WO2024071382A1

WO2024071382A1 - Pluripotential stem cell and cell differentiated and induced from stem cell having glp-1 secretion function

Info

Publication number: WO2024071382A1
Application number: PCT/JP2023/035640
Authority: WO
Inventors: 雅子岡; 晋羽藤
Original assignee: 株式会社セルージョン
Priority date: 2022-09-30
Filing date: 2023-09-29
Publication date: 2024-04-04

Abstract

The present invention addresses the problem of providing a pluripotent stem cell and a cell (especially, an endothelial cell) differentiated and induced from a stem cell, to which a new function is imparted. The pluripotent stem cell having a GLP-1 secretion function or the corneal endothelium replacement cell that is induced from a pluripotent stem cell and that has a GLP-1 secretion function according to the present invention has a function to secrete GLP-1 for a long period and thus enables development of a novel cellular therapy for diabetes or the like.

Description

Pluripotent stem cells having GLP-1 secretion function and cells differentiated from the stem cells

The present invention relates to pluripotent stem cells having the function of secreting GLP-1 (Glucagon-like peptide-1) and cells induced to differentiate from the stem cells. The present invention also relates to endothelial cells induced to differentiate and have the function of secreting GLP-1.

Corneal endothelial cell substitute from iPS cells (CECSi cells) have been developed for the treatment of bullous keratopathy (Patent Documents 1 to 3). On the other hand, CECSi cells have the advantage that they can be used to mass-produce differentiated endothelial cells with uniform quality and high adhesiveness to cells and extracellular matrix from iPS cells in a short period of time (approximately two weeks).
If it is possible to give additional functions to CECSi cells by gene editing or gene introduction while taking advantage of this characteristic, it is possible to aim to expand the application to other disease areas as well as the treatment of the cornea region. As gene editing techniques, for example, techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems have been developed. Gene introduction techniques are broadly divided into non-viral introduction methods and introduction methods using viruses, and in gene therapy, gene introduction techniques using viruses are used, and many viral vectors have been developed.

Regarding the genes to be introduced, various types of genes that are useful for human gene therapy have been attempted. For example, Patent Document 4 describes a transfected cell that expresses glucagon-like peptide-1 (GLP-1).
GLP-1 is a hormone secreted from L cells in the small intestine, which acts on the GLP-1 receptor present in the β cells of the pancreas to promote insulin secretion. GLP-1 receptor agonists are actually used to treat diabetes.

WO2013/051722 WO2016/093359 WO2019/142833 JP 2003-174897 A

The objective of the present invention is to provide pluripotent stem cells to which new functions have been imparted, and cells induced to differentiate from stem cells. In particular, the objective of the present invention is to provide corneal endothelial replacement cells to which new functions have been imparted, which are induced from pluripotent stem cells, particularly iPS cells.

In light of the above problems, the inventors attempted to impart GLP-1 secretion function to the corneal endothelial replacement cells that they had been developing. They induced the differentiation of iPS cells into corneal endothelial replacement cells, introduced the GLP-1 gene into the resulting corneal endothelial replacement cells, expressed the gene, and confirmed that the GLP-1 protein was secreted, thus completing the present invention.

That is, the present invention provides the following.
[1] A pluripotent stem cell or a cell induced to differentiate from a stem cell, which has a GLP-1 secretion function.
[2] The cell described in [1] above, wherein the cell induced to differentiate from a stem cell is an endothelial cell.
[3] The cell described in [1] or [2] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[4] The cell described in [2] above, wherein the endothelial cell is a corneal endothelial cell.
[5] The cell according to any one of [1] to [4] above, which expresses GLP-1.
[A] CECSi cells having GLP-1 secretion function.
[6] A method for producing a cell expressing GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into a cell, wherein the cell is a pluripotent stem cell or a cell induced to differentiate from a stem cell.

[7] The method described in [6] above, wherein the introduction of the nucleic acid into the cell is by gene introduction or genome editing.
[8] The method according to [6] or [7] above, wherein the cells induced to differentiate from stem cells are endothelial cells.
[9] The method according to any one of [6] to [8] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[10] The method described in [8] above, wherein the endothelial cells are corneal endothelial cells.
[B] A method for producing CECSi cells expressing GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into CECSi cells.
[11] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:
[12] The method according to [11] above, wherein the cells induced to differentiate from stem cells are endothelial cells.
[13] The method according to [11] or [12] above, wherein the pluripotent stem cell or stem cell is an iPS cell.
[14] The method described in [12] above, wherein the endothelial cells are corneal endothelial cells.
[C] (1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a CECSi cell using an expression vector containing the nucleic acid to prepare a CECSi cell containing the expression vector; and (3) culturing the CECSi cell containing the expression vector.
A method for producing a CECSi cell expressing GLP-1, comprising:
[15] A pharmaceutical composition comprising the cell according to any one of [1] to [5] and [A] above.
[16] The pharmaceutical composition according to [15], which is for treating diabetes, obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction, taste disorders, and pneumonia.

The present invention has made it possible to endow CECSi cells with the ability to secrete GLP-1. CECSi cells that have the ability to secrete GLP-1 for a long period of time will enable the development of new cell therapies for diabetes and other conditions.

FIG. 1 shows that iPS cells were induced to differentiate into CECSi cells by immunostaining for PITX2, N-Cadherin, and ZO-1. This figure shows the results of confirming GLP-1 expression in CECSi cells 48 hours after transfection with each plasmid (sig-peptide, sig-GLP-1), showing the image observed by a fluorescent microscope (A) and the amount of EGFP (GFP) in the cell culture supernatant (B). This figure shows the results of confirming GLP-1 expression in CECSi cells 65 hours after transfection with each plasmid (sig-peptide, sig-GLP-1), showing the images observed by a fluorescence microscope (A) and the amount of EGFP (GFP) in the cell culture supernatant (B). This is a diagram showing the results of confirming GLP-1 expression in CECSi cells 24 hours after transfection with each plasmid (sig-peptide, sig-GLP-1). Fluorescence microscopy images (A) and intracellular mRNA expression levels (B) are shown. n=2, mean ± standard error This is a diagram showing the results of confirming GLP-1 expression in CECSi cells 5 days after transfection with each plasmid (sig-peptide, sig-GLP-1). Fluorescence microscopy images (A) and intracellular mRNA expression levels (B) are shown. n=2, mean ± standard error The construction diagram of various plasmids (sig-peptide (a), sig-GLP-1 (b), sig-GFP (c), sig-GLP-1-GFP (d)) is shown. When constructing a pAAV vector with sig-GLP-1, IRES was used instead of T2A. This figure shows the results of confirming GLP-1 expression in CECSi cells 19 hours after transfection with each plasmid (sig-peptide (a), sig-GLP-1 (b), sig-GFP (c), sig-GLP-1-GFP (d)). Detection was performed by Western blotting using an anti-GFP antibody. Western blot images are shown. 1 shows the results of confirming GLP-1 expression in CECSi cells 13 days after infection with AAV containing each plasmid (sig-GFP, sig-GLP-1-GFP), with (A) images observed by a fluorescence microscope and (B) amounts of EGFP (GFP) in the cell culture supernatant. This figure shows the results of confirming GLP-1 expression in CECSi cells 48 hours after infection with AAV containing each plasmid (sig-peptide, sig-GLP-1), showing the image observed by a fluorescence microscope (A) and the amount of EGFP (GFP) in the cell culture supernatant (B). 1 shows the results of confirming GLP-1 expression in CECSi cells 32 days after infection with AAV containing each plasmid (sig-peptide, sig-GLP-1). 1 shows the results of co-culturing sig-GLP-1-GFP (fusion type)-secreting CECSi cells and human iPS-derived pancreatic β cells to examine whether or not c-peptide secretion is promoted. 1 shows the results of co-culturing sig-GLP-1-GFP (fusion type)-secreting CECSi cells with human iPS-derived pancreatic β cells to verify the presence or absence of c-peptide secretion promotion. The concentration (ng/ml) of c-peptide secreted from pancreatic β cells in the medium is shown. n=2, mean ± standard error This figure shows the results of examining the effect on blood glucose control when sig-GLP-1-GFP (fusion type)-introduced CECSi cells were transplanted intraperitoneally into healthy SCID mice. This is a graph showing the results of an oral glucose tolerance test (OGTT) in mice that were transplanted intraperitoneally with sig-GLP-1-GFP (separated type)-introduced CECSi cells. Suppression of blood glucose rise was confirmed in mice transplanted with ATCC CESCi cells (sig-GLP-1) that secrete sig-GLP-1 compared to controls. 1 is a graph showing the results of examining the effect on blood glucose control when sig-GLP-1-GFP (fusion type)-introduced CECSi cells are intraperitoneally transplanted into healthy SCID mice. 2 is a graph showing the results of measuring the amount of sig-GLP-1-GFP in the blood at the start of OGTT and 0.5 hours after the start.

The present invention is described below. Terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified.

In the present invention, the term "GLP-1 secretion function" refers to a function capable of producing GLP-1 in cells and then secreting it outside the cells. In the present specification, GLP-1 refers to active human glucagon-like peptide-1, i.e., GLP-1 having the amino acid sequence HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 1], GLP-1 (7-37) having the amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG [SEQ ID NO: 2], which is its active form, or a variant thereof. In the present specification, the term "variant" of GLP-1 refers to an amino acid sequence in which one or more amino acids have been changed from the native sequence by substitution, deletion or insertion of one or more amino acid residues without changing the activity of GLP-1. A "GLP-1 variant" may have one or more deleted amino acid residues, or may be a fragment of GLP-1 shorter than GLP-1(7-37) that has the same activity as the native GLP-1 sequence, for example, the activity of promoting insulin secretion by pancreatic β cells, by having an amino acid residue important for GLP-1 activity or a residue that can be substituted for one or more of the important amino acid residues. In this specification, unless otherwise specified, "GLP-1" is a concept that includes all of these.
Here, "one or more amino acid residues" may be 2, 3, 4, or 5 amino acid residues. "One or more amino acid residues" is preferably 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid residue.
In the GLP-1 secretion function, as long as the biological action expected from the secretion of GLP-1 (e.g., preinsulin (c-peptide) secretion promoting effect, insulin secretion promoting effect, glucagon secretion suppressing effect, pancreatic β cell proliferation effect, weight loss, glucose uptake effect, renal protective effect, cardiac protective effect, neuroprotection, delayed gastric emptying, appetite suppression, diuretic effect) is observed, the amount is not limited and differs depending on the type of biological action serving as an indicator of GLP-1 secretion. It is sufficient if there is a significant difference between the case where the function is exerted and the case where it is not exerted.

A preferred indicator of GLP-1 secretory function is its insulin secretion promoting effect.

1. Cells The present invention provides cells having a GLP-1 secretion function (hereinafter, also simply referred to as the cells of the present invention). Examples of the cells include pluripotent stem cells and cells obtained by inducing differentiation from the pluripotent stem cells or stem cells (hereinafter, also simply referred to as differentiated cells), in particular endothelial cells such as corneal endothelial replacement cells.

Stem cells are cells that have the ability to replicate themselves and differentiate into multiple other cell lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and skin stem cells.

Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating Stress Enduring cells) obtained from mesenchymal stem cells (MSCs), and GS cells produced from germ cells (e.g. testes) are also included in pluripotent stem cells. When simply referring to stem cells, it is intended to include pluripotent stem cells.

ES cells can be produced by culturing the inner cell population on feeder cells or in medium containing leukemia inhibitory factor (LIF). They are also available from designated institutions and can be purchased commercially. Nuclear transfer ES cells (ntES cells), a type of ES cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.

EG cells can be produced by culturing primordial germ cells in a medium containing mouse stem cell factor (mSCF), LIF, and basic fibroblast growth factor (bFGF) (Cell, 70:841-847, 1992).

iPS cells are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specific examples of iPS cells include cells in which pluripotency has been induced by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., through the expression of multiple genes selected from a group of reprogramming genes including Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. In 2006, Yamanaka et al. established induced pluripotent stem cells from mouse cells (Cell, 2006, 126(4) pp.663-676). In 2007, induced pluripotent stem cells were established from human fibroblasts, and like embryonic stem cells, they have pluripotency and the ability to self-replicate (Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106). In addition to producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp.651-654).

　Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells, T cells, etc.), liver cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

When producing artificial pluripotent stem cells, if initialization is performed by expression of several types of genes (e.g., four factors: Oct3/4, Sox2, Klf4, and Myc), the means for expressing the genes is not particularly limited. Examples of means for expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, and electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, and electroporation method), and direct protein injection methods.

It is also possible to obtain established induced pluripotent stem cells. Specifically, the iPS cells include 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 836B3, FF-I14s03, FF-I01s04, MH09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs09, QHJI14s03, QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs 14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all from iPS Academia Japan, Inc., or Kyoto University iPS Research Foundation), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), Toe (JCRB1338 strain), and Lollipop (JCRB1336 strain) (all from the National Center for Child Health and Development, National Institute of Biomedical Innovation, Department of Intractable Diseases) ATCC-DYP0730, ATCC-DYP0250, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYP0530, ATCC-DYP0530, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYP0530, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-HYR0103 ... C-DYS0100, ATCC-HYS0103, ATCC-CYS0105, KYOU-DXR0109B, ATCC-BYS0110, ATCC-BYS0111, ATCC-BYS0112, ATCC-BYS0113, ATCC-BXS0114, ATCC-BXS0115, ATCC-BXS0116, ATCC-BXS0117 (all from the non-profit American Type Culture Collection) can be used.

"Mammals" includes rodents, ungulates, felines, lagomorphs, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Felidae include dogs and cats. Lagomorphs include rabbits, etc. "Primates" refers to mammals belonging to the order Primates, and includes prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans.

The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, and most preferably human pluripotent stem cells.

The differentiated cells used in the present invention include those induced to differentiate from the above stem cells, preferably the above pluripotent stem cells. For example, endothelial cells, preferably corneal endothelial cells, more preferably corneal endothelial-like cells developed by the present inventors, so-called corneal endothelial substitute cells, can be produced and prepared by the methods described in Patent Documents 1 to 3. Preferably, they are corneal endothelial cell substitute from iPS cells (CECSi cells), which have corneal endothelial cell-like properties and functions and are characterized by enhanced expression of the NR3C2 (nuclear receptor subfamily 3, group C, member 2) gene (Patent Document 3).
An example of a method for producing corneal endothelial replacement cells is as follows.
The iPS cells are cultured for one week in a culture dish coated with iMatrix-511 (0.6 μg/cm ² ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells are seeded again on a culture dish coated with iMatrix-511 (0.3 μg/cm ² ), and differentiation induction culture of the iPS cells into corneal endothelial substitute cells is performed for 8 to 14 days using the differentiation induction medium described below (Table 1). Note that when using cryopreserved iPS cells, differentiation induction is performed after two passages and culture.

Specific examples of the corneal endothelial cell-like properties and functions possessed by the corneal endothelial substitute cells include the following characteristics (i) to (iv), and among these characteristics, the cells have at least one, preferably two, more preferably three, and even more preferably all four.
(i) Cell-cell adhesion is composed of N-cadherin.
(ii) Tight junctions are formed between the cells.
(iii) Express the Na,K-ATPase α1 subunit on the cell membrane.
(iv) Expression of the transcription factor PITX2 is observed in the cell nucleus.
Whether or not the cell-cell adhesion is composed of N-Cadherin can be confirmed by immunostaining for N-Cadherin.
Whether or not tight junctions are formed between cells can be confirmed by observing the presence of ZO-1, a protein that constitutes tight junctions, using immunostaining for ZO-1, or by directly observing the structure using an electron microscope.
Whether or not Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining of ZO-1 and Na,K-ATPase α1 subunit by immunostaining.
Whether or not the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining for PITX2.

The cells of the present invention are the above-mentioned pluripotent stem cells or their differentiated cells, particularly endothelial cells, to which a GLP-1 secretion function has been imparted. The process of imparting a GLP-1 secretion function to cells will be described in detail in "2. Cell manufacturing method" below, but specifically, this is carried out by introducing a nucleic acid encoding GLP-1 into the above-mentioned pluripotent stem cells or their differentiated cells, particularly endothelial cells. Therefore, the cells of the present invention are cells that express GLP-1. In order to secrete GLP-1 outside the cells, it is preferable that a signal peptide is attached to the GLP-1 expressed in the cells. Therefore, it is preferable that the nucleic acid encoding GLP-1 introduced into the cells in the present invention is linked to a signal sequence.

2. Method for Producing Cells The present invention provides a method for producing cells having a GLP-1 secretion function (hereinafter, also simply referred to as the method for producing the cells of the present invention).
The method for producing cells of the present invention is characterized in that it imparts a GLP-1 secretion function to cells. Examples of cells include pluripotent stem cells and their differentiated cells, particularly endothelial cells such as corneal endothelial replacement cells.
The GLP-1 secretion function may be imparted to cells at any stage. For example, the GLP-1 secretion function may be imparted to pluripotent stem cells, or to differentiated cells, particularly endothelial cells, obtained by inducing differentiation of pluripotent stem cells. Examples of endothelial cells include CECSi cells, which are corneal endothelial replacement cells developed by the present inventors and described above in the section "1. Cells".

Specifically, a nucleic acid encoding GLP-1, preferably a nucleic acid linked to a signal sequence, is inserted into an appropriate expression vector (two types of expression vectors may be used if necessary). At that time, it is incorporated into the expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or promoter. Next, this expression vector is introduced into cells to express GLP-1.

One embodiment of the method for producing the cells of the present invention is the following method.
(1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:

There is no particular limitation on the type of expression vector that can be used as long as it stably retains the inserted gene, and various types of vectors can be used. The vector can be a viral vector or a non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors. Among these, in retroviral vectors, lentiviral vectors, and adeno-associated viral vectors, the target gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Each viral vector can be prepared according to a conventional method or using a commercially available dedicated kit. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, and artificial chromosome vectors.
These vectors can also be used in gene therapy, preferably using adeno-associated virus vectors.
In the case of a viral vector, the expression vector is introduced into a cell by infection with a virus. In the case of a non-viral vector such as a plasmid, a conventional method such as electroporation, lipofection, calcium phosphate, or nucleofection can be used for introduction into a cell, and the lipofection method is preferred.

The ability to secrete GLP-1 may be imparted to cells by genome editing. "Genome editing" is a technique for intentionally modifying a target gene or genome region by site-specific cleavage of a genomic DNA strand using a nuclease, or by chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), TALENs, and CRISPR/Cas9. By using genome editing techniques, it is possible to create knockout cell lines in which a specific gene has been deleted, knock-in cell lines in which a different sequence has been artificially inserted into a specific gene locus, and the like. In the present invention, a nucleic acid encoding GLP-1 is introduced into pluripotent stem cells or their differentiated cells, particularly endothelial cells, using genome editing techniques.

The nucleic acid encoding GLP-1 that is introduced to impart GLP-1 secretion function to cells is not particularly limited as long as it can cause the cells to express the desired protein (i.e., GLP-1). Examples of the base sequence of the nucleic acid to be introduced include the following.

The gene encoding GLP-1(7-37)
CATGCTGAAGGGACCTTTACCAGTGATGGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 3)

A gene encoding GLP-1 (7-37) (linked to a sequence encoding a signal peptide on the 5' side)
TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCGCATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA (SEQ ID NO: 4)

When the cells used in the step of imparting GLP-1 secretion function to the cells are pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably carried out in a medium for culturing pluripotent stem cells. As a medium for pluripotent stem cells, a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of pluripotent stem cells. For example, DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's Examples of the medium include BMOC-3 Medium, E8 medium (Nature Methods, 2011, 8, 424-429), ReproFF2 medium (ReproCell), StemFit (registered trademark) AK medium (Ajinomoto), and a mixture of these.
The thus obtained pluripotent stem cells having the function of secreting GLP-1 can be induced to differentiate into desired differentiated cells. Differentiation induction is appropriately determined depending on the type of differentiated cells to be obtained, and is carried out using known materials and methods. For example, corneal endothelial replacement cells, which are corneal endothelial-like cells, can be obtained by inducing differentiation of pluripotent stem cells having the function of secreting GLP-1 according to the descriptions in Patent Documents 1 to 3. The obtained corneal endothelial replacement cells have the function of secreting GLP-1.

When the cells used in the step of imparting GLP-1 secretion function to cells are differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells, which have been induced to differentiate from pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably performed in a medium for culturing differentiated cells, preferably endothelial cells, particularly corneal endothelial substitute cells. As such a medium, a known medium can be used, and is not particularly limited as long as it does not inhibit the proliferation of differentiated cells. For example, DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's BMOC-3 Examples of the medium include medium and mixed mediums thereof.
The differentiation of pluripotent stem cells into differentiated cells can be carried out by known methods. For example, the differentiation of pluripotent stem cells into corneal endothelial replacement cells can be carried out according to the descriptions in Patent Documents 1 to 3.

3. Pharmaceutical composition The present invention provides a pharmaceutical composition comprising cells capable of secreting GLP-1 as an active ingredient (hereinafter, also simply referred to as the pharmaceutical composition of the present invention). The cells capable of secreting GLP-1 contained as an active ingredient in the pharmaceutical composition of the present invention are the cells described above in "1. Cells" and are cells produced by the method described above in "2. Method for producing cells".

The pharmaceutical compositions of the present invention can be prepared by mixing the cells of the present invention, which are the active ingredient, with a pharma- ceutical acceptable carrier. The term "pharma-ceutical acceptable carrier" as used herein includes diluents, adjuvants, excipients, stabilizers, vehicles, or supports (such as cell fiber (alginate hydrogel)) that are non-toxic to cells exposed thereto at the doses and concentrations used. In many cases, the carrier is an aqueous pH buffer solution, an antioxidant, a low molecular weight (less than about 10 residues) polypeptide, a hydrophilic polymer, an amino acid, a monosaccharide, a disaccharide, a chelating agent such as EDTA, a salt-forming counterion such as sodium; and a non-ionic surfactant such as TWEEN (registered trademark), polyethylene glycol (PEG), and PLURONICS (registered trademark). In particular, for compositions administered intravenously, a preferred carrier is a saline solution.

The pharmaceutical composition of the present invention contains the cells of the present invention as an active ingredient, and the cells of the present invention are capable of secreting a therapeutically effective amount of GLP-1. A therapeutically effective amount is an amount that, when the pharmaceutical composition of the present invention is administered to a subject, can provide a therapeutic effect against the above-mentioned diseases, compared to a subject not administered the pharmaceutical composition. A specific therapeutically effective amount is appropriately determined depending on the administration method, the purpose of use, and the age, weight, symptoms, etc. of the subject.

In one embodiment of the present invention, GLP-1 can be secreted in the human body using cells having GLP-1 secretion function. The secreted GLP-1 acts on the GLP-1 receptor present in the β cells of the pancreas, promoting the secretion of insulin. Diseases to which the pharmaceutical composition of the present invention can be applied include various diseases for which GLP-1 is known to be useful for the prevention and treatment of, and specific examples include, but are not limited to, diabetes (type 1 diabetes, type 2 diabetes), obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction (non-alcoholic liver disease (NASH), etc.), taste disorders, pneumonia, etc.

The present invention will be described in detail below using experimental examples and examples, but the present invention is not limited thereto. Furthermore, unless otherwise specified, the reagents and materials used are commercially available. Abbreviations used in this specification are the same as those commonly used in this field.

Experimental Example 1: Construction of an expression vector for GLP-1 A vector for mammals, pRP, was constructed using the EF1A promoter, and was added with a signal sequence (TACAGGATGCAACTCCTGTCTTGCATTCACTAAGTCTTGCACTTGTCACGAATTCG) (SEQ ID NO: 5) derived from IL2 with the start codon ATG added, and a sequence of GLP-1 (CATGCTGAAGGGACCTTTACCAGTGATGTAAGTTCTTATTTGGAAGGCCAAGCTGCCAAGGAATTCATTGCTTGGCTGGTGAAAGGCCGAGGA) (SEQ ID NO: 6). By linking to EGFP (Enhanced Green Fluorescent Protein) with a T2A linker, EGFP remains in the cell during translation, and the target protein (GLP-1) is secreted into the culture supernatant (sig-GLP-1, FIG. 6A(b)). In other words, the expression of EGFP is considered to be equivalent to the state in which GLP-1 is expressed. However, since the cleavage efficiency of the T2A linker is not 100%, some of the fusion protein is secreted into the culture supernatant. Therefore, it is possible to confirm that GLP-1 is expressed in the cells by detecting GFP in the culture supernatant. As a control, a structure in which only the signal peptide is secreted was prepared (sig-peptide, FIG. 6A(a)). In addition, an EGFP-fused signal sequence-added GLP-1 was also constructed using 3xGGGGS (SEQ ID NO: 11) instead of the T2A linker (sig-GLP-1-GFP, FIG. 6A(d)). In this case, a sequence in which the signal sequence and EGFP were directly linked was used as a control (sig-GFP, FIG. 6A(c)).
In the pAAV viral expression vector, the EF1A promoter was used as in the plasmid vector, and the target vector was constructed using IRES instead of the T2A linker (sig-GLP-1, FIG. 6A(b)). The fusion protein type was constructed using the same sequence as in the plasmid vector (EGFP fusion signal sequence-added GLP-1 using 3xGGGGS instead of the T2A linker) in the pAAV viral vector (sig-GLP-1-GFP, FIG. 6A(d)).

Experimental Example 2: Genetic transfer of GLP-1 expression plasmid into iPS cells [0223] 2 µg of each of the plasmids prepared in Experimental Example 1 was transfected into iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) using Lipofectamine ^™ Stem Transfection Reagent (Thermo Fisher STEM00001), and the EGFP fluorescence after 24 hours was observed using a fluorescence microscope (KEYENCE BZ-X810, magnification x40, exposure time 6 sec).
By introducing the plasmid into iPS cells, expression of EGFP was confirmed in the cells.

Experimental Example (Example) 3: Confirmation of GLP-1 expression in CECSi cells (plasmid vector)
3-1: Detection of fluorescent protein in cells 48 hours after plasmid introduction iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were cultured for 1 week on a culture dish coated with iMatrix-511 (0.6 μg/cm ² ) using StemFit (registered trademark) AK03N medium (Ajinomoto). Thereafter, the cells were seeded again on a culture dish coated with iMatrix-511 (0.3 μg/cm ² ) and differentiation induction culture of the iPS cells into corneal endothelial substitute cells was performed for 14 days using the differentiation induction medium in Table 1, and the cells were then frozen. The frozen cells were thawed on the 14th day after the induction of differentiation, and the state was observed under a fluorescent microscope on the 3rd day after the start of adhesion culture to confirm that the cells had been induced to differentiate into corneal endothelial replacement cells (CECSi cells), and the cells were then subjected to the subsequent experiments. The CECSi cells obtained by induction from the iPS cells purchased from ATCC may be referred to as ATCC CECSI cells.
The confirmation of induction of differentiation into CECSi cells was carried out by fluorescent immunostaining of ZO-1, N-Cadherin, and PITX2.
For cells cultured in 6 wells, the culture supernatant was removed, and the wells were washed twice with 500 μl of 1xDPBS(-). The 1xDPBS(-) was removed, 500 μl of 4% PFA was added, and the wells were left to stand at room temperature for 15 minutes. The 4% PFA was removed from the wells, and the wells were washed twice with fresh 1xDPBS(-), and blocked with antibody diluent (0.1% Triton/1% donkey serum/1xDPBS(-)) for 30 minutes or more. The blocked cells were reacted with primary antibodies at room temperature for 12 hours. Each primary antibody was diluted with antibody diluent; ZO-1 (500-fold dilution), N-Cadherin (100-fold dilution), and PITX2 (200-fold dilution). After the reaction, the cells were rinsed once with 1xDPBS(-), and then washed twice by adding 1xDPBS(-) and leaving the wells to stand for 5 minutes. Each secondary antibody was diluted with antibody diluent, and DAPI (1000-fold dilution) was added to prepare secondary antibody solutions; Alexa488-Rabbit IgG (150-fold dilution), Cy3-mouse IgG (200-fold dilution). The secondary antibody solutions were reacted with the cells and left to stand for 1.5 hours. After rinsing once with 1xDPBS(-), 1xDPBS(-) was added and left to stand for 5 minutes, this washing was repeated twice, and after removing 1xDPBS(-), the cell surface was covered with a mounting agent and observed under a fluorescent microscope (Figure 1).

5 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b))) prepared in Experimental Example 1 was introduced into the cells confirmed to be CECSi cells using Lipofectamine ^™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the EGFP fluorescence after 48 hours was confirmed using a KEYENCE BZ-X810 fluorescence microscope (exposure time: 10 seconds). The amount of EGFP present in the secretory protein contained in the culture supernatant was measured using a TECAN Spark fluorescence plate reader. Excitation was performed at a wavelength of 485 nm, and absorption at 535 nm was measured.
The results are shown in Figure 2. Figure 2A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 2B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 48 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.

3-2: Detection of fluorescent protein in cells 65 hours after plasmid introduction As in 3-1 above, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b))) prepared in Experimental Example 1 was introduced using Lipofectamine ^™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 65 hours was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 10 sec). In addition, the amount of EGFP present in the secretory protein contained in the culture supernatant was measured using a TECAN Spark fluorescent plate reader. Excitation was performed at a wavelength of 485 nm, and absorption at 535 nm was measured.
The results are shown in Figure 3. Figure 3A shows an image observed by a fluorescence microscope (fluorescence, bright field), and Figure 3B shows the result of measuring the amount of EGFP in the cell culture supernatant. Since intracellular EGFP expression was confirmed 65 hours after gene transfer, it was considered that upstream GLP-1 was also expressed and secreted into the culture supernatant. Since EGFP was also detected in the culture supernatant, it was confirmed that the fusion protein was secreted.

3-3: Confirmation of gene expression 24 hours or 5 days after plasmid introduction As in 3-1 above, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6(b))) prepared in Experimental Example 1 was introduced using Lipofectamine ^™ 3000 Transfection Reagent (Thermo Fisher L3000001), and the fluorescence of EGFP after 24 hours or 5 days was confirmed using a fluorescent microscope KEYENCE BZ-X810 (exposure time 9 sec). In addition, to confirm intracellular gene expression, mRNA was extracted from the cells using RNeasy Plus Mini kit (Qiagen 74134), and 0.5 μg of mRNA was reverse transcribed with RevaTraAce reverse transcriptase (Takara TRT-101) to prepare cDNA. The following qPCR primers were used.
GLP-1 Forward Primer: TTATTTGGAAGGCCAAGCTGC (SEQ ID NO: 7)
GLP-1 Reverse Primer: TTAGAAGACTTCCCCTGCCCT (SEQ ID NO: 8)
GAPDH Forward Primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 9)
GAPDH Reverse Primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 10)
qPCR was performed using CronoSTAR Portable 4 by the 2-step method.
The results are shown in Figures 4 and 5. Figure 4(A) shows an image (fluorescence, bright field) observed by a fluorescent microscope 24 hours after gene transfer, and Figure 4(B) is a graph showing the results of gene expression. Expression at the mRNA level was confirmed 24 hours after gene transfer. Figure 5A shows an image (fluorescence, bright field) observed by a fluorescent microscope 5 days after gene transfer, and Figure 5B is a graph showing the results of gene expression. Expression at the mRNA level was also confirmed in cells 5 days after gene transfer.

Experimental Example (Example) 4: Detection of GLP-1 in cell culture supernatant As in Experimental Example 3, 3-1, ATCC CECSi cells were used, and 2 μg of each plasmid (sig-peptide (FIG. 6A(a)), sig-GLP-1 (FIG. 6A(b)), sig-GFP (FIG. 6A(c)), sig-GLP-1-GFP (FIG. 6A(d))) prepared in Experimental Example 1 was introduced, and after 19 hours, the GLP-1 protein (GFP fusion type) secreted into the culture supernatant was detected by Western blotting. 500 μl of the culture supernatant was concentrated with VIVAspin 500 10 kDa MWCO, and the buffer was replaced with 1×DPBS(−). About 5 to 10 μl of the concentrated sample was added with 6-fold concentrated SDS sample buffer (Nacalai Tesque) to which a reducing agent (b-me) was added to make the sample 1-fold sample buffer, and the sample was adjusted. A mini-sized e-PAGEL gel (5-20%) was prepared on ATTO Pagelan Ace, the entire sample was applied, and electrophoresis was performed for 80 minutes under standard 80 conditions. After electrophoresis, the protein was transferred to the membrane using ATTO Powered Blot 2M (WSE-4125) at 25 mV for 30 minutes. The membrane after transfer was subjected to blocking treatment with Blocking One (Nacalai Tesque 03953-66) at room temperature for 2 hours, and then the primary antibody Anti GFP (Green Fluorescent Protein) pAb (MBL 598) was diluted 1000 times and reacted overnight at 4 ° C. After washing the membrane with 1x TBS / 0.05% Tween buffer, the membrane was reacted with the secondary antibody HRP-linked IgG (CST # 7074S) diluted 2000 times for 1 hour. After the reaction, the membrane was washed with 1x TBS/0.05% Tween buffer and subjected to ELC reaction using Nacalai Chemi-lumi One Ultra (Nacalai Tesque 11644). The chemiluminescence of the membrane was detected using iBright FL1000 Imaging Systems.
The results are shown in Figure 6B. The upper panel shows schematic diagrams of each peptide and the plasmids encoding them. The lower panel shows the results of Western blotting using an anti-GFP antibody.
Since the protein assumed to be sig-GLP-1-GFP was confirmed in the culture supernatant by GFP, it is assumed that sig-GLP-1 is also present in the culture supernatant.

Experimental Example (Example) 5: Confirmation of GLP-1 expression in CECSi cells (AAV vector)
5-1: Detection of fluorescent protein in cells after AAV virus infection A vector constructed by Vector Builder and an AAV virus produced using the vector were purchased (the virus is a product with a guaranteed infection efficiency of 2× ¹¹¹ ). The plasmids used in the vector construction were sig-GFP (control, FIG. 6A(c)) and sig-GLP-1-GFP (FIG. 6A(d)).
Meanwhile, iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) were induced to differentiate according to the method of Experimental Example 3. The cells were frozen 14 days after differentiation induction (frozen ATCC CECSi cells).
Frozen ATCC CECSi cells were seeded at ^5.5x104 cells in 24 wells and infected at approximately MOI: 100,000. The virus was removed the next day, and the medium was replaced with a medium (medium containing only DMEM/F12, ITS, and IGF-1 in the medium composition in Table 1), and then fluorescence observation was performed 24 hours later using a fluorescent microscope KEYENCE BZ-X810. GFP expression in the cells 13 days after virus infection is shown in Figure 7A (fluorescence, bright field). The secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 μl of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in Figure 7B.
Even on day 13 after infection, intracellular expression of EGFP was confirmed, and EGFP was also detected in the culture supernatant. Similar results were obtained on day 18.

5-2: Detection of fluorescent protein in cells after AAV virus infection The expression of fluorescent protein after AAV virus infection was examined in the same manner as in 5-1 above, except that the plasmid used for vector construction was replaced with sig-peptide (control, FIG. 6A(a)) and sig-GLP-1 (FIG. 6A(b)). The results on the second day after AAV infection are shown in FIG. 8. The state of GFP expression in cells on the second day after virus infection is shown in FIG. 8A (fluorescence, bright field). The secretory protein contained in the culture supernatant was measured by measuring the fluorescence intensity of EGFP using a TECAN Spark fluorescent plate reader. 100 μl of the culture supernatant was applied to a black clear-bottom 96-well plate, excited at a wavelength of 485 nm, and absorbance at 535 nm was measured. The results are shown in FIG. 8B.
Expression of EGFP in the cells was confirmed even on day 2 after infection, and was also detected as GFP in the culture supernatant. Expression of EGFP in the cells was also confirmed on days 3, 7, 18, 22, and 32 (FIG. 9). In other words, it can be said that GLP-1, which is located upstream of EGFP in the constructed vector, is expressed.

Experimental Example (Example) 6: Functional evaluation of GLP-1 expressed and secreted in CECSi cells (c-peptide secretion promoting effect)
The in vitro function of sig-GLP-1-GFP (fusion type, FIG. 6A(d)) was confirmed using human iPS cell-derived β cells. Human iPS cell-derived pancreatic β cells and ATCC CECSi cells secreting sig-GLP-1-GFP were prepared separately, and co-cultured at appropriate times. The methods for each are described below.
Cellartis (registered trademark) hiPS Beta Cells (ChiPS22) (Takara Bio: Y10100), which are pancreatic β cells derived from human iPS cells, were placed on a 24-well culture insert (Falcon CBP 353104) coated with the attached coating agent, and cultured for 8 days in Basal Medium 1 supplemented with supplements provided in the kit. From the 9th day, the medium was changed to Basal Medium 2 supplemented with supplements, and co-culture was initiated using cells on the 3rd day.
Frozen ATCC CECSi cells, frozen 10 days after differentiation induction, were placed in a 24-well plate at 1.1 x ¹⁰⁵ cells/well and then infected with AAV2-sigGLP-1-GFP (fusion type) viral vector and control GFP viral vector at MOI: 100,000 on the fourth day, and the viral vector was removed the next day. The medium was replaced every two days, and co-culture was performed on the fourth day after infection. When co-culture was started, the medium was replaced with Krebs buffer (KRB), and after 24 hours, the supernatant of pancreatic β cells was collected, KRB was added again, and the amount of c-peptide contained in the supernatant after 24 hours was measured using a c-peptide ELISA kit (AB Clone RKO3588). Compared to infection with the control GFP viral vector, co-culture with the AAV2-sigGLP-1-GFP (fusion type) viral vector showed an increase in the amount of c-peptide secreted into the medium (Figure 10B).
The AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the plasmid sig-GLP-1-GFP (Figure 6A(d)) or a plasmid containing only the EGFP sequence, respectively.

Experimental Example (Example) 7: Effect on glycemic control when CECSi cells expressing and secreting GLP-1 are intraperitoneally transplanted into healthy SCID mice In order to investigate the in vivo function of CECSi cells expressing and secreting GLP-1, the effect on glycemic control in healthy SCID mice was examined.
Frozen ATCC CECSI cells, ^5x105 /well, were infected with AAV2-sig-GLP-1-GFP (fusion type) virus vector and control GFP virus vector at MOI: 100000 for 24 hours. After 2 days of virus removal, the cells were collected and suspended in 100μl of physiological saline. SCID mice were fasted for 4 hours, and the prepared cells were transplanted into the abdominal cavity. Starting from the first mouse, 20% glucose solution was directly loaded into the stomach of the mouse with a sonde at 5-minute intervals to 2g/kg of body weight. Blood glucose levels were measured immediately before the glucose load, with the time set as 0 hours, and 30 minutes, 1 hour, 1.5 hours, and 2 hours after the glucose load. In the control, blood glucose increased at 30 minutes, but in the presence of sig-GLP-1-GFP, the increase in blood glucose was suppressed (Fig. 11A). Regarding the distribution of sig-GLP-1-GFP in the body, ELISA was performed using blood samples taken immediately before and 30 minutes after glucose loading, and it was confirmed that sigGLP-1 was present in the blood 30 minutes after glucose loading (Figure 11B).
The AAV2-sigGLP-1-GFP (fusion type) viral vector and the control GFP viral vector were prepared by constructing vectors using AAV serotype 2 (AAV2) and the sig-GLP-1 plasmid (Figure 6A(b)) or a plasmid carrying only the EGFP sequence, respectively.

According to the present invention, it has become possible to impart a GLP-1 secretion function to corneal endothelial substitute cells. Corneal endothelial substitute cells having a GLP-1 secretion function make it possible to develop a new cell therapy for diseases involving GLP-1, particularly diseases in which GLP-1 secretion is useful (diabetes, etc.).
This application is based on Patent Application No. 2022-158838 filed in Japan (filing date: September 30, 2022), the contents of which are incorporated in their entirety herein.

Claims

　Pluripotent stem cells or cells induced to differentiate from stem cells that have the ability to secrete GLP-1.
The cell according to claim 1, wherein the cell induced to differentiate from a stem cell is an endothelial cell.
The cell according to claim 1, wherein the pluripotent stem cell or stem cell is an iPS cell.
The cells according to claim 2, wherein the endothelial cells are corneal endothelial cells.
The cell according to claim 1, which expresses GLP-1.
A method for producing cells that express GLP-1, comprising the step of introducing a nucleic acid encoding GLP-1 into cells, the cells being pluripotent stem cells or cells induced to differentiate from stem cells.
The method according to claim 6, wherein the introduction of the nucleic acid into the cell is by gene introduction or genome editing.
The method according to claim 6, wherein the cells induced to differentiate from stem cells are endothelial cells.
The method according to claim 6, wherein the pluripotent stem cells or stem cells are iPS cells.
The method of claim 8, wherein the endothelial cells are corneal endothelial cells.
(1) inserting a nucleic acid encoding GLP-1 into an expression vector to prepare an expression vector containing the nucleic acid;
(2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to produce a cell containing the expression vector; and (3) culturing the cell containing the expression vector.
A method for producing a cell expressing GLP-1, comprising:
The method according to claim 11, wherein the cells induced to differentiate from stem cells are endothelial cells.
The method according to claim 11, wherein the pluripotent stem cells or stem cells are iPS cells.
The method of claim 12, wherein the endothelial cells are corneal endothelial cells.
A pharmaceutical composition comprising the cells according to any one of claims 1 to 5.
The pharmaceutical composition according to claim 15, which is for treating diabetes, obesity, central nervous system disorders, peripheral nervous system disorders, renal dysfunction, heart failure, ischemic heart disease, liver dysfunction, taste disorders, and pneumonia.