripotent stem cells, and three-dimensionally culturing the obtained cells in a medium containing EGF (epidermal growth factor) and a ROCK inhibitor, thereby obtaining a cell population expressing a protein related to lacrimal gland.
2. The method of claim 1, wherein the isolated cells are further CD200 negative.
3. A method for producing a lacrimal gland tissue derived from stem cells, characterized by culturing limbal stem cells in a three-dimensional medium containing EGF and a ROCK inhibitor, thereby obtaining a cell population expressing a lacrimal gland-associated protein.
4. The production method according to any one of claims 1 to 3, wherein the medium further contains TGF- β.
5. The method of any one of claims 1 to 4, wherein the related protein is one or more selected from AQP5, LYZ, CNN1, BARX2, SOX9, SOX10, RUNX1, TFCP2L1, LTF and HTN 1.
6. A method for producing a lacrimal gland organoid for transplantation, comprising a step of culturing the lacrimal gland epithelial cells obtained by the production method according to any one of claims 1 to 5.
7. A method for screening a drug for a lacrimal-gland-related disease, comprising a step of culturing the lacrimal-gland epithelial cells obtained by the production method according to any one of claims 1 to 5.
8. A tear fluid secreted from the lacrimal gland epithelial cell organoid obtained by the method of claim 6.
9. A pharmaceutical composition comprising the tear fluid of claim 8.
10. A method for selecting a cell that is easily differentiated and induced into a lacrimal tissue derived from a stem cell, the method comprising selecting the cell group formed by the production method according to any one of claims 1 to 5 using the shape of the cell group as an indicator.
11. A lacrimal organoid obtained by the method according to any one of claims 1 to 6.
12. A lacrimal organoid obtained by culturing the cell selected by the method of claim 10.
Technical Field
The present invention relates to a method for producing a stem cell-derived lacrimal gland tissue. More particularly, the present invention relates to a method for inducing lacrimal gland tissue from pluripotent stem cells and limbal stem cells, and use thereof.
Background
Human pluripotent stem cells such as human ES cells and human iPS cells are attracting attention worldwide for use in regenerative medicine. In order to apply human pluripotent stem cells to regenerative medicine, it is necessary to develop a technique for stably inducing differentiation of these stem cells into somatic cells with high efficiency, and various studies have been made on methods for inducing selective differentiation from human pluripotent stem cells into arbitrary somatic cells.
For example, non-patent
In addition, as a novel therapeutic method for severe corneal diseases such as corneal epithelial stem cell deficiency, the inventors of the present application developed a technique for producing corneal epithelial cell sheets from iPS cells, and reported that the effectiveness and the like thereof were confirmed using animal models (see
Disclosure of Invention
Problems to be solved by the invention
However, even if the lacrimal gland epithelial cells can be differentiated and induced according to the prior art, in order to apply the cells to regenerative medicine, it is necessary to form a three-dimensional structure composed of three kinds of cells, namely, ductal cells, acinar cells, and myoepithelial cells, which are characteristic of lacrimal gland tissues, from the lacrimal gland epithelial cells, and to form a lacrimal gland tissue capable of secreting tears containing various functional components under the control of the nervous system, but this has not been achieved at present.
The present invention relates to a method for producing a stem cell-derived lacrimal gland tissue that can be used in regenerative medicine and the like, and an application thereof.
Means for solving the problems
The present inventors have conducted extensive studies to solve the above-mentioned problems, and as a result, have found that a lacrimal-like tissue expressing a lacrimal-gland-related protein can be first prepared in vitro by separating a specific cell from a concentric band-like structure (self-formed ectodermal autonomous multi-zone: a self-formed ectodermal autonomous multi-zone; sea) obtained by the method described in non-patent document 2 by FACS, and performing three-dimensional induction using Matrigel (Matrigel) in combination with various growth factors.
That is, the present invention relates to the following [1] to [7 ].
[1] A method for producing a lacrimal tissue derived from stem cells, characterized by separating double positive cells SSEA4 and CD104 from a SEAM cell population (self-forming ectoderm autonomous multizone cell population) obtained from pluripotent stem cells, and three-dimensionally culturing the obtained cells in a medium containing EGF (Epidermal Growth Factor) and a ROCK inhibitor, thereby obtaining a cell population expressing a protein related to lacrimal gland.
[2] The production method according to [1], wherein the isolated cells are further CD 200-negative.
[3] A method for producing a lacrimal gland tissue derived from stem cells, characterized by culturing limbal stem cells in a three-dimensional medium containing EGF and a ROCK inhibitor, thereby obtaining a cell population expressing a lacrimal gland-associated protein.
[4] The production method according to any one of the above [1] to [3], wherein the medium further contains TGF-. beta.s.
[5] The method according to any one of [1] to [4], wherein the related protein is at least one selected from the group consisting of AQP5, LYZ, CNN1, BARX2, SOX9, SOX10, RUNX1, TFCP2L1, LTF and
[6] A method for producing a lacrimal gland-like organ for transplantation, comprising a step of culturing a lacrimal epithelial cell obtained by the production method according to any one of the above [1] to [5 ].
[7] A method for screening a drug for a lacrimal-gland-related disease, comprising a step of culturing a lacrimal-gland epithelial cell obtained by the production method according to any one of [1] to [5 ].
The present invention also includes the following [8] to [12 ].
[8] A tear fluid secreted from an organoid of lacrimal gland epithelial cells obtained by the method according to [6 ].
[9] A pharmaceutical composition comprising the tear fluid according to [8 ].
[10] A method for selecting a cell which is easily differentiated and induced into a lacrimal tissue derived from a stem cell, the method comprising selecting the shape of a cell population formed by the production method according to any one of [1] to [5] as an index.
[11] A lacrimal gland-like organ obtained by the method according to any one of the above [1] to [6 ].
[12] A lacrimal gland-like organ obtained by culturing the cells selected by the method according to [10 ].
Effects of the invention
According to the present invention, since a tissue that reliably expresses a lacrimal gland-associated protein including a functional protein to be expressed by a lacrimal gland can be obtained using a stem cell, a fundamental regenerative therapy and a next-generation therapy for a disease caused by abnormality of the lacrimal gland itself can be provided by using the obtained tissue.
In addition, the method can be applied to drug screening targeting lacrimal glands, which has not been performed so far because of difficulty in obtaining lacrimal gland cells. In addition, lacrimal fluid secreted from the obtained lacrimal gland tissue can be used as a component of a new drug.
Drawings
Fig. 1 is a graph showing the results of immunofluorescence staining of a two-dimensional tissue body (SEAM) composed of four concentric band-like structures induced by iPS cells.
Fig. 2 is a diagram showing an exemplary process of inducing iPS cells into lacrimal gland organoids.
Fig. 3 is a graph showing the results of gene expression analysis in lacrimal gland organoids derived from iPS cells.
Fig. 4 is a graph showing the results of immunofluorescence staining of lacrimal organoids derived from iPS cells.
Fig. 5 is a graph showing the results of studying induction conditions (kinds of growth factors) of lacrimal gland organoids derived from iPS cells.
Fig. 6 is a graph showing the results of studying induction conditions (TGF- β concentration) of lacrimal gland organoids derived from iPS cells.
Fig. 7 is a graph showing the results of studying induction conditions (colony shapes) of lacrimal gland organoids derived from iPS cells.
Fig. 8 is a graph showing the results of morphological and gene expression analysis of cells induced from limbal stem cells.
Fig. 9 is a graph showing the results of HE staining of a graft obtained by transplanting a lacrimal gland organoid derived from iPS cells to a nude rat.
Fig. 10 is a graph showing the results of immunofluorescence staining of a graft obtained by transplanting a lacrimal gland organoid derived from iPS cells into a nude rat, and measurement of the amount of hLTF protein in the graft extract.
Fig. 11 is a diagram showing an example of a process of forming lacrimal gland organoids derived from ES cells.
Detailed Description
The present inventors obtained a cell population (self-forming ectodermal autonomous multizone: SEAM) that reproduces the occurrence of the entire eye from iPS cells, isolated specific progenitor cells from the obtained cell population, induced the differentiation of the cells, and cultured the cells for maturation as necessary to prepare a high-purity cell population, for example, corneal epithelial cells. Here, when cells obtained from SEAM are cultured for differentiation induction and maturation, the culture is not particularly limited as long as the cells can be cultured in the presence of growth factors corresponding to a desired cell population, and the culture vessel is a vessel used for cell culture. In the above-mentioned eye cell population reproduced from iPS cells, the eye site corresponding to each cell is identified, but progenitor cells corresponding to lacrimal epithelial cells are not known. The present inventors have therefore conducted intensive studies on the induction of differentiation of progenitor cells obtained by the above-described method into lacrimal epithelial cells, and as a result, have found that by three-dimensionally culturing a progenitor cell group, which has been known to be conventionally induced to differentiate into corneal epithelial cells, in the presence of a specific growth factor, it is possible to induce differentiation into lacrimal epithelial cells and obtain a lacrimal gland-like tissue expressing a lacrimal gland-related protein such as a functional protein equivalent to the lacrimal gland. In addition, as a result of the same study on cell groups other than the cell group obtained from pluripotent stem cells such as iPS cells, it has been found that there are many progenitor cells induced to differentiate into corneal epithelial cells, but limbal stem cells induced to differentiate into corneal epithelial cells in two-dimensional culture are induced to differentiate into lacrimal epithelial cells by performing the three-dimensional culture to obtain lacrimal gland-like tissues. The detailed mechanism of differentiation and induction into lacrimal epithelial cells by three-dimensional culture is not clearly understood, and it is considered that the cells can be developed in all directions while having polarities on the luminal side and the basement membrane side by forming the scaffold of the cells into three-dimensional form, unlike in the conventional two-dimensional culture, to form a three-dimensional structure such as a duct or an acinar structure, thereby inducing characteristic lacrimal epithelial cells. However, these assumptions do not limit the present invention.
The present invention provides a method for inducing differentiation of lacrimal epithelial cells, which has the following characteristics: the specific progenitor cells derived from SEAM are cultured in three dimensions in the presence of specific factors. Specifically, a cell population expressing a lacrimal gland-associated protein can be obtained by isolating SSEA4 and CD104 double-positive cells from a SEAM cell population (self-forming ectodermal autonomous multizone cell population) obtained from pluripotent stem cells, and three-dimensionally culturing the obtained cells in a medium containing EGF (epidermal growth factor) and a ROCK inhibitor.
In the present invention, a SEAM cell population is first prepared from pluripotent stem cells.
The pluripotent stem cells in the present invention are those which have a multipotent differentiation potential to differentiate into all cells present in the body and also have a proliferative capacity. Specific examples thereof include embryonic stem cells (ES cells), embryonic stem cells (ntES cells) derived from a cloned embryo obtained by nuclear transfer, sperm stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells, and the like. Preferred are ES cells, ntES cells and iPS cells, and more preferred are ES cells and iPS cells. The pluripotent stem cells are preferably mammalian pluripotent stem cells. The mammal is not particularly limited, and examples thereof include humans, mice, rats, cows, pigs, and the like. Among them, human is preferred. By using human pluripotent stem cells, a cell population corresponding to a safe cell species applicable to human regenerative medicine can be obtained.
The SEAM cell population is a colony formed of concentric layers of different ectodermal cell species, which is obtained by two-dimensionally culturing pluripotent stem cells in a serum-free medium without using feeder cells.
Specifically, for example, the culture medium can be obtained by two-dimensionally culturing human iPS cells using a known culture medium (e.g., DMEM medium, BME medium, etc.) that can be used for culturing animal cells and a culture medium that does not contain unmodified or unpurified serum. In this case, the culture vessel is not particularly limited as long as it is a vessel used for two-dimensional cell culture, but preferably the inner surface of the vessel is coated with collagen, fibronectin, laminin fragments, or the like. The culture conditions are not particularly limited, and may be appropriately set according to the technical knowledge.
The SEAM cell population thus obtained by culture is formed by autonomous differentiation of the cells themselves without being subjected to external stimuli such as a differentiation inducer and a differentiation induction promoter, and forms a layered colony composed of ectodermal cell species. The colonies comprise concentric layers of different ectodermal cell species from the central portion toward the peripheral portion, including a first layer (neuroectodermal cells), a second layer (neural crest cells/ocular embryonic cells), a third layer (ocular ectodermal cells), and a fourth layer (surface ectodermal cells). After the autonomous differentiation, the differentiation culture may be continued, and in this case, various growth factors such as ROCK inhibitors and serum substitutes, various nutrient sources necessary for the maintenance and proliferation of cells, and various components necessary for the induction of differentiation may be added to the medium used for the autonomous differentiation. As a method for producing the above-mentioned SEAM, for example, methods described in non-patent documents 2 and 3 and
In the obtained colony, since the cell species contained in each layer are different series, the cells contained in a specific layer can be isolated by the colony and used, or the entire colony can be used as it is.
As described above, the SEAM cell population in the present specification refers to, for example, a cell population obtained as follows: the pluripotent stem cells are cultured at a concentration of 100 to 700 cells/cm2Was inoculated into a plate coated with a laminin 511E8 fragment and cultured in StemFit (registered trademark)After being maintained in nutrient medium (monosodium glutamate) for 8-10 days, the cells are cultured in a differentiation medium [ DM; GMEM medium (Life Technologies) containing 10% knockout serum replacement (KSR, Life Technologies), 1mM sodium pyruvate (Life Technologies), 0.1mM non-essential amino acids (Life Technologies), 2mM l-glutamine (Life Technologies), 1% penicillin-streptomycin solution (Life Technologies), and 55 μ M2-mercaptoethanol (Life Technologies)]Medium for 4 weeks, followed by incubation in differentiation medium [ CDM; DM medium containing 10-20 ng/mL KGF (Wako), 10 MuMY-27632 (Wako) and 1% penicillin-streptomycin solution and 1:1(v/v) mixed medium of CnT-20 or CnT-PR medium (CELLnTEC Advanced Cell Systems without EGF and FGF 2)]After 4 weeks of medium culture, the first and second layers formed were removed as desired and replaced with maintenance medium [ CEM; DMEM/F-12(2:1(v/v)) medium (Life Technologies) containing 2% B27 supplements (Life Technologies), 1% penicillin-streptomycin solution, 10-20 ng/mL KGF, 10. mu. MY-27632]Thereafter, the cells are further cultured for about 3 days to about 2 weeks, and differentiation induction is performed.
Subsequently, cells that were both SSEA4 and CD104 positive were isolated from the resulting SEAM cell population.
The invention has the following characteristics: the cell population obtained by using the expression of SSEA4 and CD104 as an index and using these markers is originally differentiated into corneal epithelial progenitor cells, but surprisingly can be further differentiated into lacrimal gland cells by performing three-dimensional culture as described later.
The isolation method is not particularly limited as long as the target cells can be isolated from the SEAM cell population using the expression of the marker as an index, and can be easily carried out by a conventional method using an antibody specific to each marker. Specifically, the separation may be performed by using, for example, separation using magnetic beads labeled with an antibody, a column immobilized with an antibody, or a cell sorter (FACS) using a fluorescent-labeled antibody. The antibody may be a commercially available antibody or an antibody prepared by a conventional method. Before the positive selection based on SSEA4 and CD104, CD 200-based negative selection may be performed with reference to the selection, in which case the cells to be separated are CD200-, SSEA4+, and CD104 +.
The isolated cells may be directly subjected to three-dimensional culture, but are preferably formed into spheres (cell aggregates). In addition, before forming spheres, the separated cells may be classified based on their characteristics and used.
The isolated cells were selected using expression of SSEA4 and CD104 as an index, but it is considered that the cells include various cells as their states. Therefore, the present inventors have studied the differentiation culture after classifying the isolated cells based on various characteristics, and as a result, have found that the degree of progression of differentiation varies depending on the shape thereof. In the present invention, for example, the ability to induce differentiation into lacrimal epithelial cells can be controlled by culturing cells classified into planar (flat) and three-dimensional (dome) shapes collectively, and it is preferable to form spheres using dome-shaped cells.
Spheres may be formed by culturing the isolated cells. The medium to be used is not particularly limited as long as it is a known medium that can be used for culturing animal cells and contains no unadjusted or unpurified serum, and it may be added with various Growth factors such as ROCK inhibitor, KGF (Keratinocyte Growth Factor), and serum replacement, if necessary. The culture vessel is preferably a culture vessel having a low adhesion surface, for example, a PrimeSurface (registered trademark) 96U plate or the like can be used, because it is preferable to suppress the specific or non-specific immobilization and to form spheres by floating the cells. The culture conditions may be appropriately set according to the general technical knowledge.
In this way, spheres of the isolated cells can be formed. The diameter of the sphere is about 10-1000 μm, preferably 100-500 μm, and the sphere has the characteristics of the expression increase of lacrimal gland epithelial cell specific markers such as BARX2, SOX9, KRT15 and the like or differentiation markers to glandular cells, and is suitable for the subsequent three-dimensional culture.
In the three-dimensional culture, any known three-dimensional culture method may be used as long as the obtained spheres can be cultured in a medium containing EGF and ROCK inhibitor. Specifically, for example, a method of culturing cells using a biocompatible material such as hydrogel, laminin, collagen, fibronectin, fibrin, vitronectin, Matrigel (registered trademark), integrin, glycosaminoglycan or the like as a cell culture carrier is exemplified.
In order to promote the differentiation induction into lacrimal epithelial cells, the medium in three-dimensional culture contains growth factors such as EGF and ROCK inhibitor. The minimal medium may be any medium (serum-free medium) that can be used for culturing epithelial cells, such as a medium used for autonomous differentiation. In addition, serum replacement may also be included. In the present specification, the term "ROCK inhibitor" refers to a substance that inhibits Rho kinase (Rho-associated, conjugated-coilcontaining protein kinase), and may be, for example, N- (4-pyridyl) -4 beta- [ (R) -1-aminoethyl-forming protein kinase]Cyclohexane-1 alpha-carboxamide (Y-27632), Fasudil (Fasudil) (HA1077), (2S) -2-methyl-1- [ (4-methyl-5-isoquinolinyl) sulfonyl]hexahydro-1H-1, 4-diazepines(H-1152), 4 beta- [ (1R) -1-aminoethyl]-N- (4-pyridyl) benzene-1 α -carboxamide (Wf-536), N- (1H-pyrrolo [2, 3-b)]Pyridin-4-yl) -4 beta- [ (R) -1-aminoethyl]Cyclohexane-1 alpha-carboxamide (Y-30141), N- (3- { [2- (4-amino-1, 2, 5-)Oxadiazol-3-yl) -1-ethyl-1H-imidazo [4,5-c]Pyridin-6-yl]Oxy } phenyl) -4- { [2- (4-morpholinyl) ethyl]Oxy } benzamide (GSK269962A), N- (6-fluoro-1H-indazol-5-yl) -6-methyl-2-oxo-4- [4- (trifluoromethyl) phenyl]-3, 4-dihydro-1H-pyridine-5-carboxamide (GSK 429286A). Examples of the "serum replacement" include albumin (e.g., lipid-rich albumin), transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc and selenium), B27 (registered trademark) supplement, N2 supplement, and the like. In the case of B27 supplement, the concentration in the medium is 0.01 to 10% by weight, preferably 0.5 to 4% by weight.
In the present invention, TGF-. beta.may be contained to promote maturation of the obtained cells. The TGF-. beta.is not particularly limited, and may be any of TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3. The concentration in the medium is 0.1 to 200ng/mL, preferably 1 to 200 ng/mL.
The medium may contain various nutrients necessary for cell growth and various components necessary for differentiation induction. For example, the nutrient source may include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, and the like, hydrocarbons such as fatty acids, fats and oils, lecithin, alcohols, and the like, nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, sodium nitrate, and the like, inorganic salts such as salt, potassium salt, phosphate, magnesium salt, calcium salt, iron salt, manganese salt, and the like (for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, manganese sulfate), various vitamins, amino acids, and the like. The contents of these components can be adjusted according to the common technical knowledge.
The culture conditions may be appropriately set according to the general technical knowledge. For example, 1 to 25% O at 36 to 38 ℃, preferably 36.5 to 37.5 ℃2、1～15％CO2Under the conditions of (1).
By the three-dimensional culture, a cell population (lacrimal epithelial cells) expressing a protein related to a lacrimal gland can be obtained. The cell population may be organoids (lacrimal organoids).
In the present invention, proteins related to lacrimal gland include functional proteins secreted into lacrimal fluid and functioning on the surface of the eye. Specific examples thereof include LTF, LYZ, HTN1, MUC1, MUC4, MUC5AC, MUC5B, MUC6, MUC7, LACRT and sIgA. LTF is an iron-binding glycoprotein and is a protein having antibacterial and antiviral activities. LYZ is a protein that breaks down the cell wall of eubacteria. HTN1 is a protein with antibacterial activity and is also used as a marker for lacrimal epithelial cells. The MUC family are glycoproteins involved in the retention of water on the ocular surface. LACRT contributes to proliferation of ocular surface cells. sIgA plays a central role in mucosal immunity. Preferably, expression of at least one of these is confirmed.
In addition to the analyses relating to the functional proteins, the gene analysis of the obtained cells confirmed the expression of AQP5, SOX9, SOX10, RUNX1, KRT14, TFCP2L1, CNN1, BARX2, PAX6, and the like. In the present invention, these proteins and the above-mentioned functional proteins are included as lacrimal gland-associated proteins or lacrimal gland-associated proteins. AQP5 is a protein having a pore existing in a cell membrane, and is one of typical proteins in the lacrimal gland that supply water of tears to acinar cells. SOX9 and SOX10 are transcription factors that play an important role in the development of lacrimal gland, and are located downstream of the signal from FGF10, contributing to the development of lacrimal gland. In particular SOX10 contributes to the differentiation of acinar cells. RUNX1 is also a transcription factor known to play an important role in the development of lacrimal glands. KRT14 is a cytoskeletal-forming protein that is observed in cells on the basal lamina side in lacrimal gland epithelial cells during the development of the lacrimal gland, and is also used as a marker for myoepithelial cells in the mature lacrimal gland. TFCP2L1 is a transcription factor required for differentiation of ductal cells of the lacrimal gland, and CNN1 is a protein expressed in myoepithelial cells of the lacrimal gland and controlling contraction of the actomyosin system. In addition, BARX2 is a transcription factor, and is expressed significantly in the process of lacrimal gland differentiation, as in KRT15, MMP2 is directly targeted, and it is known that it contributes to the formation of branches in lacrimal gland tissue, and their expression is sometimes confirmed. In the present invention, the protein whose expression is confirmed in the obtained cell population is not particularly limited as long as it is a protein related to lacrimal gland, and examples thereof include at least one selected from AQP5, LYZ, CNN1, BARX2, SOX9, SOX10, RUNX1, TFCP2L1, LTF, and
The present invention also provides a method of using the limbal stem cells as cells to be subjected to three-dimensional culture in the above-described differentiation induction method. That is, the present invention provides a method for producing a cell population expressing a lacrimal gland-associated protein by three-dimensionally culturing limbal stem cells in a medium containing an EGF and a ROCK inhibitor. It has been known so far that limbal stem cells differentiate into corneal epithelial cells by two-dimensional culture, but differentiation into lacrimal epithelial cells by three-dimensional culture was the first discovery of the present inventors. In the induction of differentiation of limbal stem cells, TGF-. beta.may be included in the culture medium to promote maturation of the cells obtained.
The limbal stem cells may be obtained by separately inducing differentiation from pluripotent stem cells, or may be collected from a living body and used after purification or the like as needed. When collected from an organism, the resulting cell population has the advantage that there is no possibility of generating a rejection reaction. The conditions for three-dimensional culture were the same as described above, except that the cells used were limbal stem cells.
The lacrimal epithelial cells obtained by the production method of the present invention can be used for research, regenerative medicine, and the like.
Specifically, for example, the lacrimal epithelial cells produced by the production method of the present invention can be expected to be further matured in vivo and further form a functional lacrimal tissue by directly transplanting an organoid, for example. Furthermore, it is expected that the regeneration can be promoted by dispersing organoids and administering cells in the form of a suspension to allow the cells to survive as a part of lacrimal gland tissue. The present invention therefore encompasses a method for producing a lacrimal epithelial cell organoid for transplantation, which comprises a step of culturing a lacrimal epithelial cell produced by the production method of the present invention. Since the culture of lacrimal gland epithelial cells can be performed in the same manner as the three-dimensional culture, a medium containing no TGF- β can be used as the medium for the three-dimensional culture in consideration of maturation in the living body after transplantation.
In addition, since the lacrimal epithelial cells produced by the production method of the present invention have proteins related to lacrimal glands reflecting living bodies, they can contribute greatly to, for example, evaluation of the efficacy of a therapeutic agent for a lacrimal-gland-related disease, analysis of the pathogenesis, and the like. Examples of the lacrimal gland-related diseases (diseases caused by abnormalities of the lacrimal gland itself) include dry eye syndrome, sjogren's syndrome, GvHD, and lacrimal gland tumors.
Specifically, for example, with respect to the lacrimal gland, it is known that tear volume is increased by increasing the expression of aquaporins (e.g., AQP 5). In addition, it is known that a decrease in the amount of Lactoferrin (LTF) is associated with dry eye symptoms, and therefore, by increasing the expression of functional proteins in tears such as lactoferrin, it is expected that dry eye symptoms will be alleviated. Therefore, it is considered that the use of the lacrimal gland-like organ obtained by the present invention for selecting a drug in which the expression and activity of the lacrimal gland-related protein are improved can be suitably used for screening of a drug for treating dry eye. Accordingly, the present invention includes a method for screening a drug for a lacrimal gland-related disease, which comprises a step of culturing a lacrimal gland epithelial cell produced by the production method of the present invention. Since the culture step can be performed in the same manner as the three-dimensional culture at the time of differentiation induction of lacrimal gland epithelial cells, a medium containing no TGF- β can be used as the medium for three-dimensional culture in consideration of the expression of lacrimal gland-associated proteins.
The screening method of the present invention is not particularly limited as long as it includes the above-mentioned culture step, and examples thereof include the following: in the lacrimal gland epithelial cells of the present invention obtained by the above-described culture step, a substance that promotes expression or activity is selected as a candidate compound for treating a lacrimal gland-related disease, using the expression of a gene encoding a lacrimal gland-related protein or the activity of a lacrimal gland-related protein as an index. Specifically, for example, in the lacrimal gland epithelial cells of the present invention after being contacted with a test substance, the expression amount of a gene encoding a lacrimal gland-associated protein or the activity value of the lacrimal gland-associated protein is measured, and in the case where the expression amount or the activity value is increased as compared with that in control cells not contacted with the test substance, the test substance can be selected as a candidate compound for treating a lacrimal gland-associated disease.
Further, since the lacrimal gland epithelial cells produced by the production method of the present invention reflect living tissues and secrete tears, tears obtained from lacrimal gland epithelial cell organs produced by the production method of the present invention can be collected in, for example, a test tube and used as a component of a new drug. Accordingly, the present invention also provides a pharmaceutical composition comprising a tear fluid obtained from the lacrimal gland epithelial cell organoid produced by the production method of the present invention. Examples of the pharmaceutical composition include tear eye drops, dry eye drops, antibacterial eye drops, antiviral eye drops, antiallergic eye drops, corneal therapeutic eye drops, steroidal anti-inflammatory eye drops, non-steroidal anti-inflammatory eye drops, asthenopia eye drops, cataract eye drops, glaucoma eye drops, and the like, and if the above tear fluid is contained, the blending of other components and the production method can be appropriately adjusted according to known techniques.
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