JP2024094292A - Sheet-shaped cell structure containing upper respiratory tract cells, and method for producing and using same - Google Patents

Abstract

[Problem] To provide a method for efficiently producing cells constituting a sheet-shaped cell structure containing upper respiratory tract cells from pluripotent stem cells. [Solution] A method for producing a sheet-shaped cell structure containing upper respiratory tract cells is provided, comprising: (1) a step (1) of dispersing a first cell population containing olfactory placode cells or olfactory epithelial stem cells to obtain single cells; (2) a step (2) of purifying the single cells obtained in the step (1) to obtain a second cell population containing olfactory placode cells or olfactory epithelial stem cells, wherein the content of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is higher than the content of the olfactory placode cells and olfactory epithelial stem cells in the first cell population; and (3) a step (3) of seeding the second cell population obtained in the step (2) on a culture vessel, culturing the second cell population, and obtaining the sheet-shaped cell structure. [Selected Figures] None

Description

The present invention relates to a sheet-shaped cell structure containing upper respiratory tract cells, as well as a method for producing and using the same. Upper respiratory tract tissues include olfactory epithelial tissue and nasal epithelial tissue.

In Non-Patent Document 1, it is reported that by co-culturing embryoid bodies formed from mouse iPS cells with primary cultured cells of olfactory epithelium or olfactory bulb collected from a mouse, nerve cells expressing some olfactory nerve cell markers are induced to differentiate. In Non-Patent Document 2, it is reported that olfactory placode cells and olfactory nerve cells are produced from human pluripotent stem cells. In Non-Patent Document 3, a method is reported in which a specimen collected from the human nasal cavity is cultured to form a cell mass, and then a sheet of nasal epithelium is obtained. In Non-Patent Document 4, a method is reported in which a specimen collected from the human nasal cavity is cultured in suspension to form a cell mass. In Patent Document 1, it is reported that mouse ES cells are cultured on feeder cells and a differentiation inducer is added, and the differentiated cells express olfactory nerve cell markers. In Patent Document 2, it is reported that a cell mass containing a preplacode is produced from human pluripotent stem cells. In addition, the inventor of the present application has reported in Patent Documents 3 and 4 that it is possible to produce three-dimensional nasal epithelial tissue and olfactory epithelial tissue from human ES cells and iPS cells. However, no method was known at all for industrially producing sheet-like upper airway tissues using human pluripotent stem cells as a starting material, suitable for evaluating regenerative medicine products and compounds for transplantation, for creating disease models for infectious or genetic diseases, or for mounting on devices such as organ-on-a-chip.

WO/2009/098149 US/2015/0125953 WO/2020/039732 WO/2022/026238

Int. J. Clin. Exp. Pathol. 2017;10(7):8072-8081 STEM CELLS AND DEVELOPMENT Volume 31, Numbers 17-18, 2022 Mbio, 2022, 13.1: e03511-21. Theranostics 2021, Vol. 11. Issue 2

However, Patent Documents 1 and 2 and Non-Patent Documents 1 to 4 do not describe the efficient production of a sheet-shaped cell structure containing upper respiratory tract cells from pluripotent stem cells. The object of the present invention is to provide a method for efficiently producing cells constituting a sheet-shaped cell structure containing upper respiratory tract cells from pluripotent stem cells, and a method for producing the sheet-shaped cell structure.

The inventors conducted extensive research to solve the above problems, and by analyzing a cell population in the process of culturing in which pluripotent stem cells differentiate into upper respiratory tract tissue, they identified multiple specific surface antigens used to separate and purify cells that constitute upper respiratory tract tissue. Furthermore, they found the timing at which the identified surface antigens can be used to effectively separate and purify cells that constitute upper respiratory tract tissue from a cell population in the process of culturing and differentiating into upper respiratory tract tissue. Furthermore, by identifying culture equipment suitable for forming a sheet-like cell structure and conditions suitable for epithelial formation of upper respiratory tract cells derived from pluripotent stem cells, they established a manufacturing method for forming a sheet-like cell structure of upper respiratory tract tissue, thereby completing the present invention.

The present invention relates to a method for producing a sheet-shaped cell structure containing upper respiratory tract cells, as exemplified below, a sheet-shaped cell structure containing upper respiratory tract cells, a method for using the cell structure as a regenerative medicine product, a method for using the cell structure as a disease model, a method for using the cell structure as a gene therapy evaluation system, and equipment, chips, systems, devices, etc. that include the cell structure.
[1] A method for producing a sheet-shaped cell structure containing upper respiratory tract cells, comprising the steps of:
(1) dispersing a first cell population containing olfactory placode cells or olfactory epithelial stem cells to obtain single cells;
(2) purifying the single cells obtained in the step (1) to obtain a second cell population containing the olfactory placode cells or olfactory epithelial stem cells, wherein the content of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is higher than the content of the olfactory placode cells and olfactory epithelial stem cells in the first cell population; and
(3) seeding the second cell population obtained in the step (2) on a culture vessel, culturing the second cell population, and obtaining the sheet-shaped cell structure;
A manufacturing method comprising:
[2] At least 20% of the first cell population in the step (1) is an ectoderm-derived cell,
The method according to [1], wherein the ectoderm-derived cells contain cells expressing at least one olfactory placode cell marker or olfactory epithelium stem cell marker selected from the group consisting of Six1, Sp8, Otx2, cytokeratin, and E-cadherin.
[3] The first cell population in the step (1) further comprises cells of the central nervous system,
The cells of the central nervous system
The production method according to [1] or [2], wherein the cells 1) express at least one central nervous system marker selected from the group consisting of Pax6, Sox1, Sox2, Sox3, Dlx5, FoxG1, N-Cadherin, MAP2, RAX, and VSX2, and 2) do not express cytokeratin.
[4] The first cell population in the step (1) comprises: 1) a non-neuroepithelial tissue portion containing the olfactory placode cells or olfactory epithelial stem cells; and 2) a neural tissue portion containing neural cells or their precursor cells,
A three-dimensional tissue or cell mass in which at least a portion of the surface of the nerve tissue section is covered with the non-neuroepithelial tissue section,
The method according to any one of [1] to [3], wherein the nervous system cells or their precursor cells include nervous system cells that constitute the central nervous system or their precursor cells.
[5] The first cell population in the step (1) includes 1) a non-neuroepithelial tissue portion containing the olfactory placode cells or olfactory epithelial stem cells, and 2) a neural tissue portion containing neural cells or their precursor cells,
the non-neuroepithelial tissue portion and the neural tissue portion are attached to the same surface of the culture substrate,
The method according to any one of [1] to [3], wherein the nervous system cells or their precursor cells include nervous system cells that constitute the central nervous system or their precursor cells.
[6] The method according to any one of [1] to [5], wherein the first cell population in the step (1) is derived from pluripotent stem cells.
[7] The first cell population in the step (1)
(a) culturing pluripotent stem cells in the presence of a Wnt signaling pathway inhibitor;
(b) culturing the cells cultured in step (a) in the presence of a BMP signaling pathway agent; and
(c) culturing the cells cultured in the step (b) in the presence of at least one substance selected from the group consisting of an FGF signaling pathway agonist and a BMP signaling pathway inhibitor, thereby producing the first cell population containing the olfactory placode cells or olfactory epithelial stem cells;
The method according to any one of [1] to [6], wherein the compound is produced by a method comprising the steps of:
[8] Prior to the step (a), the pluripotent stem cells are cultured in the absence of feeder cells,
1) at least one selected from the group consisting of a TGFβ family signaling pathway inhibitor, a sonic hedgehog signaling pathway agonist, and an FGF signaling pathway inhibitor;
2) Undifferentiated cell maintenance factor;
The method according to [7], further comprising carrying out a step (i) of culturing in a medium containing the compound.
[9] The step (2) includes purifying the first cell population to obtain the second cell population by recovering the olfactory placode cells or olfactory epithelial stem cells using a reagent having specific affinity for a cell surface marker of a target cell,
The method according to any one of [1] to [8], wherein the cell surface marker of the target cell includes at least one selected from the group consisting of EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1.
[10] The step (2) further comprises purifying the first cell population by removing the non-target cells using a reagent having specific affinity for a cell surface marker of the non-target cells to obtain the second cell population;
The cell surface marker of the non-target cells includes at least one selected from the group consisting of ATP1B2, CD82, CDH6, CDON, CELSR2, CLU, CNTFR, COL11A1, CP, CRB2, DCT, DKK3, EFNB1, FBLN2, FBN2, FN1, GPM6B, ISLR2, LRP2, LRRC4B, MMRN1, PRELP, PRSS35, S1PR3, SDK2, SLC2A1, SORCS1, TFPI, THSD7A, THY1, TMEM132B, and TSPAN18. The manufacturing method of [9].
[11] The method according to [9] or [10], wherein the step (2) comprises purifying the first cell population by fluorescence activated cell sorting (FACS) or magnetic bead assisted cell sorting (MACS) to obtain the second cell population.
[12] The step (2) further comprises cryopreserving the second cell population,
The method according to any one of [1] to [11], wherein the step (3) further comprises thawing the cryopreserved second cell population.
[13] The method according to any one of [1] to [12], wherein the step (3) comprises culturing the second cell population in the presence of at least one selected from the group consisting of a substance acting on a Sonic Hedgehog signaling pathway and a substance inhibiting a BMP signaling pathway.
[14] The method according to [13], wherein the substance acting on the Sonic Hedgehog signaling pathway includes at least one selected from the group consisting of Sonic Hedgehog protein, SAG, Purmorphamine, 20(S)-hydroxycholesterol, and GSA-10.
[15] The BMP signaling pathway inhibitor is a type I BMP receptor inhibitor,
The method for producing the present invention according to [13] or [14], wherein the type I BMP receptor inhibitor comprises at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1, DMH2, Compound 1, VU5350, OD52, E6201, Saracatinib, BYL719, and derivatives thereof.
[16] The method according to any one of [1] to [15], wherein the step (3) comprises culturing the second cell population in the presence of at least one selected from the group consisting of a ROCK inhibitor, a substance acting on the FGF signaling pathway, a substance acting on the EGF signaling pathway, a substance acting on the TGFβ family signaling pathway, a substance acting on the Wnt signaling pathway, and a substance acting on the retinoic acid signaling pathway.
[17] The TGFβ family signaling pathway inhibitor is an Alk5/TGFβR1 inhibitor,
The Alk5/TGFβR1 inhibitors include SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, A77-01, RepSox, BIBF-0775, TP0427736, TGFBR1-IN-1, SM-16, TEW-7197, LY3200882, LY2109761, R268712, IN1130, Galunisertib, AZ12799734, KRCA 0008, GSK The method according to [16], further comprising at least one selected from the group consisting of 1838705, Crizotinib, Certinib, ASP 3026, TAE684, AZD3463 and derivatives thereof.
[18] The method according to any one of [1] to [17], wherein the step (3) comprises culturing the second cell population on a substance-permeable membrane provided on the culture vessel.
[19] The method according to [7] or [8], wherein the Wnt signaling pathway inhibitor has at least one activity selected from the group consisting of 1) an inhibitory activity against a non-canonical Wnt pathway, 2) an inhibitory activity against extracellular secretion of a Wnt family protein, 3) an inhibitory activity against porcupine, and 4) an inhibitory activity against ROR.
[20] The Wnt signaling pathway inhibitor 1) has an inhibitory activity against a non-canonical Wnt pathway, and the inhibitory activity is an inhibitory activity against acylation or palmitoylation of a target protein by porcupine;
The method for producing a pharmaceutical composition according to [19], wherein the substance having inhibitory activity includes at least one selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, IWP-O1, LGK-974, Wnt-C59, ETC-131, ETC-159, GNF-1331, GNF-6231, Porcn-IN-1, RXC004, CGX1321, AZD5055, and derivatives thereof.
[21] The method according to [19], wherein the Wnt signaling pathway inhibitor comprises at least one selected from the group consisting of XAV-939, KY02111, KY03-I, and derivatives thereof.
[22] The production method described in [7] or [8], wherein one or more of the steps (a) to (c) are carried out in the presence of at least one selected from the group consisting of a TGFβ family signaling pathway inhibitor, a ROCK inhibitor, and a JNK signaling pathway inhibitor.
[23] The method according to [7] or [8], wherein step (c) is carried out in the presence of at least one selected from the group consisting of a TAK1 inhibitor, a substance acting on the FGF signaling pathway, and a substance acting on the EGF signaling pathway.
[24] A sheet-shaped cell structure comprising upper respiratory tract cells, produced by the production method according to any one of [1] to [23].
[25] A sheet-shaped cell structure comprising upper respiratory tract cells,
The upper respiratory tract cells are
1) a basal cell or a precursor cell thereof expressing at least one basal cell marker selected from the group consisting of p63, cytokeratin 5, and p75;
2) Olfactory placode cells or olfactory epithelial stem cells expressing at least one olfactory epithelial stem cell marker selected from the group consisting of Six1, Sp8, Otx2, Sox2, Dlx5, cytokeratin, and E-Cadherin; and
3) olfactory nerve cells or precursor cells thereof expressing at least one olfactory nerve cell marker selected from the group consisting of βIII tubulin, Calretinin, Ebf1, Ebf2, Ebf3, NeuroD1, and Lhx2;
A sheet-like cell structure comprising:
[26] A sheet-shaped cell structure comprising upper respiratory tract cells,
The upper respiratory tract cells are
1) ciliated cells expressing at least one or more ciliated epithelial cell markers selected from the group consisting of FoxJ1, FoxN4, p73, MYB, acetylated tubulin, and β4-tubulin; and
2) secretory cells expressing at least one secretory cell marker selected from the group consisting of mucin 1, mucin 2, mucin 3, mucin 4, mucin 5AC, mucin 5B, FoxA1, FoxA2, Nkx2.1/TTF-1, and SPDEF;
A sheet-like cell structure comprising:
[27] The sheet-shaped cell structure is
and an apical surface expressing at least one selected from the group consisting of Ezrin, Ezrin, and PKCζ;
the upper airway cells form an epithelial structure between the apical surface and a culture substrate;
the upper airway cells have epithelial cell polarity;
At least 70% of the cells constituting the sheet-shaped cell structure are localized in the epithelial structure,
The sheet-shaped cell structure according to [25] or [26], wherein the epithelial structure comprises basal cells in contact with the culture substrate.
[28] The sheet-shaped cell structure according to any one of [24] to [27], comprising upper respiratory tract cells expressing at least one virus receptor or virus infection-related factor selected from the group consisting of ACE2, Tmprss2, NRP1, CTSB, CTSL, BSG, HSPA5, DPP4, FURIN, ANPEP, Tmprss11D, ST6GAL1, ST3GAL4, CEACAM1, sialic acid α2,6Gal, and sialic acid α2,3Gal.
[29] The sheet-shaped cell structure according to any one of [24] to [28], wherein the upper respiratory tract cells are deficient or mutated in one or more genes selected from the group consisting of genes associated with genetic diseases or infectious diseases, and genes encoding viral receptors or viral infection-related factors.
[30] 1) the sheet-shaped cell structure according to any one of [24] to [29], and 2) at least one of the cells and cell populations other than upper respiratory tract cells,
A microfluidic chip or biomimetic system comprising:
[31] A method for screening a compound for treating a disease, using an apparatus, chip, system, or equipment containing the sheet-shaped cell structure according to any one of [24] to [29].
[32] A screening kit for a compound for treating a disease, comprising an apparatus, chip, system, or equipment containing the sheet-shaped cell structure according to any one of [24] to [29].
[33] A method for using an instrument, chip, system, or device containing the sheet-shaped cell structure described in any one of [24] to [29] as a disease model of the upper respiratory tract.
[34] A method for evaluating the efficacy or toxicity of a target substance using an instrument, chip, system, or device containing the sheet-shaped cell structure described in any one of [24] to [29].
[35] A kit for evaluating the efficacy or toxicity of a target substance, comprising an instrument, chip, system, or device containing the sheet-shaped cell structure described in any one of [24] to [29].
[36] A method for searching for disease-specific biomarkers using an instrument, chip, system, or device containing the sheet-shaped cell structure described in any one of [24] to [29].
[37] A composition for transplantation comprising an instrument, chip, system, or equipment containing the sheet-shaped cell structure according to any one of [24] to [29].
[38] Use of an instrument, chip, system, or device containing the sheet-shaped cell structure according to any one of [24] to [29] in the isolation and culture of viruses or microorganisms.
[39] An upper respiratory tract tissue model animal or human tissue model animal, to which the sheet-shaped cell structure according to any one of [24] to [29] has been transplanted.
[40] A method for purifying olfactory placode cells or olfactory epithelial stem cells contained in a sample, comprising:
and recovering the olfactory placode cells or olfactory epithelial stem cells from the sample using a reagent having a specific affinity for a cell surface marker of the olfactory placode cells or olfactory epithelial stem cells;
The cell surface marker of the olfactory placode cell or olfactory epithelial stem cell comprises at least one selected from the group consisting of EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1.

According to the present invention, it is possible to efficiently produce a sheet-shaped cell structure containing upper respiratory tract cells from pluripotent stem cells. In addition, according to the present invention, it is possible to provide a transplant composition for regenerative medicine using a sheet-shaped cell structure containing upper respiratory tract cells, a disease model, a screening method for disease therapeutic drugs, a screening kit, a method and an evaluation kit for evaluating the pharmacological effect or toxicity of a compound, and the like. In another aspect, the sheet-shaped cell structure containing upper respiratory tract cells produced according to the present invention can be used for research on infectious diseases including influenza virus, novel coronavirus (SARS-CoV-2), and seasonal coronaviruses, for the isolation and culture of viruses, and the like.

FIG. 1 is a schematic diagram showing a sheet-shaped cell structure containing upper respiratory tract cells according to this embodiment. FIG. 2 is a schematic diagram showing a sheet-shaped cell structure containing upper respiratory tract cells according to this embodiment. FIG. 3A is a schematic diagram (top view, cross-sectional view) showing a sheet-shaped cell structure containing upper respiratory tract cells according to this embodiment. FIG. 3B is a schematic diagram (top view, cross-sectional view) showing a sheet-shaped cell structure containing upper respiratory tract cells according to this embodiment. Figure 4 shows a schematic diagram (top) illustrating the process for producing a cell population containing olfactory epithelial stem cells from human iPS cells in Experiment 1, and bright field and fluorescent images (bottom) of an inverted microscope of a cell population containing olfactory epithelial stem cells on day 23 after the start of culture. FIG. 5 shows staining images of the cell population containing olfactory epithelial stem cells in Experiment 1 after immunostaining with each marker. 6 is a schematic diagram showing the process of producing a cell population containing olfactory epithelial stem cells from human iPS cells (top). The bottom is a bright-field image of the cell population 20 days after the start of culture in Experiment 2, observed with an inverted microscope. FIG. 7 is a two-dimensional plot of the results of single-cell analysis of olfactory epithelium organoids in Experiment 2. 8 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 2. The results of the analysis of the expression levels of each of the DLX5, Pax6, Sp8, and EpCAM genes in each cell are shown. 9 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 2. The results of the analysis of the expression levels of each gene, SIX1, OTX2, SOX2, and Ki67, per cell are shown. 10 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 2. The results of the analysis of the expression levels of each gene, Ebf2, NeuroD1, Lhx2, and Calretinin, per cell are shown. 11 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 2. The results of the analysis of the expression levels of the SOX1 and Vsx2 genes in each cell are shown. FIG. 12 is a table showing the expression fold of cell surface marker genes in a cell population containing olfactory epithelial stem cells. 13 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 3. The results of the analysis of the expression levels of each gene, EpCAM, E-Cadherin, P-Cadherin, and CLDN4, per cell are shown. 14 is a two-dimensional plot diagram of the single-cell analysis results in the olfactory epithelium organoid of Experiment 3. The results of the expression level analysis of each gene of CLDN6, CLDN7, LAMB1, and FREM2 per cell are shown. 15 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 3. The results of the expression level analysis of each gene, TACSTD2, IGSF1, F11R, and FRAS1, per cell are shown. 16 is a two-dimensional plot diagram of the results of single-cell analysis of the olfactory epithelium organoid in Experiment 3. The diagram shows the results of the analysis of the expression level of the SORL1 gene in each cell. FIG. 17 shows a schematic diagram (top) illustrating the process of purifying olfactory placode cells or olfactory epithelial stem cells from the olfactory epithelial organoids of the first cell population in Experiment 4, and the results of FACS analysis of the purified cell population (bottom). FIG. 18 is a schematic diagram (top) showing the process of purifying olfactory placode cells or olfactory epithelial stem cells from the olfactory epithelial organoids of the first cell population produced using a microwell plate in Experiment 5, and the results of FACS analysis of the purified cell population (bottom). Figure 19 is a schematic diagram (top) showing the process of producing a sheet-shaped cell structure containing upper respiratory tract cells through steps 1, 2, and 3 in experiment 6, and bright-field observation images (bottom) taken with an inverted microscope when the second cell population was cultured in medium of each condition in step 3. FIG. 20 shows staining images of olfactory epithelial stem cells, basal cells, olfactory nerve progenitor cells, and immature olfactory nerve cells after immunostaining with each marker in Experiment 6. FIG. 21 shows staining images of olfactory epithelial stem cells, basal cells, olfactory nerve progenitor cells, and immature olfactory nerve cells after immunostaining with each marker in Experiment 6. FIG. 22 shows staining images of olfactory epithelial stem cells, basal cells, olfactory nerve progenitor cells, and immature olfactory nerve cells after immunostaining with each marker in Experiment 7. Figure 23 is a schematic diagram (top) showing the process of producing a sheet-shaped cell structure containing upper respiratory tract cells through steps 1, 2, and 3 in experiment 8, and a bright-field observation image (bottom) taken with an inverted microscope when the second cell population was cultured in step 3. FIG. 24 shows staining images of olfactory epithelial stem cells, basal cells, secretory cells, and ciliated cells after immunostaining with each marker in Experiment 8. FIG. 25 shows an image of a sheet-shaped cell structure containing upper respiratory tract cells in Experiment 9, observed with a scanning electron microscope. Figure 26 shows a schematic diagram (top row) of the process for producing a cell mass containing non-neuroepithelial tissue, including olfactory epithelium-like tissue, from pluripotent stem cells in Experiment 10, and bright field and fluorescent images (bottom row) taken with an inverted microscope of a cell population containing olfactory epithelium stem cells on day 23 after the start of culture. FIG. 27 shows staining images of the cell population containing olfactory epithelial stem cells in Experiment 10 after immunostaining with each marker. 28 is a schematic diagram (upper part) showing the process of preparing an upper airway cell sheet from human iPS cells, manufacturing a cell sheet-scaffold complex with collagen sponge, and performing an animal transplantation test in Experiment 11. The lower part shows an oblique illumination bright-field observation image and a fluorescent tube image of a sample prepared by fixing rat nasal tissue one week after the transplantation test in Experiment 11.

The following is a detailed description of the embodiment of the present invention (hereinafter referred to as "the present embodiment"). Note that the present invention is not limited to the following embodiment. In this specification, the notation in the form "A-Z" means the upper and lower limits of a range (i.e., A or more and Z or less). When no unit is stated for A and only a unit is stated for Z, the unit of A and the unit of Z are the same. In this specification, the expression "comprises" includes the concept of "consisting of" or "consisting only of".

1. Definitions In this specification, the definitions of terms are as follows.
"Stem cells" refer to undifferentiated cells that have differentiation and proliferation capabilities (especially self-renewal capabilities). Stem cells include pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on their differentiation capabilities. "Pluripotent stem cells" refer to stem cells that can be cultured in vitro and have the ability to differentiate into all cells that constitute a living body (multipotency). All cells are cells derived from the three germ layers of ectoderm, mesoderm, and endoderm. "Multipotent stem cells" refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, although not all types. "Unipotent stem cells" refer to stem cells that have the ability to differentiate into specific tissues and cells.

Pluripotent stem cells can be induced 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). Multi-lineage differentiating stress ending cells (Muse cells) obtained from mesenchymal stem cells (MSCs), and GS cells produced from germ cells (e.g. testes) are also included in pluripotent stem cells. Human embryonic stem cells are established from human embryos within 14 days of fertilization.

Embryonic stem cells were first established in 1981, and have been used to create knockout mice since 1989. Human embryonic stem cells were established in 1998, and are being used in regenerative medicine. ES cells can be produced by culturing the inner cell population on feeder cells or in a medium containing leukemia inhibitory factor (LIF). Methods for producing ES cells are described in, for example, International Publication No. 96/22362, International Publication No. 02/101057, U.S. Patent No. 5,843,780, U.S. Patent No. 6,200,806, and U.S. Patent No. 6,280,718, etc. Embryonic stem cells are available from designated institutions, and can also be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Both are mouse embryonic stem cells; EB5 cells are available from the National Research and Development Agency RIKEN, and the D3 line is available from the American Type Culture Collection (ATCC). Nuclear transfer ES cells (ntES cells), which are 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).

"Induced pluripotent stem cells" are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specific examples of induced pluripotent stem 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 in mouse cells (Cell, 2006, 126(4) pp.663-676). In 2007, artificial 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 artificial pluripotent stem cells by direct reprogramming through gene expression, artificial pluripotent stem cells can also be induced from somatic cells by adding 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 induced pluripotent stem cells, in the case of initializing 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 cell lines; for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells, 1383D2 cells, and 1383D6 cells established at Kyoto University are available from Kyoto University and iPS Academia Japan Inc. An example of an established induced pluripotent stem cell line is the hiPS β-actin GFP cell line, which can be purchased from Merck.

The pluripotent stem cells may be genetically modified. Genetically modified pluripotent stem cells can be produced, for example, by using homologous recombination techniques. Examples of genes on chromosomes that can be modified include cell marker genes, histocompatibility antigen genes, and disease-related genes caused by disorders of nervous system cells. Modification of a target gene on a chromosome can be performed using the methods described in Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8, Gene Targeting, Creation of Mutant Mice Using ES Cells, Yodosha (1995), etc.

Specifically, for example, the genomic gene of the target gene to be modified (e.g., a cell marker gene, a gene for a histocompatibility antigen, or a disease-related gene) is isolated, and a target vector for homologous recombination of the target gene is prepared using the isolated genomic gene. The target vector thus prepared is introduced into stem cells, and cells in which homologous recombination has occurred between the target gene and the target vector are selected, thereby producing stem cells in which genes on chromosomes have been modified.

Methods for isolating the genomic gene of the target gene include known methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997). Genomic DNA Library Screening System (manufactured by Genome Systems) and Universal GenomeWalker Kits (manufactured by Clontech) can be used.

The preparation of a target vector for homologous recombination of a target gene and efficient selection of a homologous recombinant can be performed according to the methods described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8, Gene Targeting, Preparation of Mutant Mice Using ES Cells, Yodosha (1995), etc. Either a replacement type or an insertion type target vector can be used. Selection methods include positive selection, promoter selection, negative selection, and poly A selection. Methods for selecting the desired homologous recombinant from the selected cell lines include Southern hybridization and PCR for genomic DNA.

As pluripotent stem cells, pluripotent stem cells that have been subjected to genome editing can also be used. "Genome editing" is a technique for intentionally modifying a target gene or genome region by the principles of site-specific cleavage of genomic DNA strands using nucleases, or chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), TALENs, CRISPR/Cas3, 9, 13, etc. By using genome editing technology, 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, etc.

Disease-specific pluripotent stem cells may be used as pluripotent stem cells. "Disease-specific pluripotent stem cells" refer to pluripotent stem cells that have a genetic mutation or genetic background involved in the onset of a disease. Disease-specific pluripotent stem cells can be produced by establishing induced pluripotent stem cells from patients or close relatives who have the target disease using the above-mentioned methods, or by modifying the genome of already established pluripotent stem cells using genome editing techniques such as zinc finger nuclease (ZFN), TALEN, and CRISPR.

As the pluripotent stem cells, low immunogenic pluripotent stem cells having the function of suppressing immune rejection reactions that occur during allogeneic transplantation may be used. The "low immunogenic pluripotent stem cells" may be pluripotent stem cells established from a homozygous donor of human leukocyte antigen (HLA), or may be pluripotent stem cells in which one or more immune-related genes, such as the HLA gene group, B2M gene, CIITA gene, RFX5 gene, RFXAP gene, RFXANK gene, etc., have been deleted or functionally modified by genome editing technology, or may be pluripotent stem cells that express CD47, PD-L1 gene, HLA-G gene, etc. Universal donor cells are also included in low immunogenic pluripotent stem cells. Such pluripotent stem cells can be produced by methods described, for example, in International Publication No. WO 1995/017911, International Publication No. WO 98/42838, International Publication No. WO 2016/183041, International Publication No. WO 2012/145384, International Publication No. WO 2013/158292, etc.

"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, including 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.

"Cell adhesion" includes adhesion between cells (cell-cell adhesion) and adhesion between cells and extracellular matrix (substrate) (cell-substrate adhesion). Cell adhesion also includes adhesion of cells to culture equipment, etc., that occurs in an in vitro artificial culture environment. The bond formed in cell-cell adhesion is a cell-cell junction, and the bond formed in cell-substrate adhesion is a cell-substrate junction. Examples of types of cell adhesion include anchoring junctions, communicating junctions, and occluding junctions.

Examples of cell-cell junctions include "tight junctions" and "adherence junctions." Tight junctions are relatively strong cell-cell junctions that can occur in epithelial cells. The presence of tight junctions between cells can be detected by techniques such as immunohistochemistry using antibodies against components of tight junctions (anti-claudin antibodies, anti-ZO-1 antibodies, etc.).

"Suspension culture" refers to culturing cells while maintaining a state in which they exist suspended in the culture solution. In other words, suspension culture is performed under conditions that do not allow cells to adhere to the cultureware and feeder cells, etc. on the cultureware (hereinafter referred to as "cultureware, etc."), and is distinguished from culture performed under conditions that allow cells to adhere to the cultureware, etc. (adherent culture). More specifically, suspension culture refers to culture under conditions that do not allow strong cell-substrate bonds to be formed between the cells and the cultureware, etc. Those skilled in the art can easily distinguish whether the cultured cells are in a suspension culture state or an adherent culture state, for example, by rocking the cultureware during microscopic observation.

"Adherent culture" refers to culturing cells while maintaining a state in which the cells are attached to culture equipment, etc. Here, cells adhering to culture equipment, etc. means, for example, that strong cell-substrate bonds, which are a type of cell adhesion, are formed between the cells and the culture equipment, etc.

In cell aggregates during suspension culture, cells adhere to each other through a surface. In cell aggregates during suspension culture, strong cell-substrate bonds are not formed between the cells and the culture equipment, etc., and cell-substrate bonds are hardly formed, or even if they are formed, their contribution is small. Internal cell-substrate bonds may exist inside cell aggregates during suspension culture. "Plane attachment between cells" refers to cells adhering to each other through a surface. More specifically, "plane attachment between cells" refers to the proportion of the surface area of a cell that is attached to the surface of another cell, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. The cell surface can be observed by staining with a membrane-staining reagent (e.g., DiI), immunostaining with cell adhesion factors (e.g., E-cadherin, N-cadherin, etc.), etc.

The cultureware used in the adhesion culture is not particularly limited as long as it is capable of adhesion culture, and a person skilled in the art can appropriately determine the cultureware. Examples of such cultureware include flasks, tissue culture flasks, dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, and biofunctional chips such as organ-on-chips, cultureware, etc. As cell-adhesive cultureware, those whose surfaces have been artificially treated for the purpose of improving adhesion to cells or inducing or maintaining the properties of cells in a state favorable for the present invention can be used. Examples of artificial treatments include coating treatments with extracellular matrices, polymers, etc., and surface treatments such as gas plasma treatment and positive charge treatment. Examples of extracellular matrices to which cells are attached include basement membrane preparations, laminin, entactin, collagen, gelatin, etc. Examples of polymers include polylysine, polyornithine, etc. The culture surface of the cultureware may be flat or uneven.

"Laminin" is a heterotrimeric molecule consisting of α, β, and γ chains, and is an extracellular matrix protein with isoforms that differ in the composition of the subunit chains. Specifically, laminin has about 15 isoforms, which are heterotrimeric combinations of five types of α chains, four types of β chains, and three types of γ chains. The names of laminins are determined by combining the numbers of the α chains (α1-α5), β chains (β1-β4), and γ chains (γ1-γ4). For example, a laminin made up of a combination of α5 chains, β1 chains, and γ1 chains is called laminin 511.

The cultureware used in performing suspension culture is not particularly limited as long as it is capable of performing suspension culture, and a person skilled in the art can appropriately determine the cultureware. Examples of such cultureware include flasks, flasks for tissue culture, dishes, Petri dishes, dishes for tissue culture, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, spinner flasks, and roller bottles. These cultureware are preferably non-adhesive to cells in order to enable suspension culture. As a non-adhesive cultureware, a cultureware whose surface has not been artificially treated to improve adhesion to cells can be used. As a non-adhesive cultureware, a cultureware whose surface has been artificially treated to reduce adhesion to cells can also be used. The culture surface of the cultureware may be flat, U- or V-bottom, or may have irregularities. Examples of treatments that reduce adhesion to cells include superhydrophilic treatments using coatings such as 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, Poly(2-hydroxyethyl methacrylate) (Poly-HEMA), and polyethylene glycol (PEG), as well as low protein adsorption treatments.

"Shaking culture" is a culture method in which the culture medium is stirred by shaking the culture equipment, promoting the supply of oxygen to the culture medium and the exchange of materials with the surrounding cells. Agitation culture, flow path culture, etc. can also be performed.

In order to protect the cell aggregates from physical stresses such as shear forces that occur during suspension culture, and to increase the local concentrations of growth factors and cytokines secreted by the cells and promote tissue development, the cell aggregates can be embedded in a gel or encapsulated in a substance-permeable capsule before suspension culture (Nature, 2013, 501.7467:373). The encapsulated cell aggregates may be cultured with shaking. The gel or capsule used for embedding may be made of either a biological or synthetic polymer. Examples of gels or capsules used for such purposes include Matrigel (Corning), Cultrex UltiMatrix (R&D Systems), PuraMatrix (3D Matrix), VitroGel 3D (TheWell Bioscience), collagen gel (Nitta Gelatin), alginate gel (PG Research), Cell-in-a-Box (Austrianova), and Matrimix (Nippi).

"Culture equipment" refers to materials that serve as a scaffold for cells or tissues when culturing cells. Cells are usually attached to the culture equipment when used. Materials that can be used for the culture equipment include inorganic materials such as alumina, ceramics, glass, silica, zirconia, titanium, and calcium phosphate; proteins such as gelatin and collagen; polysaccharides such as alginic acid and cellulose; and synthetic polymer materials such as polyester, polypropylene, polystyrene, polycarbonate, polycaprolactone, and silicone rubber. Culture equipment can be in the form of, for example, a membrane, bead, fiber, hollow fiber, mesh, or sponge. Commercially available culture equipment can also be used. Examples of cultureware used for such purposes include Transwell (Corning), ad-MED Vitrigel 2 (Kanto Chemical), 3D Insert (Biotek), Zelets (Hilex Corporation), NanoECM (Nanofiber Solutions), SpongeCol collagen sponge (Advanced Biomatrix), CERAHIVE (CoorsTek), and Cellbed (Japan Vilene).

The medium used for culturing cells can be prepared as a basal medium using a medium normally used for culturing animal cells. Examples of the basal medium include Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow Minimum Essential Medium (Glasgow MEM), Improved MEM Zinc Option, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle Minimum Essential Medium (Eagle MEM), and Alpha Modified Eagle Minimum Essential Medium (Eagle MEM). Examples of such medium include αMEM, Dulbecco's Modified Eagle Medium (DMEM), F-12 medium, DMEM/F12, IMDM/F12, Ham's medium, RPMI 1640, Fischer's medium, and mixtures thereof.

For culturing pluripotent stem cells, a medium for culturing pluripotent stem cells based on the above-mentioned basal medium, a medium for preferably known embryonic stem cells or induced pluripotent stem cells, a medium for culturing pluripotent stem cells under feeder-free conditions (feeder-free medium), etc. Many synthetic media have been developed and are commercially available as feeder-free media, for example, Essential 8 medium. Essential 8 medium contains, in DMEM/F12 medium, the following additives: L-ascorbic acid-2-phosphate magnesium (64 mg/l), sodium selenium (14 μg/l), insulin (19.4 mg/l), NaHCO ₃ (543 mg/l), transferrin (10.7 mg/l), bFGF (100 ng/mL), and a TGFβ family signaling pathway active substance (TGFβ1 (2 ng/mL) or Nodal (100 ng/mL)) (Nature Methods, 8, 424-429 (2011)). Commercially available feeder-free media include, for example, Essential 8 (manufactured by Thermo Fisher Scientific), S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Thermo Fisher Scientific), hESF9, mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), mTeSR Plus (manufactured by STEMCELL Technologies), StemFit (manufactured by Ajinomoto Co., Inc.), and ReproMed iPSC. Examples of such medium include Cellartis DEF-CS Xeno-Free Culture Medium (manufactured by ReproCELL), NutriStem XF (manufactured by Biological Industries), NutriStem V9 (manufactured by Biological Industries), Cellartis DEF-CS Xeno-Free Culture Medium (manufactured by Takara Bio Inc.), Stem-Partner SF (manufactured by Kyokuto Pharmaceutical Co., Ltd.), PluriSTEM Human ES / iPS Cell Medium (manufactured by Merck), and StemSure hPSC Medium Δ (manufactured by Fujifilm Wako Pure Chemical Industries).

The serum-free medium may contain a serum substitute. Examples of serum substitutes include those that contain albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO 98/30679. Commercially available products may be used as serum substitutes. Examples of commercially available serum substitutes include Knockout Serum Replacement (manufactured by Thermo Fisher Scientific) (hereinafter sometimes referred to as "KSR"), Chemically-defined Lipid Concentrated (manufactured by Thermo Fisher Scientific), Glutamax (manufactured by Thermo Fisher Scientific), B27 Supplement (manufactured by Thermo Fisher Scientific), and N2 Supplement (manufactured by Thermo Fisher Scientific).

The serum-free medium used in suspension culture and adherent culture may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, etc., as appropriate.

To avoid the complication of preparation, a serum-free medium containing an appropriate amount (e.g., about 0.5% to about 30%, preferably about 1% to about 20%) of commercially available KSR (manufactured by Thermo Fisher Scientific) may be used as the serum-free medium (e.g., a medium containing 1x chemically-defined lipid concentrated, 5% KSR and 450 μM 1-monothioglycerol added to a 1:1 mixture of F-12 medium and IMDM medium). Alternatively, a serum-free medium containing N2 Supplement, B27 Supplement and HEPES added to DMEM/F-12 medium or Advanced DMEM/F-12 medium may be used. Additionally, an example of a medium equivalent to KSR is the medium disclosed in JP2001-508302.

"Serum medium" refers to a medium containing unconditioned or unpurified serum. The medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-monothioglycerol, pyruvic acid, buffers, inorganic salts, and the like. Serum is usually collected from fetuses, newborns, adults, and the like of cows, horses, donkeys, goats, and rabbits. The most commonly used serum medium is a medium using serum derived from fetal cows, but is not limited to this.

The culture in the present invention is preferably carried out under xeno-free conditions. "Xeno-free" means conditions in which components derived from organisms other than the organism of the cells to be cultured are excluded. The culture in the present invention is more preferably carried out under animal-free conditions. "Animal-free" means conditions in which components derived from animals are excluded.

The medium used in the present invention is preferably a medium whose components are chemically defined (CDM) in order to avoid contamination with chemically undefined components.

"Basement membrane structure" means a thin membrane-like structure composed of extracellular matrix. In living organisms, basement membranes are formed on the basal side of epithelial cells. Components of basement membranes include type IV collagen, laminin, heparan sulfate proteoglycan (perlecan), entactin/nidogen, cytokines, growth factors, etc. Whether or not basement membranes are present in tissues derived from living organisms and in cell populations produced by the production method of the present invention can be detected by, for example, tissue staining such as PAM staining, and immunohistochemistry using antibodies against components of basement membranes (anti-laminin antibodies, anti-type IV collagen antibodies, etc.).

The term "basement membrane preparation" refers to a preparation containing basement membrane components that have the function of controlling epithelial cell-like cell morphology, differentiation, proliferation, movement, functional expression, etc., when desired cells having basement membrane formation ability are seeded and cultured thereon. In the present invention, when cells are cultured for adhesion, they can be cultured in the presence of a basement membrane preparation. Here, "basement membrane components" refer to thin membrane-like extracellular matrix molecules that exist between the epithelial cell layer and the interstitial cell layer, etc., in animal tissues. A basement membrane preparation can be prepared, for example, by removing cells having basement membrane formation ability that are adhered to a support via a basement membrane from the support using a solution capable of dissolving the lipids of the cells, an alkaline solution, or the like. Examples of basement membrane preparations include products commercially available as basement membrane preparations, such as Matrigel (manufactured by Corning), Geltrex (manufactured by Thermo Fisher Scientific), and Cultrex Basement Membrane Extract (manufactured by R&D Systems), which contain extracellular matrix molecules known as basement membrane components (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.).

For cell or tissue culture, basement membrane preparations such as Matrigel (Corning) extracted and solubilized from tissues or cells such as Engelbreth-Holm-Swarm (EHS) mouse sarcoma can be used. Similarly, as basement membrane components for cell culture, human solubilized amniotic membrane (BioLamina), human recombinant laminin produced in HEK293 cells (BioLamina), human recombinant laminin fragments (Nippi), human recombinant vitronectin (Thermo Fisher Scientific), etc. can also be used. From the viewpoint of avoiding contamination with components from different biological species and from the viewpoint of avoiding the risk of infectious diseases, the basement membrane preparation preferably uses recombinant proteins with known components.

In this specification, "medium containing substance X" and "in the presence of substance X" respectively mean a medium to which exogenous substance X has been added or a medium containing exogenous substance X, and in the presence of exogenous substance X. Exogenous substance X is distinguished from endogenous substance X, for example, in which cells or tissues present in the medium endogenously express, secrete, or produce the substance X. Substance X in the medium may have undergone a slight change in concentration due to decomposition of substance X or evaporation of the medium.

In this specification, the start of culture in a medium with a concentration of substance X of Y preferably refers to the point in time when the concentration of substance X in the medium becomes uniform at Y, but when the culture vessel is sufficiently small (for example, a 96-well plate or culture in 200 μL or less of culture medium), the start of culture at concentration Y is interpreted as the point in time when the medium addition operation, half-volume medium replacement operation, or full-volume medium replacement operation described below is performed to achieve concentration Y. In addition, when the concentration of substance X in the medium is Y, this includes the case where the average concentration of X over a certain culture period is Y, the period during which substance X is contained at a concentration of Y is 50% or more of the culture period, the period during which substance X is contained at a concentration of Y is equal to or longer than the shortest period of the culture periods expected in each process, etc.

In this specification, "in the absence of substance X" means a medium to which exogenous substance X has not been added, a medium that does not contain exogenous substance X, or a state in which exogenous substance X is not present.

As used herein, "human protein X" means that protein X has the amino acid sequence of protein X that is naturally expressed in the human body. Human protein X preferably undergoes post-translational modifications such as phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, etc. equivalent to protein X that is naturally expressed in the human body.

"Isolated" means that the target component or factors other than the cell have been removed, and that the protein is no longer in a naturally occurring state. Therefore, "isolated protein X" does not include endogenous protein X that is produced from the cells or tissues to be cultured and is contained in the cells, tissues, and medium. The purity of "isolated protein X" (the percentage of protein X weight in the total protein weight) is usually 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 99% or more, and most preferably 100%.

In this specification, the term "derivative" refers to a group of compounds that are produced by replacing a part of a molecule of a specific compound with another functional group or another atom. In this specification, the term "modified form" of a protein refers to a protein that has been mutated, such as by deletion, addition, or substitution of amino acid residues, to the extent that the properties of the original protein can be maintained. The number of mutated amino acids is not particularly limited, but may be 1 to 4, 1 to 3, 1 to 2, or 1. The "modified form" of a protein may be a protein that has an amino acid sequence that is at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to the original protein. The mutated amino acid in the modified form may be a non-natural type. The term "protein fusion" or "fusion protein" refers to a protein in which an amino acid sequence required for the function of the same or another protein is added to a certain protein. Fusion proteins may be either artificially created by techniques such as genetic engineering, or naturally generated by chromosomal translocation, etc. Examples of fusion proteins include GST tag fusion proteins to which glutathione-S-transferase has been added for efficient purification and detection, fluorescent protein fusion proteins such as GFP to which a fluorescent protein has been added to reveal the localization of a protein, and Fc fusion proteins to which an IgG Fc domain has been added to improve protein stability, etc.

In this specification, "after time A (day A)" includes time A (day A) and refers to anything after time A (day A). "within time B (day B)" includes time B (day B) and refers to anything before time B (day B).

"Feeder cells" refers to cells other than the cells that are allowed to coexist when the cells are cultured. Examples of feeder cells used in the culture of pluripotent stem cells to maintain the undifferentiated state include mouse fibroblasts (MEF), human fibroblasts, and SNL cells. It is preferable that the feeder cells have been subjected to a growth-inhibiting treatment. Examples of the growth-inhibiting treatment include treatment with a growth inhibitor (e.g., mitomycin C) and gamma-ray irradiation. Feeder cells used in the culture of pluripotent stem cells to maintain the undifferentiated state of pluripotent stem cells contribute to maintaining the undifferentiated state of pluripotent stem cells by secreting humoral factors (preferably factors for maintaining the undifferentiated state) and creating a scaffold (extracellular matrix) for cell adhesion.

In this specification, "in the absence of feeder cells" refers to culturing in the absence of feeder cells. Examples of "in the absence of feeder cells" include conditions where no feeder cells are added, or conditions where feeder cells are substantially not included (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less).

A "cell aggregate" or "cell mass" refers to a mass formed by the gathering of cells, in which the cells are adhered to each other. Embryoid bodies, spheres, spheroids, and organoids are also included in cell aggregates. In cell aggregates, cells are preferably adhered to each other on their surfaces. In some embodiments, in a part or all of a cell aggregate, cells are adhered to each other, for example, forming adhesion junctions. In some embodiments, the cell aggregate may contain an extracellular matrix. In some embodiments, two or more cell aggregates can be further artificially adhered or aggregated to each other. Masses in which cell populations are further adhered or aggregated to each other, and assembloids are also included in cell aggregates. The shape of the cell aggregate is not limited to a spherical shape, but may be, for example, a bisphere, a beaded shape, a collection of spheres, a string-like or branched shape (shapes described in Scientific reports, 11:21421 (2021), Japanese Patent Application No. 2021-078154), a sheet-like shape, etc.

"Uniform cell aggregates" means that the size of each cell aggregate is constant when multiple cell aggregates are cultured. When the size of a cell aggregate is evaluated by the length of the maximum diameter, uniform cell aggregates mean that the variance of the length of the maximum diameter is small. More specifically, this means that the maximum diameter of 75% or more of the multiple cell aggregates is within ±100% of the average maximum diameter of the multiple cell aggregates, preferably within ±50% of the average maximum diameter, and more preferably within ±20% of the average maximum diameter.

"Cell population" refers to a cell group consisting of two or more cells. A cell population may be composed of one type of cell, or may be composed of multiple types of cells. The cells constituting the cell population may be suspended in a medium or may be attached to a culture vessel or the like. The cells constituting the cell population may be single cells, or at least a part of the cell population may be formed by cell adhesion between the cells. Here, "single cells" refer to, for example, cells that have almost no adhesion between cells (e.g., surface adhesion). In some embodiments, being dispersed into single cells refers to a state in which cell-cell bonds (e.g., adhesive bonds) have almost no bond between the cells. The cell population may include cell aggregates, cell clumps, and tissues described below.

"Tissue" refers to a structure of a cell population in which multiple types of cells with different morphologies or properties are arranged three-dimensionally in a certain pattern. In one aspect of this embodiment, the above tissue can also be understood as a type of cell aggregate or cell mass.

"Ectoderm" refers to the outermost of the three germ layers formed after fertilization of an egg during the early development of an organism. As development progresses, the ectoderm divides into neuroectoderm and superficial ectoderm, and the neuroectoderm further divides into neural tube and neural crest. Various organs of the body are formed from these ectoderm, and organs formed from the ectoderm are said to be derived from the ectoderm. For example, the neural tube forms organs or tissues of the central nervous system, such as the brain and spinal cord. For example, some central nervous system cells, facial bones and cartilage, sensory nerve cells, autonomic nerve cells, pigment cells, mesenchymal cells, etc. are formed from the neural crest. Upper respiratory tract tissues including olfactory epithelium, epidermis, inner ear, anterior pituitary gland, etc. are formed from the superficial ectoderm. Placodes and placode-derived tissues are derived from the superficial ectoderm. In the process of differentiation into ectoderm, pluripotent stem cells express genes known as ectoderm markers, such as Pax3, Otx2, and Sox1.

"Endoderm" refers to the innermost of the three germ layers that are formed after fertilization of an egg during the early development of an organism. For example, the digestive system, urinary tract, pharynx, trachea, bronchi, and lungs are formed from the endoderm. In the process of differentiating into endoderm, pluripotent stem cells express genes known as endoderm markers, such as SOX17, HNF-3β/FoxA2, Klf5, GATA4, GATA6, and PDX-1.

"Mesoderm" refers to the germ layer formed between the ectoderm and endoderm. Organs or tissues such as the circulatory system, skeleton, and muscles are formed from the mesoderm. In the process of differentiating into the mesoderm, pluripotent stem cells express genes known as mesoderm markers, such as T/Brachury, SMA, ABCA4, Nkx2.5, and PDGFRα.

"Nervous tissue" refers to tissue composed of nervous system cells such as the cerebrum, midbrain, cerebellum, spinal cord, retina, sensory nerves, and peripheral nerves during development or adulthood. In this specification, "neuroepithelial tissue" refers to nervous tissue that has formed an epithelial structure with a layered structure. The amount of neuroepithelial tissue in nervous tissue can be evaluated by bright-field observation using an optical microscope, etc.

The "central nervous system" refers to the area where nervous tissue is accumulated and which is the center of information processing. In vertebrates, the central nervous system includes the brain and spinal cord. The central nervous system is derived from the ectoderm.

"Neural cells" refers to cells of ectoderm-derived tissues other than epidermal cells. In other words, neural cells include neural progenitor cells, neurons (nerve cells), glial cells, neural stem cells, neuronal progenitor cells, glial progenitor cells, and other cells. Neural cells also include cells that constitute retinal tissue (retinal cells), retinal progenitor cells, retinal layer-specific nerve cells, neural retina cells, and retinal pigment epithelial cells, which will be described later. Neural cells can be identified by using nestin, βIII tubulin (Tuj1), PSA-NCAM, N-cadherin, and other markers.

Neurons are functional cells that form neural circuits and contribute to signal transmission. Neurons can be identified based on the expression of immature neuronal markers such as TuJ1, Dcx, and HuC/D, and/or mature neuronal markers such as Map2 and NeuN.

Examples of glial cells include astrocytes, oligodendrocytes, and Muller glia. An example of an astrocyte marker is GFAP. An example of an oligodendrocyte marker is O4. An example of a Muller glia marker is CRALBP.

Neural stem cells are cells that have the ability to differentiate into neurons and glial cells (pluripotency) and the ability to proliferate while maintaining pluripotency (sometimes called self-renewal). Neural stem cell markers include Nestin, Sox2, Musashi, the Hes family, CD133, etc. However, these markers are markers for progenitor cells in general and are not considered to be specific to neural stem cells. The number of neural stem cells can be evaluated by neurosphere assays, clonal assays, etc.

Neuronal progenitor cells are cells that have the ability to proliferate, produce nerve cells, and do not produce glial cells. Markers for neuronal progenitor cells include Tbr2 and Tα1. Cells that are positive for immature nerve cell markers (TuJ1, Dcx, HuC/D) and proliferation markers (Ki67, pH3, MCM) can also be identified as neuronal progenitor cells. Glial progenitor cells are cells that have the ability to proliferate, produce glial cells, and do not produce nerve cells.

Neural precursor cells are a collection of precursor cells including neural stem cells, neuronal precursor cells, and glial precursor cells. Neural precursor cells have the ability to proliferate and produce neurons and glia. Neural precursor cells can be identified using markers such as Nestin, GLAST, Sox2, Sox1, Musashi, and Pax6. Cells that are positive for neural cell markers and proliferation markers (Ki67, pH3, MCM) can also be identified as neural precursor cells.

"Non-neuroepithelial tissue" refers to tissues having an epithelial structure other than neuroepithelial tissue. Epithelial tissue is formed from any of the germ layers, including ectoderm, mesoderm, endoderm, and trophectoderm. Epithelial tissue includes epithelium, mesothelium, and endothelium. Examples of tissues included in non-neuroepithelial tissue include epidermis, corneal epithelium, nasal epithelium, olfactory epithelium, oral epithelium, tracheal epithelium, bronchial epithelium, airway epithelium, renal epithelium, renal cortical epithelium, and placental epithelium. Epithelial tissues are usually connected by various intercellular junctions, forming tissues with a single layer or stratified layer structure. The presence or absence of these epithelial tissues and the amount present can be confirmed by observation with an optical microscope or by immunohistochemistry using antibodies against epithelial cell markers (anti-E-Cadherin antibody, anti-N-Cadherin antibody, anti-EpCAM antibody, etc.).

In the present invention, "epithelial polarity" refers to the bias in the distribution of spatially formed components and cell functions within epithelial cells. For example, corneal epithelial cells are localized in the outermost layer of the eyeball, and express apical-specific proteins such as membrane-bound mucins (MUC-1, 4, 16) that retain tears on the apical side. In addition, corneal epithelial cells express basal-specific proteins such as α6 integrin and β1 integrin that adhere to the basement membrane on the basal side.

Whether or not epithelial cell polarity is present in epithelial cells and epithelial tissue in tissues derived from living organisms and in cell populations produced by the manufacturing method of this embodiment can be detected by immunohistochemistry or other techniques using phalloidin, apical markers (anti-MUC-1 antibody, anti-PKC-zeta antibody, etc.), and basal markers (anti-α6 integrin antibody, anti-β1 integrin antibody, etc.).

In the present invention, "trans-epithelial electrical resistance (TEER)" refers to electrical resistance caused by the restriction of ion permeation between the apical surface side and the basement membrane side by tight junctions formed by epithelial cells. The value of trans-epithelial electrical resistance reflects the barrier performance, substance permeability, etc. of epithelial tissue. The value of trans-epithelial electrical resistance can be measured using, for example, devices such as cellZscope (manufactured by CellSeed), EVOM, EVOM2 (manufactured by WPI), Millicell ERS-2 Resistance Measurement System (manufactured by Merck), and trans-epithelial electrical resistance (TEER) measuring device (manufactured by Tsuruga Electric Co., Ltd.) and electrodes attached to the device. The value of trans-epithelial electrical resistance is expressed, for example, as unit area resistance: Ω·cm ² .

"Placode" refers to the primordium of an organ that is formed by thickening of a part of the epidermal ectoderm mainly during the development of vertebrates. Tissues derived from the placode include the lens, olfactory epithelium, inner ear, trigeminal nerve, and pituitary gland. Markers of the placode and its precursor tissue, the preplacode and preplacode region, include Six1, Six4, Dlx2, Dlx5, and Eya2.

The "olfactory epithelium placode" is a thickened structure formed in the epidermal ectoderm region during embryogenesis, which expresses olfactory epithelium progenitor cell markers and represents the primordium of the organ that will become the olfactory epithelium in the future. Known genes expressed in the olfactory epithelium placode and olfactory epithelium progenitor cell markers include Pax6, Otx2, FoxG1 (also known as Bf1), FoxD4, Sox2, Sox3, Pou2f1, Sp8, Chd7, Aldh1a1, Aldh1a3, Gata3, Dmrt4, N-Cadherin, E-Cadherin, EpCAM, CK18, PDGFRβ, etc.

The term "respiratory system" refers to the organs involved in external respiration, which takes in oxygen from outside the body and expels carbon dioxide to the outside. Examples of organs included in the respiratory system include the nasal cavity, pharynx, larynx, trachea, bronchi, lungs, and thorax. Anatomically, the respiratory system is divided into the upper and lower respiratory tract. The nostrils, nasal cavity, nasopharynx, pharynx, and larynx are classified as the upper respiratory tract. The trachea, bronchi, bronchioles, and lungs are classified as the lower respiratory tract. The nasal epithelium and olfactory epithelium are upper respiratory tract tissues.

The "nasal cavity" is one of the tissues of the upper respiratory tract, and refers to the space that exists behind the openings (nostrils) in the external nose, which is located in the center of the face. The nasal cavity begins at the nostrils and connects to the upper part of the pharynx. The nasal cavity is mainly composed of a part called the olfactory region, which contains olfactory epithelium and detects odorous substances in the air, a part called the respiratory region that is covered by nasal epithelium, and a part that has properties similar to skin and is covered by keratinized squamous stratified epithelium.

"Nasal epithelium" refers to the epithelial tissue found in the nasal cavity, and mainly refers to the area other than the olfactory epithelium, which will be described later. The functions of the nasal epithelium are to warm the air taken in and to remove foreign bodies by the mucus secreted on the surface and the action of cilia on the cell surface. The nasal epithelium is mainly derived from ectodermal non-neural epithelium, and is composed of cells such as secretory cells, ciliated cells, club cells, basal cells, salt cells, neuroendocrine cells, and isolated chemosensory epithelial cells.

"Oral epithelium" refers to the epithelial tissue and cells that form the oral cavity. Examples of oral epithelium include oral mucosal epithelium, salivary gland epithelium, and odontogenic epithelium. Oral mucosal epithelium is usually a mucosal tissue made of stratified squamous epithelium, and contains basal cells, Merkel cells, melanin-producing cells, etc. on the basement membrane in contact with connective tissue, with spinous cells, granular cells, and a stratum corneum formed on the upper layer. Oral mucosal epithelium can be detected as tissue that is positive for cytokeratin 7, 8, 13, 14, and 19, for example.

"Secretory cells" refers to cells that secrete mucus or serous fluid in respiratory tissues. Goblet cells and glandular cells belong to the secretory cells. Genes and markers expressed in secretory cells and their precursor cells include, for example, mucin 1, mucin 2, mucin 3, mucin 4, mucin 5AC, mucin 5B, FoxA1, FoxA2, FOXA3, Nkx2.1/TTF-1, SPDEF, etc. In addition, genes, transcripts, and markers expressed in secretory cells that the present inventors found in WO/2022/026238 include CEACAM5, LY6D, ADH1C, MSMB, PSCA, TSPAN1, BPIFB1, FAM3D, SCGB1A1, CYP2F1, ADH7, CRYM, CP, GSTA1, STEAP4, ALDH3A1, FMO3, and CXCL8.

"Ciliated cells" (also called "ciliated epithelial cells") are cells that form epithelial tissue and have cilia on the cell surface. Ciliated cells are present in epithelial tissues such as the respiratory system, ventricles, and fallopian tubes, and are known to transport liquids, humoral factors, foreign bodies that have entered the body, eggs, etc. in a specific direction by the movement of their cilia. Examples of genes and markers expressed in ciliated cells and their precursor cells include FoxJ1, FoxN4, p73, acetylated tubulin, β4 tubulin, DNAI1, DNAH5, and SNTN. The presence of ciliated cells and their precursor cells can be confirmed by observing the ciliary structure on the cell surface after staining the tissue or cells with hematoxylin-eosin (HE) or the like under a microscope. For example, see J. Microsc., 211 (2003), pp. The presence of ciliated cells can also be observed by measuring the degree of ciliary beating frequency (CBF) using the method described in 103-111.

"Ciliary beating" refers to the movement of cilia on the cell surface, and in mammals, including humans, it has functions such as eliminating pathogens such as bacteria and viruses in the respiratory system and transporting eggs in the fallopian tubes. Ciliary beating is usually a wave movement consisting of a positive stroke and a negative stroke. Whether a cell is performing ciliary beating can be detected, for example, by using a high-magnification lens to take video footage under a microscope.

"Club cells" (also called "Clara cells") are cells that form epithelial tissue and have functions such as secreting mucus or serous components, metabolizing compounds through cytochrome P450, secreting cytokines, and controlling immune functions. Genes and markers expressed in club cells include SCGB1A1, SCGB3A2, CYP2f1, CYP2f2, uroplakin3a, Cldn10, Aox3, Pon1, and Cckar. In addition, "Hillock cells," which express cytokeratin 4 and cytokeratin 13 and are thought to be involved in barrier function and immune responses, are also included as a subpopulation of club cells.

"Basal cells" refers to cells that exist in the basal layer of the epithelium. Basal cells have the ability to divide, and as tissue stem cells, contribute to the long-term maintenance of biological tissues and repair in the event of injury. Genes and markers expressed in basal cells of the nasal epithelium and their precursor cells include cytokeratin 5, cytokeratin 14, cytokeratin 15, p63, ΔNp63, p75, etc.

Chloride cells (also called "ionocytes") are cells that express CFTR and have the function of controlling the viscosity of mucosal mucus. Genes and markers expressed in chloride cells and their precursor cells include CFTR, Foxi1, vacuolar proton ATPase (vacuolar H+-ATPase, V-ATPase), Ascl3, Tfcp2l1, DMRT2, etc.

Neuroendocrine cells are cells that secrete hormones and peptides and have the function of controlling the development and homeostasis of the living body. Genes and markers expressed in neuroendocrine cells and their precursor cells include Ascl1, NeuroD1, Chromogranin A, Synaptophisin, neuron-specific enolase (NSE), CALCA, etc.

Isolated chemosensory epithelial cells are cells that have functions such as sensing chemical substances and controlling host defense reactions. Tuft cells, brush cells, and microvilli cells also represent the same type of cells. Genes and markers expressed in isolated chemosensory epithelial cells include ChAT/Trpm5, Pou2f3/Skn-1a, bitter taste receptor Tas2r, Gnat3, Plcb2, GNG13, etc.

The "olfactory epithelium" is epithelial tissue in the nasal cavity, and refers to the olfactory organ that detects odor information in living organisms. The olfactory epithelium is classified as upper respiratory tract tissue. The olfactory epithelium is one of the tissues derived from the placode, and is composed of cells such as olfactory nerve cells that express olfactory receptors and detect volatile molecules in the air, supporting cells that support the olfactory nerve cells, basal cells that are the stem and progenitor cells of the olfactory epithelium, and Bowman's gland cells that secrete mucus. The olfactory epithelium is morphologically classified into the superficial layer, intermediate layer, and basal layer, with supporting cells in the superficial layer, olfactory nerve cells in the intermediate layer, and basal cells in the basal layer. Bowman's glands form branched tubular alveolar glands scattered throughout the olfactory epithelium.

"Olfactory epithelium precursor tissue" includes the olfactory epithelium placode. Genes and markers expressed in the olfactory epithelium and olfactory epithelium placode include the placode and pre-placode markers mentioned above, as well as Pax6, Otx2, FoxG1 (also known as Bf1), Sox2, Pou2f1, Sp8, Chd7, N-Cadherin, E-Cadherin, EpCAM, CK18, PDGFRβ, etc. In addition, the present inventors have found in WO/2022/026238 that genes, transcripts, and markers expressed in the precursor tissue of the olfactory epithelium include HIST1H4C, ZNF367, A2M, MGP, HIST1H1A, HIST1H2BH, NUSAP1, HIST1HIC, MCAM, RGS5, ESCO2, C11orf88, ZMYD10, MELK, HJURP, TOP2A, C These include EP152, CAV1, HMGB2, CENPW, OR2B11, SLC17A6, GADL1, SCN4B, SMIM35, AC245595.1, LINC00642, GNG13, JAKMIP1, LINGO2, ANO2, C7orf57, NXPH3, STOM, KRT74, NXNL2, SCGB2A1, GABBR2, TMPRSS6, and HYDIN.

"Olfactory system tissue" refers to fetal or adult olfactory system tissue, such as tissue in which one or at least multiple types of cells constituting the olfactory epithelium (e.g., olfactory nerve cells, supporting cells, basal cells, Bowman's gland cells, olfactory ensheathing cells, and their precursor cells, etc.), cells constituting the olfactory bulb, cells constituting the olfactory cortex, etc., are arranged three-dimensionally in layers. The presence of each cell can be confirmed by known methods, such as the presence or absence of expression of cell-specific genes, or the degree of expression, etc.

Olfactory receptor neuron (ORN) refers to the nerve cell that receives the sense of smell located in the intermediate layer of the olfactory epithelium of the nasal mucosa. Olfactory neurons capture volatile molecules in the air with olfactory receptors located in the cilia on their surface. Olfactory neurons are bipolar sensory cells. In olfactory neurons, olfactory information captured by olfactory receptors expressed on the peripheral side is transmitted to nerve axons called olfactory filaments on the central side. Several dozen olfactory filaments gather together to form a bundle, and the olfactory nerve refers to all of these bundles. The olfactory filaments reach the olfactory bulb in the brain through the ethmoid foramen in the cribriform plate of the ethmoid bone, where they synapse with mitral cells and transmit olfactory information to the olfactory center in the brain. Genes and markers expressed in olfactory neurons and their precursor cells include cyclic nucleotide-sensitive channel α2 subunit (CNGA2), cyclic nucleotide-sensitive channel α4 subunit (CNGA4), cyclic nucleotide-sensitive channel β1b subunit, olfactory specific G protein (Golf) adenylate cyclase, Olfactory Marker protein (OMP), NCAM, OCAM (NCAM-2), Ebf1, Ebf2, Ebf3, NeuroD, PGP9.5, Neuron Specific Enolase (NSE), Growth Associated Protein 43 (GAP-43/B50), vimentin, Lhx2, Id3, β-Tubulin III (Tuj), Calretinin, TrkB, Ctip2, Uncx, and various olfactory receptors (ORs). In addition, genes, transcripts, and markers expressed in olfactory nerve cells discovered by the present inventors in WO/2022/026238 include NHLH1, GNG8, LHX9, OLIG2, TSHZ1, SPOCK2, PCDH8, INSM1, CARTPT, TUBA1A, DOC2B, MDM1, OLIG1, KCNMA1, ARHGDIG, TRIB3, ELAVL4, LINC02384, and FNDC5.

"Supporting cells" (sustentacular cells) refer to pseudostratified columnar epithelium-like cells present at the most apical side of the olfactory epithelium. Supporting cells are involved in the functioning of other cells such as olfactory nerve cells, as well as their survival and maintenance of epithelial structure. Microvillar cells of the olfactory epithelium are a subtype of supporting cells. Genes and markers expressed in supporting cells include Notch2, Notch3, carbonyl reductase 2 (Cbr2), S-100, Ezrin, Reep6, Sox2, Tyro3, CYP2A6, SUS-1, SUS-4, EPAS1, etc. In addition, genes, transcripts, and markers expressed in supporting cells discovered by the present inventors in WO/2022/026238 include SEC14L3, GPX6, LINC02159, UCN3, ATP13A5, KLKB1, TMEM211, FGF20, CYP2A13, SCARA5, SLC14A1, ERMN, METTL7A, NOS2, B3GNT7, SLC5A8, PPP1R16B, CES1, DUOXA1, and SLCO1A2.

Basal cells in the olfactory epithelium refer to cells that exist in the basal layer of the olfactory epithelium. Morphologically, basal cells are divided into globular basal cells (GBC) and horizontal basal cells (HBC). Of these two types of cells, globular basal cells are active precursor cells and stem cells that constantly undergo cell division and supply new olfactory nerve cells. On the other hand, horizontal basal cells are dormant stem cells whose cell cycle of cell division is normally stopped and that are activated when large-scale damage to the olfactory epithelium occurs. Genes and markers expressed in horizontal basal cells include p63, cytokeratin 5, cytokeratin 14, ICAM-1, etc. Genes and markers expressed in globular basal cells include GAP43, GBC-1, Lgr5, Ascl1, LSD1, SEC8, c-Kit/CD117, Sox2, Hes1, Id, Bmi1, etc. In addition, genes, transcripts, and markers expressed in basal cells of the olfactory epithelium discovered by the present inventors in WO/2022/026238 include S100A2, NPPC, ADIRF, POSTN, ATF3, MYC, KRT17, FOSB, MEG3, CXCL14, SULT1E1, CD44, FHL2, FXYD3, TRIM29, CLDN1, SERPINF1, and IGF1.

"Bowman's gland cells" refers to cells that make up the Bowman's gland (olfactory gland). The Bowman's gland is a branched, thin duct-like tissue that exists in the olfactory epithelium, and has functions such as secreting mucus that protects the olfactory epithelium. Genes and markers expressed in Bowman's gland cells include Sox9, E-Cadherin, Aquaporin5, Ascl3, and cytokeratin 18.

Olfactory ensheathing glia (OEG) refers to a type of glial cell present in the olfactory epithelium and olfactory bulb. Olfactory ensheathing glia release neurotrophic factors such as BDNF and NGF, and are involved in the constant regeneration of the olfactory nerve. Genes and markers expressed in olfactory ensheathing glia include p75NTR, S100β, Sox10, GFAP, BLBP, Aquaporin1, and Integrin α7.

The "lateral olfactory epithelium" refers to the peripheral region of the olfactory epithelium, which is continuous with non-neural epithelium other than the olfactory epithelium. In the present invention, the "medial olfactory epithelium" refers to the central region of the olfactory epithelium surrounded by the lateral olfactory epithelium. It is known that the ratio of cells constituting the lateral olfactory epithelium and the medial olfactory epithelium is different (Development, 2010, 137: 2471-2481). Genes highly expressed in the lateral olfactory epithelium include Pbx1/2/3, Meis1, and βIVTubulin. Genes highly expressed in the medial olfactory epithelium include Tuj1, Ascl1, and Sox2.

"Gonadotropin-releasing hormone positive neurons" are neurons that are mainly present in the hypothalamus of the central nervous system and secrete gonadotropin releasing hormone (GnRH), which is involved in controlling the organs of the reproductive system. Gonadotropin-releasing hormone positive neurons develop in the olfactory epithelium during fetal development and then migrate to the central nervous system. Genes and markers expressed in gonadotropin-releasing hormone positive neurons include gonadotropin releasing hormone (GnRH), Pou2f1, Dlx5, Meis1, Pbx1, Pknox1, Zeb1, etc.

The "olfactory pit" is the tissue that becomes the basis of the olfactory system tissues when the olfactory epithelium placode invaginates into the inside of the embryo during development. The olfactory pit is mainly composed of tissues and cells that will make up the future olfactory epithelium, as well as tissues and cells of the nasal epithelium that will form continuously with the olfactory epithelium.

"Bone tissue" refers to the cells that make up bone and the bone matrix that is composed of calcium phosphate, type I collagen, etc. The cells that make up bone include osteocytes, osteoclasts, osteoblasts, etc.

In the present invention, "mesenchymal cells" are non-epithelial cells that are derived mainly from the mesoderm and neural crest and form connective tissue. Some of these cells are multipotent somatic stem cells called mesenchymal stem cells. Whether or not mesenchymal cells are present in the first cell population and the second cell population in the production method of the present invention, and in the sheet-shaped cell structure produced by the production method of the present invention, can be detected by a method such as immunohistochemistry using antibodies against mesenchymal cell markers such as nestin, vimentin, cadherin-11, laminin, and CD44. The presence or absence of mesenchymal stem cells can be detected by immunohistochemistry or other techniques using antibodies against mesenchymal stem cell markers such as CD9, CD13, CD29, CD44, CD55, CD59, CD73, CD105, CD140b, CD166, VCAM-1, STRO-1, c-Kit, Sca-1, Nucleostemin, CDCP1, BMPR2, BMPR1A, and BPMR1B.

In the present invention, "niche" or "stem cell niche" refers to a microenvironment involved in the proliferation, differentiation, and maintenance of properties of stem cells. Examples of niches in the body include the hematopoietic stem cell niche, hair follicle stem cell niche, intestinal epithelial stem cell niche, muscle stem cell niche, pituitary niche, and basal cell niche. In these niches, stem cells specific to each tissue and supporting cells that provide the niche exist. In addition, stem cells are maintained by cytokines, chemokines, extracellular matrices, cell adhesion factors, intercellular signaling factors, and the like provided by the supporting cells.

A "receptor protein" is a protein found in the cell membrane, cytoplasm, or nucleus that binds to substances such as hormones, cytokines, cell growth factors, and compounds, and induces various cellular responses.

"Olfactory receptor" refers to a receptor protein that is expressed in olfactory nerve cells and other cells and is involved in the detection of compounds, etc.

A "pathogen" is an entity that has the ability to cause disease in a host organism. Viruses, bacteria, fungi, protozoa, parasites, proteins such as abnormal prion proteins, and nucleic acids are all considered pathogens.

A "viral receptor" is a host factor that a virus binds to when it infects a cell or tissue. A viral receptor is usually a protein or sugar chain. For example, angiotensin-converting enzyme 2 (ACE2) is a viral receptor for SARS-CoV-2, and it has been reported that it binds to the spike protein of SARS-CoV-2. Sialic acid α2,6Gal has been reported to be a receptor for human seasonal influenza viruses (H1N1, H3N2, etc.). Sialic acid α2,3Gal has been reported to be a receptor for avian influenza viruses (H5N1, etc.). Other viral receptors are described, for example, in Nature reviews Molecular cell biology 4.1 (2003): 57-68, etc.

"Viral infection-related factors" refer to host factors that affect the virus' infection of cells and tissues. For example, TMPRSS2 is a type of protease that has been reported to cleave SARS-CoV-2 proteins and convert them into active forms when the virus infects cells.

"Pattern recognition receptors (PRRs)" refer to receptors that have the function of recognizing foreign substances such as pathogens. Examples of pattern recognition receptors include lectins, complements, membrane receptors, and cytoplasmic proteins. Pattern recognition receptors recognize pathogen-specific molecular patterns (pathogen-associated molecular patterns; PAMPs) and exert their functions. Examples of pathogen-specific molecular patterns include nucleic acids (DNA, RNA), sugar chains, and proteins, and pattern recognition receptors bind to their respective specific targets.

"Cytokine" refers to a group of proteins that the body uses to transmit information between cells. Examples of cytokines include chemokines, growth factors, hematopoietic factors, interferons, interleukins, and tumor necrosis factors.

"Immunity" is a system that the body has to recognize and eliminate or neutralize non-self substances such as pathogens and viruses, abnormal and harmful cells that are of self-origin such as cancer cells, and foreign substances. "Immune response" is a physiological reaction exhibited by the tissues and cells of the immune system when the immune system is functioning.

"Virus isolation" refers to the proliferation and identification of a specific virus present in a specimen in which the presence of the virus is suspected. Examples of specimens include tissues, excretions, and secretions of animals, including humans, plants, and other living organisms, as well as environmental samples. Cultured cells, animals, etc. are used for virus isolation depending on the type and characteristics of the target virus.

[2. Method for producing a sheet-shaped cell structure containing upper respiratory tract cells]
The present invention provides a method for producing a sheet-shaped cell structure containing upper respiratory tract cells.
One aspect of the present invention is
A method for producing a sheet-shaped cell structure containing upper respiratory tract cells, comprising the steps of:
(1) dispersing a first cell population containing olfactory placode cells or olfactory epithelial stem cells to obtain single cells;
(2) purifying the single cells obtained in the step (1) to obtain a second cell population containing the olfactory placode cells or olfactory epithelial stem cells, wherein the content of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is higher than the content of the olfactory placode cells and olfactory epithelial stem cells in the first cell population; and
(3) seeding the second cell population obtained in the step (2) on a culture vessel, culturing the second cell population, and obtaining the sheet-shaped cell structure;
including.

The first cell population in step (1) is
(a) culturing pluripotent stem cells in the presence of a Wnt signaling pathway inhibitor;
(b) culturing the cells cultured in step (a) in the presence of a BMP signaling pathway agent; and
(c) culturing the cells cultured in the step (b) in the presence of at least one substance selected from the group consisting of an FGF signaling pathway agonist and a BMP signaling pathway inhibitor, thereby producing the first cell population containing the olfactory placode cells or olfactory epithelial stem cells;
It is preferably produced by a process comprising:

That is, another preferred embodiment of the method for producing a sheet-shaped cell structure containing upper respiratory tract cells comprises:
A method for producing a sheet-shaped cell structure containing upper respiratory tract cells, comprising the steps of:
(a) culturing pluripotent stem cells in the presence of a Wnt signaling pathway inhibitor;
(b) culturing the cells cultured in step (a) in the presence of a BMP signaling pathway agent; and
(c) culturing the cells cultured in the step (b) in the presence of at least one substance selected from the group consisting of an FGF signaling pathway agonist and a BMP signaling pathway inhibitor to produce a first cell population containing olfactory placode cells or olfactory epithelial stem cells;
(1) dispersing the first cell population to obtain single cells;
(2) purifying the single cells obtained in the step (1) to obtain a second cell population containing the olfactory placode cells or olfactory epithelial stem cells, wherein the content of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is higher than the content of the olfactory placode cells and olfactory epithelial stem cells in the first cell population; and
(3) seeding the second cell population obtained in the step (2) on a culture vessel, culturing the second cell population, and obtaining the sheet-shaped cell structure;
Below, the method for producing a first cell population (step (i) and steps (a) to (c)) will be explained, and then the method for producing a sheet-shaped cell structure from the first cell population (steps (1) to (3)) will be explained.

<2-1. Method for producing a first cell population>
<Step (i)>
Prior to explaining steps (a) to (c), step (i), which is the culture for maintaining the undifferentiated state of pluripotent stem cells, will be described.
In the manufacturing method according to this embodiment,
Prior to the step (a) described below, the pluripotent stem cells are cultured in the absence of feeder cells,
The strain may further comprise a step (i) of culturing the strain in a medium comprising:

By culturing pluripotent stem cells in the presence of at least one selected from the group consisting of a TGFβ family signaling pathway inhibitor, a sonic hedgehog signaling (Shh) pathway agonist, and an FGF signaling pathway inhibitor, and then initiating step (i), the state of the pluripotent stem cells changes, making them more likely to differentiate and less likely to die, thereby improving the efficiency of producing a cell population (first cell population) containing olfactory placode cells or olfactory epithelial stem cells.

A cell population containing olfactory placode cells or olfactory epithelial stem cells produced from pluripotent stem cells can also be produced by the methods described in the examples of WO 2020/039732 and WO 2022/026238.

In this embodiment, the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells. Induced pluripotent stem cells are available from a designated institution, and can also be purchased commercially. For example, the human induced pluripotent stem cell line 201B7 is available from Kyoto University. The HC-6 #10 line, the 1231A3 line, and the 1383D2 line are available from the National Research and Development Agency, RIKEN. The hiPS β-actin GFP line is available from Merck.

The TGFβ family signaling pathway (i.e., the TGFβ superfamily signaling pathway) is a signaling pathway that uses transforming growth factor β (TGFβ), Nodal/Activin, BMP, etc. as ligands and is transmitted within cells by substances of the Smad family.

The term "TGFβ family signaling pathway inhibitor" refers to a substance that inhibits the TGFβ family signaling pathway, i.e., a signaling pathway transmitted by substances of the Smad family, and specific examples include TGFβ signaling pathway inhibitors, Nodal/Activin signaling pathway inhibitors, and BMP signaling pathway inhibitors. As the TGFβ family signaling pathway inhibitor, TGFβ signaling pathway inhibitors are preferred.

There are no particular limitations on the TGFβ signaling pathway inhibitor, so long as it is a substance that inhibits the signaling pathway caused by TGFβ, and it may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes that code for TGFβ (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding between TGFβ receptors and TGFβ, and substances that inhibit physiological activities caused by signaling via TGFβ receptors (e.g., TGFβ receptor inhibitors, Smad inhibitors, etc.). An example of a protein known as a TGFβ signaling pathway inhibitor is Lefty.

As a TGFβ signaling pathway inhibitor, compounds well known to those skilled in the art can be used. Specifically, SB431542 (sometimes abbreviated as "SB431") (4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzoamide), SB505124 (2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5 -yl]-6-methylpyridine), SB525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazoI-4-yl]quinoxaIne), LY2157299 (4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyr azol-3-yl]-6-quinolinecarboxamide), LY2109761 (4-[5,6-dihydro-2-(2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-[2-(4-morpholinyl)ethoxy]-quinoline), GW788388 (4-{4-[3-(pyridin-2-yl)-1H- pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide), LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]quinoline), SD-208 (2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl amine), EW-7197 (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methaneamine), A83-01 (3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole), Re pSox (2-[5-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthalidine), SM16 (4-[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazo[2-yl]bicyclo[2.2.2]octane-1-carboxamide), R268712 (4-[2-Fluoro-5-[3-(6-m ethyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol), IN1130 (3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazoI-2-yl]methyl]benzamide), Galunisertib (4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl) -4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide, AZ12799734 (4-({4-[(2,6-dimethylpyridin-3-yl)oxy]pyridin-2-yl}amino)benzenesulfonamide), A77-01 (4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoline), KRCA 0008 (1,1-[(5-Chloro-2,4-pyrimidinediyl)bis[imino(3-methoxy-4,1-phenylene)-4,1-piperazinediyl]]bisethanene), GSK 1838705 (2-[[2-[[1-[(Dimethylamino)ethanol]-5-(methyloxy)-2,3-dihydro-1H-indol-6-yl]amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino]-6-fluoro-N-methylbenzamide), Crizotinib (3-[(1R)-1-(2,6-Dichloro-3-flu orophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-2-aminopyridine), Ceritinib (5-Chloro-N2-[2-isopropyloxy-5-methyl-4-(4-piperidyl)phenyl]-N4-(2-isopropylsulfonylphenyl)pyrimidine-2,4-diamine), ASP 3026 (N2-[2-Methoxy-4-[4-(4-methyl-1-piperazinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfone), TAE684 (5-Chloro-N2-[2-methoxy-4-[4-(4-m ethyl-1-piperidinyl)-1-piperidinyl]phenyl]-N4-[2-[(1-methylethyl)sulfonyl]phenyl]-2,4-pyrimidinediamine, AZD3463 (N-[4-(4-amino-1-piperidinyl)- 2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)-2-pyrimidinamine, TP0427736 (6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole) , TGFBR1-IN-1 (5-(1,3-benzothiazol-6-yl)-N-(4-hydroxyphenyl)-1-(6-methylpyridin-2-yl)pyrazole-3-carboxamide), TEW-7197 (2-fluoro-N-[[5-(6-methylpyridine 2-[4-[[4-[1-cyclopropyl-3-(oxan-4-yl)pyrazole-4-yl]methyl]aniline; LY3200882 (2-[4-[[4-[1-cyclopropyl-3-(oxan-4-yl)pyrazole-4-yl]methyl]aniline); yl]oxypyridin-2-yl]amino]pyridin-2-yl]propan-2-ol), BIBF-0775 ((3Z)-N-Ethyl-2,3-dihydro-N-methyl-2-oxo-3-[phenyl[[4-(1-piperidinylmethyl)phenyl] Alk5/TGFβR1 inhibitors such as SIS3 (1-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-3-(1-methyl-2-phenyl-1H-pyrrolidine) Examples of the inhibitor include SMAD3 inhibitors such as 4-[1,1'-biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinoleic acid ethyl ester (ITD-1), and receptor degradation promoters such as these compounds. These substances may be used alone or in combination. SB431542 is a compound known as an inhibitor of the TGFβ receptor (ALK5) and Activin receptor (ALK4/7) (i.e., a TGFβR inhibitor). SIS3 is a TGFβ signaling pathway inhibitor that inhibits phosphorylation of SMAD3, an intracellular signaling factor under the control of the TGFβ receptor. ITD-1 is a proteasome degradation promoter for the TGF-β type II receptor. Those skilled in the art are aware that the above compounds have activity as TGFβ signaling pathway inhibitors (for example, as described in Expert Opinion on Investigational Drugs, 2010, 19.1: 77-91, etc.).

The TGFβ signaling pathway inhibitor preferably includes an Alk5/TGFβR1 inhibitor. The Alk5/TGFβR1 inhibitor is preferably SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, RepSox, SM16, R268712, IN1130, Galunisertib, AZ12799734, A77-01, KRCA 0008, GSK 1838705, Crizotinib, Certinib, ASP 3026, TAE684, AZD3463, TP0427736, and more preferably SB431542, RepSox, or A83-01.

The concentration of the TGFβ signaling pathway inhibitor in the medium can be appropriately set depending on the substance used within a range that can achieve the above-mentioned effects. When SB431542 is used as the TGFβ signaling pathway inhibitor in step (a), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 10 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, and particularly preferably about 1 μM to about 10 μM. When a TGFβ signaling pathway inhibitor other than SB431542 is used, it is desirable to use it at a concentration that shows the same TGFβ signaling pathway inhibitory activity as SB431542 at the above concentration. The TGFβ signaling pathway inhibitory activity of SB431542 and the like can be determined by methods well known to those skilled in the art, for example, by detecting phosphorylation of Smads by Western blotting (Mol Cancer Ther. (2004) 3, 737-45, etc.).

A substance acting on the Shh signaling pathway is a substance that can enhance signal transduction mediated by Shh. Examples of substances acting on the Shh signaling pathway include proteins belonging to the Hedgehog family (e.g., Shh, Ihh), Shh receptors, Shh receptor agonists, Smo agonists, Purmorphamine (9-cyclohexyl-N-[4-(morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), GSA-10 (Propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinone-3-carboxamido)benzoate), Hh-Ag1.5, 20(S)-Hydroxycholesterol, SAG (Smoothened Agonist: N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), 20(S)-hydroxy Cholesterol (3S,8S,9S,10R,13S,14S,17S)-17-[(2R)-2-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol) and the like. These substances may be used alone or in combination. Those skilled in the art are aware that the above compounds have activity as Shh pathway acting substances (for example, as described in Molecular BioSystems, 2010, 6.1: 44-54.).

The substance acting on the Shh signaling pathway preferably includes at least one selected from the group consisting of SAG, Purmorphamine, and GSA-10, and more preferably includes SAG. The concentration of the substance acting on the Shh signaling pathway in the medium can be appropriately set depending on the substance used within a range in which the above-mentioned effects can be achieved. In step (i), SAG is usually used at a concentration of about 1 nM to about 2000 nM, preferably about 10 nM to about 1000 nM, more preferably about 10 nM to about 700 nM, even more preferably about 50 nM to about 700 nM, particularly preferably about 100 nM to about 600 nM, and most preferably about 100 nM to about 500 nM. In addition, when a substance acting on the Shh signaling pathway other than SAG is used, it is desirable to use it at a concentration that shows the same Shh signaling promoting activity as SAG at the above-mentioned concentration. Shh signaling promoting activity can be determined by methods well known to those skilled in the art, for example, a reporter gene assay focusing on the expression of the Gli1 gene (Oncogene (2007) 26, 5163-5168, etc.).

In the present invention, the FGF signaling pathway refers to a signaling pathway that transmits physiological responses induced by signaling molecules belonging to the fibroblast growth factor (FGF) family. The FGF signaling pathway includes both an FGF receptor-dependent pathway in which an FGF family molecule binds to an FGF receptor and activates a downstream intracellular signaling pathway, and an FGF receptor-independent pathway in which an FGF family molecule induces a physiological response without binding to an FGF receptor.

The FGF signaling pathway inhibitor is not particularly limited as long as it can suppress signaling mediated by FGF, and may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that directly bind to and trap FGF, substances that inhibit the kinase activity of FGF receptors, substances that induce the degradation of FGF receptors, substances that inhibit intracellular signaling activated by FGF, and substances that suppress gene expression induced by FGF stimulation. Examples of substances that have the above-mentioned effects include anti-FGF antibodies, GP369 (anti-FGFR2 antibodies), and SU5402 (3-[4-Methyl-2-[(2-oxo-1H-indol-3-ylidene)methyl]-1H-pyrrol-3-yl]propanoic acid. acid), BGJ398 (3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-[6-[[4-(4-ethylpiperazin-1-yl)phenyl]amino]pyrimidin-4-yl]-1-methylurea), PD173074 (1-(Tert-Butyl)-3-(2-((4-(diethylamino)butyl) amino)-6-(3,5-dimethylphenyl)pyrido[2,3-d]pyrimidin-7-yl)urea), AZD4547 (rel-N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-1H-pyrazol-3-yl]-4-[(3R,5S)-3,5-dimethyl-1-piperazinyl]benzamide), Erdafitinib (N-(3,5-dimethylphenyl)-N-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine), BYL719 ((2S)-N1-[4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimeth 2-Methyl-1-[[2-Methyl-3-(trifluoromethyl)phenyl]Methyl]-6-(4-Morpholinyl)-1H-benzimidazole-4-carboxylate acid), Taselisib (2-Methyl-2-[4-[2-(5-Methyl-2-propan-2-yl-1,2,4-triazol-3-yl)-5,6-dihydroiMidazo[1,2-d][1,4]benzoxazepin-9-yl]pyrazol-1-yl]propanamide), Capivasertib (4-Amino-N-[(1S)-1-(4-chlorophenyl)-1,2-dihydro-1,2-dihydro-1,4 ... orophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinecarboxamide, Sapanisertib (2-(((1R,4R)-4-((((4-chlorophenyl)(phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetic acid), Dabrafenib (N-[3-[5-(2-amino-4-pyrimidinyl)-2-(tert-butyl)-4-thiazolyl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide), Binimetinib (5-[(4-bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-b enzimidazole-6-carboxamide), trametinib (N-[3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-3,4,6,7-tetrahydro-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl]acetamide), DGY-09-192, and derivatives thereof.

The FGF signaling pathway inhibitor used in step (i) is preferably a substance having inhibitory activity against any one of fibroblast growth factor receptors (FGFR) 1 to 4, and more preferably a substance having inhibitory activity against fibroblast growth factor receptor 1 (FGFR1). More preferably, it is a receptor tyrosine kinase inhibitor against fibroblast growth factor receptor 1 (FGFR1). The receptor tyrosine kinase inhibitory activity against fibroblast growth factor receptor 1 (FGFR1) can be measured by, for example, a method such as the cell-free caliper mobility shift assay and ATP competitive inhibition assay described in Oncotarget. 2014; 5: 4543-4553. The substance having inhibitory activity against fibroblast growth factor receptor 1 (FGFR1) preferably includes at least one selected from the group consisting of PD173074, ponatinib, sulfatinib, ON123300, SU5402, lenvatinib, danusertib, PD-166866, brivanib, sorafenib, SSR128129E and derivatives thereof, more preferably includes PD173074. In step (i), PD173074 is usually used at a concentration of about 1 nM to about 2000 nM, preferably about 10 nM to about 1000 nM, more preferably about 10 nM to about 700 nM, even more preferably about 20 nM to about 700 nM, particularly preferably about 30 nM to about 500 nM, and most preferably about 50 nM to about 300 nM. In addition, when using an FGF signaling pathway inhibitor other than PD173074, it is desirable to use it at a concentration that exhibits FGF signaling pathway inhibitory activity or fibroblast growth factor receptor 1 (FGFR1) receptor tyrosine kinase inhibitory activity equivalent to the above-mentioned concentration of PD173074. FGF signaling pathway inhibitory activity can be measured by methods well known to those skilled in the art, and fibroblast growth factor receptor 1 (FGFR1) receptor tyrosine kinase inhibitory activity can be measured by methods such as the cell-free caliper mobility shift assay and ATP competitive inhibition assay described in Oncotarget. 2014; 5: 4543-4553.

The medium used in step (i) contains an undifferentiated state maintenance factor to enable the undifferentiated state maintenance culture. The undifferentiated state maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. In the case of primed pluripotent stem cells (e.g., human ES cells, human iPS cells), undifferentiated state maintenance factors commonly used by those skilled in the art include FGF signaling pathway acting substances, TGFβ family signaling pathway acting substances, insulin, etc. Specific examples of FGF signaling pathway acting substances include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). In addition, examples of TGFβ family signaling pathway acting substances include TGFβ signaling pathway acting substances and Nodal/Activin signaling pathway acting substances. Examples of TGFβ signaling pathway acting substances include TGFβ1 and TGFβ2. Examples of substances acting on the Nodal/Activin signaling pathway include Nodal, Activin A, and Activin B. These substances may be used alone or in combination. When culturing human pluripotent stem cells (e.g., human ES cells, human iPS cells), if the medium in step (i) does not contain an FGF signaling pathway inhibitor, it is preferable to contain bFGF as an undifferentiated maintenance factor. Alternatively, the cells may be cultured for a certain period of time in a medium that does not contain an FGF signaling pathway inhibitor but contains bFGF, and then the medium may be changed to one that contains an FGF signaling pathway inhibitor but does not contain bFGF.

The undifferentiation maintenance factor is usually a mammalian undifferentiation maintenance factor. Examples of mammals include those mentioned above. Since the undifferentiation maintenance factor may have cross-reactivity between mammalian species, any mammalian undifferentiation maintenance factor may be used as long as it can maintain the undifferentiated state of the pluripotent stem cells to be cultured. The undifferentiation maintenance factor is preferably an undifferentiation maintenance factor of the same mammalian species as the cells to be cultured. For example, human undifferentiation maintenance factors (e.g., bFGF, FGF4, FGF8, EGF, BMP, Nodal, Activin A, Activin B, TGFβ1, TGFβ2, etc.) are used for culturing human pluripotent stem cells. The undifferentiation maintenance factor is preferably isolated. Culture conditions for pluripotent stem cells that maintain efficient differentiation ability into three-dimensional tissues including organoids, and the form of the undifferentiation maintenance factor to be added are described in, for example, Stem Cell Reports Vol. 17 1-19 October 11, 2022, a combination of BMP4, TGFβ1, Activin A, Nodal, and TGFβ3 added simultaneously is also preferred.

As long as the undifferentiated state maintenance factor has the ability to maintain the undifferentiated state of the pluripotent stem cells to be cultured, it can be one produced by any host or one artificially synthesized. The undifferentiated state maintenance factor used in the present invention is preferably one that has been modified in the same way as that which occurs in the body, and more preferably one that has been produced in cells of the same type as the pluripotent stem cells to be cultured under conditions that do not contain any heterologous components.

One embodiment of the production method according to the present invention includes a step of providing an isolated factor for maintaining undifferentiation. One embodiment of the production method according to the present invention includes a step of exogenously (or extrinsically) adding the isolated factor for maintaining undifferentiation to the medium used in step (i). The factor for maintaining undifferentiation may be added in advance to the medium used in step (i).

The concentration of the undifferentiation maintenance factor in the medium used in step (i) is a concentration that can maintain the undifferentiated state of the pluripotent stem cells being cultured, and can be appropriately set by a person skilled in the art. For example, when bFGF is used as the undifferentiation maintenance factor in the absence of feeder cells, the concentration is usually about 4 ng/mL to about 500 ng/mL, preferably about 10 ng/mL to about 200 ng/mL, and more preferably about 30 ng/mL to about 150 ng/mL. When BMP4, TGFβ1, Activin A, Nodal, and TGFβ3 are added simultaneously as factors for maintaining undifferentiation, the concentrations are usually about 0.01 ng/mL to about 5 ng/mL for BMP4, about 0.01 ng/mL to about 5 ng/mL for TGFβ1, about 0.1 ng/mL to about 100 ng/mL for Activin A, about 10 ng/mL to about 500 ng/mL for Nodal, and about 0.1 ng/mL to about 50 ng/mL for TGFβ3.

Step (i) is carried out in the absence of feeder cells. The culture of pluripotent stem cells in step (i) may be carried out under either suspension culture or adherent culture conditions, but is preferably carried out by adherent culture. When culturing pluripotent stem cells in the absence of feeder cells, a suitable matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold in place of feeder cells. The pluripotent stem cells are cultured in an adherent manner in a culture vessel whose surface is coated with a matrix scaffold.

Matrices that can be used as scaffolds include laminin (Nat Biotechnol. 28, 611-615 (2010)), laminin fragments (Nat Commun 3, 1236 (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974 (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin. Laminin 511 is preferably used as the matrix (Nat Biotechnol 28, 611-615 (2010)).

The laminin fragment is not particularly limited as long as it has adhesive properties to pluripotent stem cells and enables the maintenance culture of pluripotent stem cells under feeder-free conditions, but is preferably an E8 fragment. The laminin E8 fragment was identified as a fragment with strong cell adhesive activity among fragments obtained by digesting laminin 511 with elastase (EMBO J., 3: 1463-1468, 1984, J. Cell Biol., 105: 589-598, 1987). As the laminin fragment, the E8 fragment of laminin 511 is preferably used (Nat. Commun. 3, 1236 (2012), Scientific Reports 4, 3549 (2014)). The laminin E8 fragment does not need to be a product of elastase digestion of laminin, and may be a recombinant product. It may be produced in a genetically modified animal (such as a silkworm). In order to avoid contamination with unidentified components, recombinant laminin fragments are preferably used. The E8 fragment of laminin 511 is commercially available and can be purchased from, for example, Nippi Corporation.

From the viewpoint of avoiding contamination with unidentified components, it is preferable that the laminin or laminin fragment used in the present invention is isolated. In the culture of pluripotent stem cells in the absence of feeder cells in step (i), the pluripotent stem cells are cultured in an adherent manner in a culture vessel whose surface is preferably coated with isolated laminin 511 or the E8 fragment of laminin 511, more preferably with the E8 fragment of laminin 511.

The medium used in step (i) is not particularly limited as long as it is a medium (feeder-free medium) that allows the maintenance of undifferentiated culture of pluripotent stem cells under feeder-free conditions. Many synthetic media have been developed and are commercially available as feeder-free media, such as Essential 8 medium. Essential 8 medium contains, in DMEM/F12 medium, the following additives: L-ascorbic acid-2-phosphate magnesium (64 mg/l), sodium selenium (14 μg/l), insulin (19.4 mg/l), NaHCO ₃ (543 mg/l), transferrin (10.7 mg/l), bFGF (100 ng/mL), and a TGFβ family signaling pathway active substance (TGFβ1 (2 ng/mL) or Nodal (100 ng/mL)) (Nature Methods, 8, 424-429 (2011)). Commercially available feeder-free media include, for example, Essential 8 (manufactured by Thermo Fisher Scientific), S-medium (manufactured by DS Pharma Biomedical Co., Ltd.), StemFlex Medium, StemPro (manufactured by Thermo Fisher Scientific), hESF9, mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), mTeSR Plus (manufactured by STEMCELL Technologies), StemFit (manufactured by Ajinomoto Co., Inc.), and ReproMed iPSC. Medium (manufactured by ReproCELL), NutriStem XF (manufactured by Biological Industries), NutriStem V9 (manufactured by Biological Industries), Cellartis DEF-CS Xeno-Free Culture Medium (manufactured by Takara Bio), Stem-Partner SF (manufactured by Kyokuto Pharmaceuticals), PluriSTEM Human ES/iPS Cell Medium (manufactured by Merck & Co., Inc.), StemSure hPSC MediumΔ (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), ciKIC iPS medium (manufactured by Kanto Chemical Co., Ltd.), StemMACS iPS-Brew XF, Examples of suitable medium include hPSC Growth Medium DXF (manufactured by Promocell), hPSC Growth Medium DXF (manufactured by System Biosciences), etc. Examples of suitable medium include hPSC Growth Medium DXF (manufactured by System Biosciences ... StemFit medium contains bFGF as a component for maintaining undifferentiated state (Scientific Reports (2014) 4, 3594).

The pluripotent stem cells in step (i) may be in either a primed state or a naive-like state, but are preferably in a primed state. Pluripotent stem cells in a naive-like state can be obtained by treating and culturing pluripotent stem cells in a primed state, for example, by the method described in Cell, 2014, 158.6: 1254-1269., or by treating and culturing them in a commercially available medium such as ReproNaive (manufactured by ReproCELL), NaiveCult Induction Kit (manufactured by STEMCELL Technologies), RSeT Medium (manufactured by STEMCELL Technologies), AlphaSTEM Culture System (manufactured by Minerva Biotechnology), etc.

The medium used in step (i) may be a serum medium or a serum-free medium. From the viewpoint of avoiding contamination with chemically undefined components, the medium used in step (i) is preferably a serum-free medium. The medium may contain a serum substitute.

The culture time of the pluripotent stem cells in step (i) is not particularly limited as long as it is within a range that can achieve the effect of improving the quality of the cell aggregates that can be formed in the subsequent step (a), but is usually 0.5 to 144 hours, preferably 2 to 96 hours, more preferably 6 to 72 hours, even more preferably 12 to 60 hours, and particularly preferably 18 to 54 hours, for example 48 hours. That is, step (i) is started 0.5 to 144 hours, preferably 18 to 54 hours, before the start of step (a), and step (a) is performed after step (i) is completed. As a preferred embodiment of the implementation of step (i), for example, an FGF signaling pathway inhibitor is added 54 to 42 hours before the start of step (a), and a TGFβ signaling pathway inhibitor and an Shh signaling pathway activator are added 30 to 18 hours before the start of step (a).

In a preferred embodiment of step (i), human pluripotent stem cells are cultured as adherent cells in a serum-free medium containing bFGF in the absence of feeder cells. The adherent culture is preferably performed in a culture vessel whose surface is coated with laminin 511, E8 fragment of laminin 511, or vitronectin. The adherent culture is preferably performed using StemFit as a feeder-free medium.

In a preferred embodiment of step (i), human pluripotent stem cells are cultured in suspension in a serum-free medium containing bFGF in the absence of feeder cells. In the suspension culture, the human pluripotent stem cells may form aggregates of human pluripotent stem cells.

In step (i) and the steps described below, culture conditions such as culture temperature and _CO2 concentration can be appropriately set. The culture temperature may be, for example, about 30° C. to about 40° C., and preferably about 37° C. The _CO2 concentration may be, for example, about 1% to about 10%, and preferably about 5%.

In the preparation of pluripotent stem cells used in the production method according to the present invention, as well as in step (i) and subsequent steps, it is also preferable to use an automatic culture device, an automatic culture robot, or a lab automation device in order to eliminate the influence of differences in technique between operators, prevent contamination by foreign matter originating from the operator, stabilize production quality, and increase production volume. Examples of the above-mentioned devices that can be used in the implementation of the present invention include, but are not limited to, an automatic culture device (manufactured by Panasonic Corporation, Kawasaki Heavy Industries, Ltd., Hitachi, Ltd., iACE2 (ACC-200)) and the robot described in SLAS TECHNOLOGY: Translating Life Sciences Innovation, 2020, 2472630320972109 (manufactured by Robotic Biology Institute, Inc., LabDroid). When implementing the present invention using the above-mentioned equipment, a person skilled in the art can adjust or modify the manufacturing method and steps described in the present invention to suit the equipment used.

In the preparation of pluripotent stem cells used in the production method of the present invention, and in step (i) and subsequent steps, it is also preferable to carry out an intermediate evaluation of the product from the viewpoint of managing and stabilizing the production quality. The intermediate evaluation may be either a random inspection performed by consuming a part of the cell mass being produced, or a non-invasive inspection, but is preferably a non-invasive inspection. Examples of non-invasive inspection include optical observation using a microscope, measurement using an optical coherence tomography (OCT), and quantification of metabolites, nucleic acids, and secreted proteins contained in the medium collected during medium replacement. One aspect of optical observation using a microscope includes optically observing and detecting the epithelial structure, upper respiratory tract tissue, nasal epithelium, olfactory epithelium, etc. formed on the surface of the cell mass, and quantifying and evaluating them using the method described in International Publication No. 2017/090741. The evaluation criteria for the optical observation can be appropriately set, for example, by visual judgment by a person skilled in the art, or by a method such as model construction by machine learning of a captured image. Examples of evaluation criteria that can be used include the percentage of the surface area of the cell mass covered with epithelium, the thickness of the surface epithelium, the percentage of the surface epithelium, and the amount of dead cells or debris around the cell mass. Evaluation methods using an optical coherence tomography can be performed by, for example, acquiring tomographic images, 3D images, etc. using Cell3iMager Esteier (manufactured by SCREEN Holdings, Inc.), and quantifying the tomographic area, volume, sphericity, surface roughness, internal cavity volume, and thickness of the surface epithelium of the cell mass. Examples of analyses that can be performed using the collected culture medium include exosome analysis and metabolomics analysis.

<Step (a)>
In step (a), pluripotent stem cells are cultured in the presence of a Wnt signaling pathway inhibitor.

The Wnt signaling pathway is a signaling pathway that uses Wnt family proteins as ligands and mainly Frizzled as a receptor. Examples of the signaling pathway include the canonical Wnt pathway and the non-canonical Wnt pathway. The canonical Wnt pathway is transmitted by β-catenin. Examples of the non-canonical Wnt pathway include the planar cell polarity (PCP) pathway, the Wnt/JNK pathway, the Wnt/Calcium pathway, the Wnt-RAP1 pathway, the Wnt-Ror2 pathway, the Wnt-PKA pathway, the Wnt-GSK3MT pathway, the Wnt-aPKC pathway, the Wnt-RYK pathway, and the Wnt-mTOR pathway. In the non-classical Wnt pathway, there are common signaling factors that are also activated in signaling pathways other than Wnt, but in the present invention, these factors are also considered to be components of the Wnt signaling pathway, and inhibitors of these factors are also included in the Wnt signaling pathway inhibitors.

The Wnt signaling pathway inhibitor is not limited as long as it can suppress signaling induced by Wnt family proteins. The inhibitor may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of the inhibitor include substances that inhibit Wnt processing and extracellular secretion, substances that act directly on Wnt (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding Wnt (e.g., antisense oligonucleotides, siRNAs, etc.), substances that inhibit the binding of Wnt receptors to Wnt, and substances that inhibit physiological activities resulting from signaling by Wnt receptors. In this embodiment, the Wnt signaling pathway inhibitor preferably has at least one activity selected from the group consisting of 1) inhibitory activity against the non-classical Wnt pathway, 2) inhibitory activity against the extracellular secretion of Wnt family proteins, 3) inhibitory activity against porcupine, and 4) inhibitory activity against ROR.

Proteins known to be inhibitors of the Wnt signaling pathway include proteins belonging to the secreted frizzled related protein (sFRP) class (sFRP1-5, Wnt inhibitory factor-1 (WIF-1), Cerberus), proteins belonging to the Dickkopf (Dkk) class (Dkk1-4, Kremen), APCDD1, APCDD1L, proteins belonging to the Draxin family, IGFBP-4, Notum, and proteins belonging to the SOST/Sclerostin family.

As the Wnt signaling pathway inhibitor, compounds well known to those skilled in the art can be used. Examples of inhibitors of the classical Wnt signaling pathway include Frizzled inhibitors, Dishevelled (Dvl) inhibitors, Tankyrase (TANK) inhibitors, casein kinase 1 inhibitors, catenin-responsive transcription inhibitors, p300 inhibitors, CREB-binding protein (CBP) inhibitors, BCL-9 inhibitors, and TCF degradation inducers (Am. J. Cancer Res. 2015; 5 (8): 2344-2360). Examples of inhibitors of the non-classical Wnt pathway include porcupine (PORCN) inhibitors, calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors, TGF-β-activated kinase 1 (TAK1) inhibitors, Nemo-Like Kinase (NLK) inhibitors, LIM Kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, Rac inhibitors, c-Jun NH 2-terminal kinase (JNK) inhibitors, protein kinase C (PKC) inhibitors, and methionine aminopeptidase inhibitors. Examples of such inhibitors include MetAP2 inhibitors, calcineurin inhibitors, nuclear factor of activated T cells (NFAT) inhibitors, and ROCK inhibitors. In addition, examples of inhibitors of the Wnt signaling pathway include KY02111 (N-(6-chloro-2-benzothiazolyl)-3,4-dimethoxybenzenepropanamide) and KY03-I (2-(4-(3,4-dimethoxyphenyl)butanamide)-6-iodobenzothiazole), although the mechanism of action has not been reported. These substances may be used alone or in combination. In one aspect of this embodiment, the Wnt signaling pathway inhibitor preferably includes at least one selected from the group consisting of XAV-939 (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), KY02111, KY03-I, and derivatives thereof.

PORCN inhibitors, which are Wnt signaling pathway inhibitors, are substances that inhibit the activity of porcupine, an acyltransferase localized in the endoplasmic reticulum. Porcupine is an enzyme that catalyzes the reaction of adding palmitoleic acid to a serine residue in a motif conserved among Wnt family proteins using palmitoleoyl-CoA as a substrate. As described in Nature volume 607, pages 816-822 (2022), for example, PORCN inhibitors are substances that bind to the 332nd serine residue of porcupine and inhibit its enzyme activity.

The activity of a test substance as a PORCN inhibitor can be evaluated by a double screening system that combines a luciferase reporter system using HEK293-STF cells that constitutively express Wnt3A, as described in J. Med. Chem. 2015, 58, 15, 5889-5899, with a luciferase reporter system using HEK293-STF/Wnt cells that do not express Wnt3A and a conditioned medium containing Wnt. If a test substance has activity as a PORCN inhibitor, the secretion of Wnt from the cells is inhibited while the activity of Wnt in the conditioned medium is not affected, so that the reporter activity is reduced in a system using cells that constitutively express Wnt3A, and the activity is not reduced in a system using a Wnt-containing conditioned medium. If the test substance has any activity other than a PORCN inhibitor, such as an inhibitory activity of Tankyrase, a downstream factor of the intracellular Wnt signaling pathway, the reporter activity will be reduced in both a system using cells that constantly express Wnt3A and a system using a Wnt-containing conditioned medium. Alternatively, the activity of the test substance as a PORCN inhibitor can be measured by quantifying and comparing the amount of Wnt secreted into the medium when cells that constantly express Wnt3A, such as HEK293-STF cells, are treated with the test substance, using a method such as ELISA.

Examples of PORCN inhibitors include IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthioeno[3,2-d]pyrimidin-2-yl)thio]-acetamide), IWP-3 (2-[[3-(4-fluorophenyl)-3,4,6,7-tetrahydro- 4-oxothioeno[3,2-d]pyrimidin-2-yl]thio]-N-(6-methyl-2-benzothiazolyl)-acetamide, IWP-4 (N-(6-methyl-2-benzothiazolyl)-2-[[3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothioeno[3,2-d]pyr IWP-L6 (N-(5-phenyl-2-pyridinyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthio[3,2-d]pyrimidin-2-yl)thio]-acetamide), IWP-12 (N-(6-methyl-2-benzothiazolyl)- 2-[(3,4,6,7-tetrahydro-3,6-dimethyl-4-oxothioeno[3,2-d]pyrimidin-2-yl)thio]acetamide), IWP-O1 (1H-1,2,3-Triazole-1-acetamide,5-phenyl-N-(5-phenyl-2-pyridinyl)-4-(4-pyridinyl)-), IWP-29 (N-(4-ethoxyphenyl)-2-{[4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetamide), LGK-974 (2-(2',3-dimethyl-2,4'-bipyridin-5-yl)-N-(5-(pyrazine-2-yl)pyridin-2-yl)a acetamide), Wnt-C59 (2-[4-(2-Methylpyridin-4-yl)phenyl]-N-[4-(pyridin-3-yl)phenyl]acetamide), ETC-131, ETC-159 (1,2,3,6-Tetrahydro-1,3-dimethyl-2,6-dioxo-N-(6-phenyl-3-pyridazine yl)-7H-purine-7-acetamide), GNF-1331 (N-(6-methoxy-1,3-benzothiazol-2-yl)-2-[(4-propyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]acetamide), GNF-6231 (N-[5-(4-Acetyl-1-pipera Znyl)-2-pyridinyl]-2'-fluoro-3-methyl[2,4'-bipyridine]-5-acetamide, Porcn-IN-1 (N-[[5-fluoro-6-(2-methylpyridin-4-yl)pyridin-3-yl]methyl]-9H-carbazole-2-carboxamide), WIC1 ( Examples of such substances include N-[4-(4-Ethyl-1-piperazinyl)phenyl]-2-oxo-2H-1-benzopyran-3-carboxamide), RXC004, CGX1321, AZD5055, and derivatives thereof. These substances may be used alone or in combination. In addition, as the PORCN inhibitor, for example, compounds described as Wnt regulators or PORCN inhibitors in US9045416B2, WO2013169631A2, US9783550B2, WO2016/055786, etc. may be used.

Examples of JNK inhibitors include JNK-IN-8 ((E)-3-(4-(dimethylamino)but-2-enamido)-N-(3-methyl-4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide), SP600125 (Anthra[1-9-cd]pyrazol-6(2H)-one), DB07268 (2-[[2-[(3-Hydroxyphenyl)amino]-4-pyrimidinyl]amino]be nzamide), Tanzisertib (trans-4-[[9-[(3S)-Tetrahydro-3-furanyl]-8-[(2,4,6-trifluorophenyl)amino]-9H-purin-2-yl]amino]cyclohexanol), Bentamapimod (1,3-Benzothiazol-2-yl)[2-[[4-[(morpholin-4-yl)methyl]benzyl]oxy]pyrimidin-4-yl]acetonitrile, TCS JNK 6o (N-(4-amino-5-cyano-6-ethoxy-2-pyridinyl)-2,5-dimethoxybenzeneacetamide), SU3327 (5-[(5-nitro-2-thiazolyl)thio]-1,3,4thiadiazole-2-amine), CEP1347 ((9S,10R,12R)-5-16-B is[(ethylthio)methyl]-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester), c-JUN peptide, AEG3482 (6-Phenylimidazo[2,1-b]-1,3,4-thiadiazole-2-sulfonamide), TCS JNK 5a (N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thienyl-2-yl)-1-naphthalenecarboxamide), BI-78D3 (4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-2,4-dihydro-5-[(5-nitro-2-thiazolyl)thio]-3H-1,2,4-triazol-3-one), IQ-3 (11H-Indeno[1,2-b]quinoxalin-11-one O-(2-furanylcarbonyl)oxime), SR 3576 (3-[4-[[[(3-Methylphenyl)amino]carbonyl]amino]-1H-pyrazol-1-yl]-N-(3,4,5-trimethoxyphenyl)benzamide), IQ-1S (11H-Indeno[1,2-b]quinoxalin-11-one oxime sodium salt), JIP-1 (153-163), CC-401 (3-[3-[2-(1-Piperidinyl)ethoxy]phenyl]-5-(1H-1,2,4-triazol-5-yl)-1H-indazole dihydrochloride) and derivatives thereof. These substances may be used alone or in combination. The fact that the above compounds have activity as JNK inhibitors is described, for example, in J. Enzyme Inhib. Med. Chem. 2020; 35(1): 574-583, and is known to those skilled in the art.

Examples of Rac inhibitors include EHT1864 (5-(5-(7-(Trifluoromethyl)quinolin-4-ylthio)pentyloxy)-2-(morpholinomethyl)-4H-pyran-4-one dihydrochloride), NSC23766 (N6-[2-[[4-(Diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride), EHop-016 (N4-(9-Ethyl-9H-carbazol-3-yl)-N2-[3-(4-morpholinyl)propyl]-2,4-pyrimidinediamine), 1A-116 (N-(3,5-Dimethylphenyl)-N'-[2-(trifluoromethyl)phenyl]guanidinium ne), ZCL278 (2-(4-broMo-2-chlorophenoxy)-N-(4-(N-(4,6-diMethylpyriMidin-2-yl)sulfaMoyl)phenylcarbaMothioyl)acetamide), MBQ-167 (9-ethyl-3-(5-phenyl-1H-1,2,3-triazol-1-yl)-9H-carbazol ole), KRpep-2d (actinium; [(2S)-1-[[(2S)-1-[[(2S)-1-[[(3R,8R,11S,14S,20S,23S,26S,29S,32S,35S,38S)-8-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino-5-carbamimidamido-1-oxopenta n-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]carbamoyl]-29-[(2R)-butan-2-yl]-20-(carboxyl ethyl)-26-(hydroxymethyl)-23,32-bis[(4-hydroxyphenyl)methyl]-35-(2-methylpropyl)-2,10,13,19,22,25,28,31,34,37-decaoxo-11-propan-2-yl-5,6-dithia-1,9,12,18,21,24,27,30,33,36-deca zatricyclo[36.3.0.014,18]hentetracontan-3-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino] -5-carbamimidamido-1-oxopentan-2-yl]azanide), ARS-853 (1-[3-[4-[2-[[4-Chloro-2-hydroxy-5-(1-methylcyclopropyl)phenyl]amino]acetyl]-1-piperazinyl]-1-azetidinyl]-2-propen-1-one), Sa Examples of such compounds include lirasib (2-(((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)thio)benzoic acid), ML141 (4-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-yl)benzenesulfonamide) and derivatives thereof. These substances may be used alone or in combination. The fact that the above compounds have activity as Rac inhibitors is described, for example, in Cancer research, 2018, 78.12: 3101-3111, and is known to those skilled in the art.

The Wnt signaling pathway inhibitor preferably includes at least one selected from the group consisting of a PORCN inhibitor, KY02111, and KY03-I, and more preferably includes a PORCN inhibitor. It is also preferable that the Wnt signaling pathway inhibitor includes a substance having inhibitory activity against the non-classical Wnt pathway of Wnt. More preferably, the Wnt signaling pathway inhibitor includes a substance having inhibitory activity against the Wnt/Planar Cell Polarity (PCP) pathway. The PORCN inhibitor used in the present invention preferably includes at least one selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, Wnt-C59, ETC-159, and GNF-6231, more preferably includes IWP-2 or Wnt-C59, and even more preferably includes IWP-2.

In one aspect of this embodiment, the Wnt signaling pathway inhibitor has: 1) an inhibitory activity against a non-canonical Wnt pathway, the inhibitory activity being an inhibitory activity against acylation or palmitoylation of a target protein by porcupine;
The substance having inhibitory activity preferably includes at least one selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, IWP-O1, LGK-974, Wnt-C59, ETC-131, ETC-159, GNF-1331, GNF-6231, Porcn-IN-1, RXC004, CGX1321, AZD5055, and derivatives thereof.

The concentration of the Wnt signaling pathway inhibitor in the medium can be appropriately set depending on the substance used within a range that can achieve the above-mentioned effects. From the viewpoint of improving the production efficiency of cells that constitute upper respiratory tract tissue, for example, when IWP-2, a type of PORCN inhibitor, is used as the Wnt signaling pathway inhibitor, its concentration is usually about 10 nM to about 50 μM, preferably about 10 nM to about 30 μM, more preferably about 100 nM to about 10 μM, and most preferably about 0.5 μM. When Wnt-C59, a type of PORCN inhibitor, is used, its concentration is usually about 10 pM to about 1 μM, preferably about 100 pM to about 500 nM, and more preferably about 50 nM. When KY02111 is used, its concentration is usually about 10 nM to about 50 μM, preferably about 10 nM to about 30 μM, more preferably about 100 nM to about 10 μM, and even more preferably about 5 μM. When using a Wnt signaling pathway inhibitor other than the above, it is desirable to use it at a concentration that shows the same Wnt signaling pathway inhibitory activity as the above concentration. The effect of the Wnt signaling pathway inhibitor can be measured by a person skilled in the art, for example, by Western blot using anti-phosphorylated LRP-6, anti-phosphorylated Dvl, and anti-β-Catenin antibodies using L-Wnt-STF cells described in Nat. Chem. Biol. 2009 Feb; 5(2): 100-107, luciferase reporter assay, or the like. Those skilled in the art can measure the effect of the PORCN inhibitor by a method such as cell migration/scratch assay using human dermal lymphatic endothelial cells (HD-LEC) described in Scientific Reports volume 9, Article number: 4739 (2019).

The Wnt signaling pathway inhibitor is usually added within 48 hours, preferably within 24 hours, more preferably within 12 hours, and even more preferably simultaneously with the start of culture of the pluripotent stem cells in step (a).

The medium in step (a) preferably further contains a TGFβ signaling pathway inhibitor. The TGFβ signaling pathway inhibitor used in step (a) may be the same as that exemplified in step (i). The TGFβ signaling pathway inhibitors in steps (i) and (a) may be the same or different. The TGFβ signaling pathway inhibitor used in step (a) of the present application is preferably SB431542, RepSox, or a combination of SB431542 and RepSox.

The concentration of the TGFβ signaling pathway inhibitor in the medium can be appropriately set within a range that can achieve the above-mentioned effects. When SB431542 is used as the TGFβ signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 500 nM to about 10 μM. When RepSox is used as the TGFβ signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 500 nM to about 10 μM. In addition, when a TGFβ signaling pathway inhibitor other than SB431542 and RepSox is used, it is desirable to use it at a concentration that shows the same TGFβ signaling pathway inhibitory activity as SB431542 and RepSox at the above concentrations. In a preferred embodiment of the present invention, when step (a) is carried out by adding SB431542 and RepSox simultaneously, for example, SB431542 is added at about 1 μM and RepSox is added at about 1 μM.

In order to suppress differentiation into mesendoderm and improve the efficiency of producing placode regions, upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue in step (a) and the subsequent steps, it is also preferable to add an inhibitor of transforming growth factor-β-activated kinase 1 (TAK1). TAK1 is a serine-threonine protein kinase of the MAP kinase kinase kinase (MAPKKK) family that mediates signal transduction activated by TGFβ, bone morphogenetic protein (BMP), interleukin 1 (IL-1), TNF-α, etc.

The TAK1 inhibitor is not limited as long as it can suppress signal transduction mediated by TAK1. The TAK1 inhibitor may be any of a nucleic acid, a protein, and a low molecular weight organic compound. Examples of such substances include substances that inhibit the binding of TAK1 to a substrate, substances that inhibit the phosphorylation of TAK1, substances that promote the dephosphorylation of TAK1, substances that inhibit the transcription or translation of TAK1, and substances that promote the degradation of TAK1.

Examples of TAK1 inhibitors include (5Z)-7-Oxozeaenol ((3S,5Z,8S,9S,11E)-3,4,9,10-tetrahydro-8,9,16-trihydroxy-14-methoxy-3-methyl-1H-2-benzoxacyclotetradecin-1,7(8H)-dione), N-Des(aminocarbonyl)AZ-TAK1 Inhibitor (3-amino-5-[4-(4-morpholinylmethyl)phenyl]-2-thiophenecarbamide), Takinib (N1-(1-propyl-1H-benzimidazol-2-yl)-1,3-benzenedicarbamide), NG25 (N-[4-[(4-ethyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-4-methyl-3-(1H-pyrrolo[2,3-b]pyridin-4-yloxy)-benzamide) trihydrochloride) and their derivatives and analogues. These substances may be used alone or in combination of multiple types.

The TAK1 inhibitor is preferably (5Z)-7-Oxozeaenol. When (5Z)-7-Oxozeaenol is used as the TAK1 inhibitor in step (1), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 25 μM, and even more preferably about 500 nM to about 10 μM. When a TAK1 inhibitor other than (5Z)-7-Oxozeaenol is used, it is preferably used at a concentration that exhibits the same TAK1 inhibitory activity as the above-mentioned concentration of (5Z)-7-Oxozeaenol. The activity as a TAK1 inhibitor can be determined by a person skilled in the art, for example, by a method such as the kinase assay described in J. Biol. Chem. 2003 May 16; 278 (20): 18485-90.

From the viewpoint of controlling the proportion of olfactory epithelial stem cells and their precursor cells contained in the cell mass to be produced, the TAK1 inhibitor can be added at any stage of step (a) or the subsequent steps. In one embodiment, if it is intended to increase the proportion of cells constituting the olfactory epithelium in the obtained cell population, it is preferable to add the TAK1 inhibitor simultaneously with the start of step (a). If it is intended to increase the proportion of cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium in the obtained cell population, it is preferable to add the TAK1 inhibitor simultaneously with the start of step (c) described below, or within a certain period of time from the start of step (c), for example, within 14 days from the start of step (c).

The medium used in step (a) is not particularly limited as long as it is as described in the definition section above. The medium used in step (a) may be a serum medium or a serum-free medium. From the viewpoint of avoiding contamination with chemically undefined components, a serum-free medium is preferably used in the present invention. In order to avoid the complication of preparation, it is preferable to use a serum-free medium to which an appropriate amount of a serum substitute such as commercially available KSR has been added. The amount of KSR added to the serum-free medium is usually about 1% to about 30%, and preferably about 2% to about 20%, in the case of human ES cells, for example. Examples of serum-free media include a medium in which 5% KSR, 450 μM 1-monothioglycerol, and 1x Chemically Defined Lipid Concentrate are added to a 1:1 mixture of IMDM and F-12, or a medium in which 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol are added to GMEM.

At the start of step (a), the cells may be in either an adherent state or a suspended state. In a preferred embodiment, the pluripotent stem cells are dispersed into single cells and then reaggregated to form suspended cell aggregates. For this purpose, it is preferred to carry out an operation of dispersing the pluripotent stem cells obtained in step (i) into single cells (also referred to as step (ii)) before the start of step (a). The "dispersed cells" obtained by the dispersion operation are preferably single cells, but may also include cell clumps consisting of a small number of cells, for example, 2 to 100 or less, or may include cell clumps consisting of 2 to 50 cells or less. The "dispersed cells" may, for example, include 70% or more single cells and 30% or less cell clumps, and preferably include 80% or more single cells and 20% or less cell clumps. In another embodiment, the pluripotent stem cells may be dispersed into single cells, and then seeded on a cell-adhesive culture vessel, and cultured in an adherent state.

Methods for dispersing pluripotent stem cells include mechanical dispersion treatment, cell dispersion treatment, and cell protective agent addition treatment, and these treatments may be performed alone or in combination. A preferred method for dispersing cells is to perform cell dispersion treatment simultaneously with cell protective agent addition treatment, followed by mechanical dispersion treatment.

Examples of the cell protective agent used in the cell protective agent addition treatment include FGF signaling pathway active substances, heparin, Rho-associated protein kinase (ROCK) inhibitors, myosin inhibitors, polyamines, integrated stress response (ISR) inhibitors, caspase inhibitors, cell adhesion promoters, serum, or serum substitutes. A preferred cell protective agent is a ROCK inhibitor. In order to suppress cell death of pluripotent stem cells (especially human pluripotent stem cells) induced by dispersion, it is preferable to add a ROCK inhibitor from the start of the culture in the first step. ROCK inhibitors include Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide, dihydrochloride), Fasudil (HA1077) (1-(5-isoquinolinylsulfonyl) homopiperazine, hydrochloride), and H-1152 (5-[[( 2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl]sulfonyl]-4-methyl-isoquinone, dihydrochloride), HA-1100 (Hydroxyfasudil) ([1-(1-Hydroxy-5-isoquinonesulfonyl) homopiperazine, hydrochloride), Chroman 1 ((3S)-N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl]-6-methoxy-3,4-dihydro-2H-chromene-3-carboxamide), Belumosudil (KD025, 2-[3-[4-[(1H-Indazol-5-yl)amino]qui nazolin-2-yl]phenoxy]-N-isopropylacetamide, HSD1590 ([2-Methoxy-3-(4,5,10-triazatetracyclo[7.7.0.02,6.012,16]hexadeca-1(9),2(6),3,7,10,12(16)-hexaen-11-yl)phenyl]boronic acid), CRT0066854 ((S)-3-phenyl-N1-(2-pyridin-4-yl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)propane-1,2-diamine), RKI1447 (1-(3-hydroxybenzyl)-3-(4-( pyridin-4-yl) thiazol-2-yl) urea), Ripasudil (4-Fluoro-5-[[(2S)-hexahydro-2-methyl-1H-1,4-diazepin-1-yl] sulfonyl] isoquinone), GSK269962A (N-[3-[2-(4-amino-1,2,5-oxa diazol-3-yl)-1-ethylimidazo[4,5-c]pyridin-6-yl]oxyphenyl]-4-(2-morpholin-4-ylethoxy)benzamide, GSK429286A (N-(6-fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-4-(4-(trifluoromethyl)phenyl) (R)-4-(1-aminoethyl)-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide), LX7101 (N,N-dimethylcarbamic acid 3-[[[4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinyl]carbonyl]amino]phenyl ester, AT13148 ((alphaS)-alpha-(aminomethyl)-alpha-(4-chlorophenyl)-4-(1H-pyrazol-4-yl)benzenemethanol), SAR407899 (6-(piperidin-4-yloxy)isoquinolin-1(2H)-one hydrochloride), GSK180736A (4-(4-fluorophenyl)-N-(1H-indazol-5-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide), hydroxyfasudil (1-(1-hydroxy-5-isoquinolinesulfonyl) homopiperazine, HCl), bdp5290 (4-chloro-1-(4-piperidinyl)-N-[3-(2-pyridinyl)-1H-pyra zol-4-yl]-1H-pyrazole-3-carboxamide), sr-3677 (N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazole-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamidehydrochloride), CCG-222740 (N-(4-Chlorophenyl)-5,5-difluoro-1-(3-(furan-2-yl)benzoyl)piperidine-3-carboxamide), ROCK inhibitor-2 (N-[(1R)-1-(3-methoxyphenyl)ethyl]-4-pyridin-4-ylbenzamide), Rho-Kinase-IN-1 (N-[1-[(4-methylsulfanylphenyl)methyl]piperidin-3-yl]-1H-indazol-5-amine), ZINC00881524 (N-(4,5-dihydropyridine) ydronaphtho[1,2-d]thiazol-2-yl)-2-(3,4-dimethylphenyl)acetamide, SB772077B ((3S)-1-[[2-(4-amino-1,2,5-oxadiazole-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride), Verosudil (N-(1,2-dihydro-1-oxo-6-isoquinolinyl)-alpha-(dimethylamino)-3-thiopheneacetamide), GSK-25 (4-(4-chloro-2-fluorophenyl)-2-(2-chloropyridin-4-yl)-1-(6-fluoro-1H-indazol-5-yl)-6-methyl-4H-pyrimidine-5-carboxamide) and derivatives thereof. Examples of the cell adhesion promoter include adhesamine and adhesamine-RGDS derivative (manufactured by Nagase & Co., Ltd.). A prepared cell protective agent can also be used as the cell protective agent. Examples of the prepared cell protective agent include RevitaCell Supplement (manufactured by Thermo Fisher Scientific), CloneR, and CloneR2 (manufactured by Stemcell Technologies). These substances may be used alone or in combination. In the step (a), when Y-27632, which is a ROCK inhibitor, is added as the cell protective agent, it is usually added to the culture environment at a concentration of about 10 nM to about 10 mM, preferably about 100 nM to about 1 mM, and more preferably about 1 μM to about 100 μM. In step (a), when Chroman 1, a ROCK inhibitor, is added as a cell protective agent, it is usually added to the culture environment at a concentration of about 10 pM to about 1 mM, preferably about 100 pM to about 100 μM, more preferably about 1 nM to about 10 μM. When a substance other than Y-27632 and Chroman 1 is used as a cell protective agent, the concentration to be added can be determined, for example, by a colony formation assay using human ES cells described in Nature Biotechnology volume 25, pages 681-686 (2007).

The cell dispersion liquid used in the cell dispersion liquid treatment may be a solution containing at least one of enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, and papain, and a chelating agent such as ethylenediaminetetraacetic acid. Commercially available cell dispersion liquids, such as TripLE Select, TripLE Express (manufactured by Thermo Fisher Scientific), Accutase, Accumax (manufactured by Innovative Cell Technologies), etc., may also be used. A preferred cell dispersion liquid for the treatment of the pluripotent stem cells obtained after step (i) in step (ii) is phosphate buffered saline (PBS) supplemented with 5 mM EDTA, but is not limited thereto.

Mechanical dispersion methods include pipetting or scraping with a scraper. The dispersed cells are suspended in the above-mentioned medium.

One method for dispersing pluripotent stem cells is, for example, treating a colony of pluripotent stem cells with ethylenediaminetetraacetic acid or Accumax in the presence of a ROCK inhibitor, and then dispersing the colony by pipetting.

When suspension culture is performed in step (a), a suspension of dispersed pluripotent stem cells is seeded in a non-cell-adhesive culture vessel. When the culture vessel is non-adhesive, the cells are cultured in suspension, and multiple pluripotent stem cells gather together to form cell aggregates.

When performing suspension culture, dispersed pluripotent stem cells may be seeded in a relatively large culture vessel such as a 10 cm dish, so that multiple cell aggregates are simultaneously formed in one culture vessel. On the other hand, from the viewpoint of preventing the occurrence of variation in size for each cell aggregate, it is preferable to seed a certain number of dispersed pluripotent stem cells in each well of a multi-well plate (U-bottom, V-bottom) such as a non-cell-adhesive 96-well microplate. When this is subjected to static culture, the cells rapidly aggregate, so that one cell aggregate can be formed in each well. For example, the culture vessel can be made non-cell-adhesive by processing such as coating the surface of the culture vessel with a superhydrophilic polymer. Examples of non-cell-adhesive multi-well plates include PrimeSurface 96V-bottom plates (MS-9096V, manufactured by Sumitomo Bakelite Co., Ltd.). Centrifugation may be performed to form cell aggregates more rapidly. A uniform population of cell aggregates can be obtained by recovering the cell aggregates formed in each well from multiple wells. If the cell aggregates are uniform, the production efficiency for each well and each replicate can be more stable in subsequent steps, allowing cells that make up upper respiratory tract tissue to be produced more reproducibly.

Another aspect for forming cell aggregates from dispersed pluripotent stem cells is a method using a culture vessel in which one well is divided into a plurality of microwells and two or more cell aggregates are formed. In other words, in one aspect of this embodiment, the suspension culture of any one or more of the steps (a), (b), and (c) may be performed in a culture vessel in which at least one well is formed, the well is divided into a plurality of microwells, and the suspension culture is performed so that one cell aggregate is formed in each of the microwells, and the number of cell aggregates corresponding to the number of divided microwells may be prepared for each well. As the microwell, a culture vessel having a plurality of mortars, downward pyramids, concaves, grids, or protuberances on the bottom surface on which cells settle in one place to promote the formation of aggregates, or a culture vessel having a process to allow cells to adhere to only a part of the bottom surface to facilitate the formation of aggregates may be used. The culture area per well of a culture device having microwells is not particularly limited, but from the viewpoint of efficient production of cell masses, the bottom area is preferably greater than 1 ^cm2 (equivalent to a 48-well plate), more preferably greater than ² cm2 (equivalent to a 24-well plate), and even more preferably greater than 4 ^cm2 (equivalent to a 12-well plate). Examples of the culture equipment include, but are not limited to, the embryoid body formation plate AggreWell (manufactured by StemCell Technologies), PAMCELL (manufactured by ANK), spheroid microplate (manufactured by Corning), NanoCulture Plate/Dish (manufactured by Organogenix), Cell-able (manufactured by Toyo Gosei), EZSPHERE (manufactured by AGC Technoglass), SPHERICALPLATE 5D (manufactured by Mito Kogyo Co., Ltd.), TASCL (manufactured by Sims Bio), and microwell bags (e.g., those described in Scientific reports, 2022, 12.1: 1-11).

As a cultureware for forming cell aggregates from dispersed pluripotent stem cells, it is also preferable to use a three-dimensional cell culture vessel that allows the medium of the entire plate to be replaced at once while the cell aggregates remain in each well. An example of such a three-dimensional cell culture vessel is the PrimeSurface 96 slit well plate (manufactured by Sumitomo Bakelite Co., Ltd.). This plate has narrow openings (slits) at the top of each of the 96 wells through which the medium can enter and exit. The slits are set to a width that makes it difficult for cell aggregates to pass through, so that the medium of the entire plate can be replaced at once while preventing adhesion between the cell aggregates, improving the efficiency of the operation and the quality of the cell aggregates.

The concentration (cell density) of pluripotent stem cells in step (a) can be appropriately set so as to form cell aggregates more uniformly and efficiently. For example, when human pluripotent stem cells (e.g., human iPS cells obtained from step (i)) are cultured in suspension using a 96-well microwell plate, a liquid prepared so as to obtain about 1 x 10 ³ to about 1 x 10 ⁵ cells, preferably about 3 x 10 ³ to about 5 x 10 ⁴ cells, more preferably about 4 x 10 ³ to about 2 x 10 ⁴ cells, even more preferably about 4 x 10 ³ to about 1.8 x 10 ⁴ cells, and particularly preferably about 8 x 10 ³ to about 1.5 x 10 ⁴ cells per well is added to each well, and the plate is left to form cell aggregates. For example, when human pluripotent stem cells (e.g., human iPS cells obtained in step (i)) are cultured in suspension using EZSPHERE SP Dish 35 mm Type 905, a culture ware having about 260 microwells per dish, a ^liquid prepared so that the number of cells per dish is usually about 1 x ¹⁰ to about 1 x 10, preferably about 3 x ¹⁰ to about 5 x ¹⁰ , more preferably about 4 x ¹⁰ to about 2 x ¹⁰ , even more preferably about 4 x ¹⁰ to about 1.6 x ¹⁰ , and particularly preferably about 8 x ¹⁰ to about 1.2 x ¹⁰ , is added to the dish, and the dish is allowed to stand to form cell aggregates. For example, when human pluripotent stem cells (e.g., human iPS cells obtained in step (i)) are cultured in suspension using AggreWell 800, 6-well plate, a cultureware having about 1800 microwells per well, a solution prepared so that each well contains usually about 1×10 ⁴ to about 1×10 ⁸ cells, preferably about 3×10 ⁴ to about 5×10 ⁷ cells, more preferably about 4×10 ⁴ to about 2×10 ⁷ cells, even more preferably about 4×10 ⁴ to about 1.6×10 ⁷ cells, and particularly preferably about 8×10 ⁴ to about 1.2×10 ⁷ cells, is added to each well, and the plate is centrifuged to form cell aggregates. The number of cells can be determined by counting with a hemocytometer or an automatic cell counter.

The time required for suspension culture to form cell aggregates can be appropriately determined depending on the pluripotent stem cells used, but it is desirable that the time be as short as possible in order to form uniform cell aggregates. The process from the dispersed cells until the formation of cell aggregates can be divided into a process in which the cells aggregate and a process in which the aggregated cells form aggregates. The time from the time the dispersed cells are seeded (i.e., the start of suspension culture) until the cells aggregate is preferably within about 24 hours, more preferably within about 12 hours, in the case of human pluripotent stem cells (human iPS cells, etc.), for example. The time from the time the dispersed cells are seeded (i.e., the start of suspension culture) until the cell aggregate is formed is preferably within about 72 hours, more preferably within about 48 hours, in the case of human pluripotent stem cells (human iPS cells, etc.), for example. The time until the cell aggregate is formed can be appropriately adjusted by adjusting the tool for aggregating the cells or the centrifugation conditions, etc.

When pluripotent stem cells are rapidly assembled to form cell aggregates, epithelial-like structures can be reproducibly formed in cells induced to differentiate from the formed cell aggregates. Examples of experimental procedures for forming cell aggregates include a method of confining cells in a small space using a small well plate (e.g., a plate with a well bottom area of about 0.1 to ^2.0 cm2 calculated as a flat bottom) or a micropore, and a method of aggregating cells by centrifuging for a short time using a small centrifuge tube. Examples of small well plates include 24-well plates (area of about 1.88 ^cm2 calculated as a flat bottom), 48-well plates (area of about 1.0 ^cm2 calculated as a flat bottom), 96-well plates (area of about 0.35 ^cm2 calculated as a flat bottom, inner diameter of about 6 to 8 mm), and 384-well plates. A preferred example is a 96-well plate. As for the shape of a small well plate, the shape of the bottom of the well when viewed from above can be a polygon, a rectangle, an ellipse, or a perfect circle, and a perfect circle is preferred. In this embodiment, "the shape of the bottom surface of the well is a perfect circle" is not limited to a geometric perfect circle, but also includes a shape of an approximately perfect circle. The same applies to other shapes. As the shape of the small well plate, the shape of the bottom surface when the well is viewed from the side is preferably a structure in which the outer periphery is high and the inside is low and concave, for example, a U-bottom, a V-bottom, or an M-bottom is included, preferably a U-bottom or a V-bottom, and most preferably a V-bottom is included. As the small well plate, a cell culture dish (e.g., a 60 mm to 150 mm dish, a culture flask) having an uneven or concave bottom surface may be used. It is preferable to use a cell non-adhesive bottom surface, preferably a cell non-adhesive coated bottom surface, as the bottom surface of the small well plate.

As another method for forming cell aggregates, it is also preferable to use a three-dimensional printer or 3D printer. Cell aggregates of the desired shape can be prepared by suspending a dispersed single cell or a spheroid composed of multiple cells in a biocompatible ink (bioink) and outputting it using a bio 3D printer (e.g., BIO X manufactured by Celllink, etc.), or by piercing a cell population with a needle and stacking it (Spike manufactured by Cyfuse, etc.).

The formation of cell aggregates can be determined based on the size and cell number of the cell aggregates, the macroscopic morphology, the microscopic morphology and its uniformity determined by tissue staining analysis, the expression and uniformity of differentiated and undifferentiated markers, the control of expression of differentiation markers and their synchrony, and the reproducibility of differentiation efficiency between aggregates.

At the start of step (a), in a preferred embodiment, adhesion culture is performed. The pluripotent stem cells on the culture vessel after step (i) may be used as they are in step (a), or the pluripotent stem cells may be dispersed into single cells by the treatment of step (ii) and then seeded again on the adhesive culture vessel. When reseeding is performed after dispersing the pluripotent stem cells into single cells, an appropriate extracellular matrix or synthetic cell adhesion molecule may be coated on the surface of the culture vessel as a scaffold. The scaffold allows the pluripotent stem cells to be cultured in the culture vessel with the surface coated. The extracellular matrix is preferably Matrigel or laminin. Examples of synthetic cell adhesion molecules include synthetic peptides containing cell adhesive domains such as poly-D-lysine and RGD sequences. The number of cells seeded in the adhesion culture in step (a) is usually about 1 x 10 ¹ to about 1 x 10 ⁶ cells per well, preferably about 1 x 10 2 to about 1 x 10 ⁵ cells, more preferably about ² x 10 2 to about 5 x 10 ⁴ cells, even more preferably about 1 x 10 ³ to about 2 x 10 ⁴ cells, and particularly preferably about 2 x 10 ³ to about 1 x 10 ⁴ cells, when ^seeded in a cell-adhesive flat-bottom 96-well plate. A liquid prepared to achieve the above-mentioned seeding number is added to each well, making it possible to carry out adhesion culture. When using culture equipment of different sizes, the seeding number of the above-mentioned cells can be appropriately changed depending on the culture area and the amount of medium.

It is also preferable to use a temperature-responsive cell culture substrate as an adhesive culture substrate. Adherent cells, including cells produced by the method described in the present application, generally have the property of easily adhering to a certain degree of hydrophobic surfaces, and adhesion is inhibited on hydrophilic surfaces. For this reason, by using a substrate having a surface that changes from moderately hydrophobic to hydrophilic or vice versa in response to temperature, it becomes possible to control the adhesion of adherent cells to the surface by temperature change. As a result, it becomes possible to recover, for example, epithelial cells as a sheet from the culture substrate while maintaining cell-cell adhesion without using protease or chelating agent. A culture substrate having the above-mentioned properties is generally called a "temperature-responsive cell culture substrate". A temperature-responsive cell culture substrate is produced, for example, by immobilizing poly-N-isopropylacrylamide (PIPAAm) on the surface. Examples of such a temperature-responsive cell culture substrate include UpCell (manufactured by CellSeed Co., Ltd.) and the substrate described in Patent Publication No. 5846584.

It is also preferable to use a culture vessel having a micropattern applied thereto as the adhesive culture vessel. The micropattern on the culture vessel can be composed of a cell adhesive region and a cell non-adhesive region, and it is preferable that cells are cultured in the cell adhesive region. The cell adhesive region is preferably surrounded by the cell non-adhesive region on the same surface. The shape of the cell adhesive region can be appropriately set, and can be set to, for example, a circular, elliptical, triangular, square, rectangular, pentagonal, hexagonal, octagonal, star-shaped, or other shape. The shape of the cell adhesive region is preferably, but not limited to, any of a circular, square, and rectangular shape. The cell adhesive region and the cell non-adhesive region may be formed as a single region on one culture vessel, or may be formed as a plurality of regions. It is preferable that the cell adhesive region has been subjected to a cell adhesive treatment (gas plasma treatment, etc.) or has been treated with a substance that promotes cell adhesion. A preferred cell adhesion promoting substance in this embodiment is preferably laminin, and more preferably laminin 511 to 521, but is not limited thereto. Laminin may be an E8 fragment. The cell adhesive region may be the temperature-responsive cell culture substrate or may be treated with a temperature-responsive polymer. The cell non-adhesive region is preferably in a state where no cell adhesive treatment has been performed, or has been treated to inhibit cell adhesion. Preferred cell adhesion inhibition treatments in this embodiment include, for example, superhydrophilic treatments using coatings such as 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, Poly(2-hydroxyethyl methacrylate) (Poly-HEMA), polyethylene glycol (PEG), and the like, and low protein adsorption treatments. The area of the cell adhesive region can be appropriately set, and is, for example, 0.1 cm ² to 100 cm ² , preferably 0.3 cm ² to 80 cm ² , and more preferably 0.5 cm ² to 60 cm ² , but is not limited thereto. The number of pluripotent stem cells to be seeded on the culture substrate having the micropattern is, for example, 1 x 10 ³ cells/cm ² to 1 x 10 ⁷ cells/cm ² per area of the cell adhesive region, preferably 1 x 10 ⁴ cells/cm ² to 1 x 10 ⁶ cells/cm ² , but is not limited thereto.

Examples of micropatterned cultureware include CYTOOChip (manufactured by CYTOOC) and ibidi Micropatterning (manufactured by ibidi). Cultureware can also be prepared using a PDMS mold and matrix. Alternatively, cultureware coated with an extracellular matrix, a substrate that promotes cell adhesion, etc. can be processed with a laser or the like using a cell processing device (Model: CPD-017, manufactured by Kataoka Seisakusho Co., Ltd.) to create cell adhesive regions and cell non-adhesive regions of any shape. When culturing the pluripotent stem cells obtained in step (i) on a micropatterned cultureware, the method can be carried out by referring to a previously reported method (Nature protocols, 11(11), 2223-2232.).

It is also preferable that the culture equipment has a flow path (microflow path) for perfusion of the medium, and cells may be cultured in a perfusion environment in step (a) and the subsequent steps. Such a culture equipment is also called a microfluidic chip. The culture equipment (e.g., microfluidic chip) may be equipped with a sensor. The flow path of the culture equipment may be equipped with a valve. The culture equipment may be connected by a flow path to other culture equipment for culturing cells or tissues other than the cells to be cultured in the manufacturing method of the present invention. This makes it possible to reproduce the interaction between the upper respiratory tract tissue and other cells or tissues. Examples of other cells or tissues to be co-cultured with the upper respiratory tract tissue include, but are not limited to, tissues that promote the growth, differentiation, maturation, and survival of the upper respiratory tract tissue, such as mesenchymal cells, as well as cells and tissues of the brain, head and neck, blood vessels, bone, muscle, fat, thyroid, liver, adrenal gland, testis, ovary, and breast. Examples of other cells to be co-cultured with the upper respiratory tract tissue include, but are not limited to, cell lines, primary culture cells, stem cells, and differentiated cells derived from stem cells. Methods for perfusing the medium include, but are not limited to, the use of a magnetic stirrer, a peristaltic pump, a syringe pump, a diaphragm valve, etc.

The cultureware may have a membrane that is permeable to oxygen or culture medium (sometimes referred to as a "membrane having substance permeability"). The cultureware may be capable of forming a concentration gradient of compounds, growth factors, etc. The membrane is, for example, a porous membrane. In a cultureware having such a membrane, cells can be cultured on one side separated by the membrane using the manufacturing method of the present invention, and other cells or tissues, feeder cells, etc. can be cultured on the other side. This allows cells that constitute upper respiratory tract tissue or their precursor cells and cell populations containing these to be cultured without contamination with other cells or tissues. The cultureware may be used as a cultureware in subsequent steps.

In step (a) and the subsequent steps, when a medium exchange operation is performed, examples include an operation of adding new medium without discarding the original medium (medium addition operation), an operation of discarding about half the original medium (about 30 to 90% of the volume of the original medium, for example about 40 to 60% of the volume of the original medium) and adding about half the new medium (about 30 to 90% of the volume of the original medium, for example about 40 to 60% of the volume of the original medium) (half medium exchange operation), and an operation of discarding about the entire amount of the original medium (90% or more of the volume of the original medium) and adding about the entire amount of new medium (90% or more of the volume of the original medium) (full medium exchange operation).

When adding a specific component at a certain point, for example, after calculating the final concentration, an operation (half medium exchange operation) may be performed in which about half of the original medium is discarded and about half of a new medium containing the specific component at a concentration higher than the final concentration is added. When diluting a component contained in the original medium to reduce the concentration at a certain point, for example, the medium exchange operation may be performed multiple times a day, preferably multiple times within an hour (for example, 2 to 3 times). Also, when diluting a component contained in the original medium to reduce the concentration at a certain point, the cells or cell aggregates may be transferred to another culture vessel. The tools used for the medium exchange operation are not particularly limited, but examples include a pipetter, Pipetman (registered trademark), a multichannel pipette, and a continuous dispenser. For example, when a 96-well plate is used as the culture equipment, a multichannel pipette may be used.

The incubation time in step (a) is usually about 8 hours to 6 days, preferably about 12 hours to 48 hours.

From the viewpoint of controlling the direction of cell differentiation and promoting the survival and proliferation of cells or tissues, step (a) and any one or more of the subsequent steps can be performed in the presence of a substance acting on the Wnt signaling pathway. As described above, examples of Wnt signaling pathways include the classical Wnt pathway and the non-classical Wnt pathway.

The substance acting on the Wnt signaling pathway is not limited as long as it can activate signaling induced by Wnt family proteins. The substance acting on the Wnt signaling pathway may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that promote the autosecretion of Wnt, substances that stabilize Wnt and inhibit its degradation, recombinant Wnt proteins, partial sequence peptides of Wnt and their derivatives or derivatives, substances that act on and activate Wnt receptors, substances that activate the intracellular signaling mechanism of Wnt, intracellular signaling factors of Wnt and their variants (such as β-Catenin S33Y), and substances that activate gene expression downstream of the Wnt response sequence. Proteins known as substances acting on the Wnt signaling pathway include Wnt and R-spondin.

When using a recombinant Wnt protein as a substance acting on the Wnt signaling pathway, the origin is not particularly limited, and Wnt proteins derived from various organisms can be used as long as they have activity on the target cells. Among them, Wnt proteins derived from mammals are preferable. Examples of mammals include humans, mice, rats, cows, pigs, rabbits, etc. More preferably, the recombinant protein is derived from the same species as the pluripotent stem cells used in the production of the cell population according to the present invention. Examples of mammalian Wnt proteins include, but are not limited to, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. For example, as described in Molecular Cancer volume 8, Article number: 90 (2009), among Wnt proteins, Wnt1, Wnt2, Wnt2B, Wnt3, Wnt8A, Wnt8B, Wnt10A, and Wnt10B are substances acting on the Wnt signaling pathway that mainly activate the Wnt canonical pathway. In addition, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 are substances acting on the Wnt signaling pathway that mainly activate the Wnt non-canonical pathway. In producing the cell population according to the present invention, the Wnt proteins may be used alone or in combination. Recombinant Wnt proteins that can be used to produce the cell population of the present invention are available from, for example, R&D Systems and Fujifilm Wako Pure Chemical Industries, Ltd. The Wnt protein used in the production of the cell population of the present invention is preferably a Wnt signaling pathway active substance that activates the Wnt canonical pathway.

In producing the cell population according to the present invention, a substance that stabilizes the recombinant Wnt protein in the medium and maintains its activity can also be added. An example of a substance having the above-mentioned activity is afamin. Afamin is a glycoprotein that belongs to the albumin family, and is known to inhibit the aggregation and inactivation of the highly hydrophobic Wnt protein and maintain its activity. Afamin recombinant protein is available, for example, from R&D Systems. A mixture of afamin and recombinant Wnt protein is available from MBL Life Sciences.

Examples of R-spondins that can be used as Wnt signaling pathway active substances include proteins belonging to the R-spondin family consisting of R-spondin1, R-spondin2, R-spondin3 and R-spondin4. The R-spondin family is a secretory protein, and as described in, for example, Nature volume 485, pages 195-200 (2012), it inhibits the activity of the seven-transmembrane ubiquitin ligase ZNRF/43/RNF43, which negatively controls the amount of Wnt receptor on the cell membrane surface, and is involved in the activation and control of the Wnt signaling pathway. In the production of the cell population according to the present invention, multiple types of R-spondins may be used in combination. Recombinant R-spondin proteins that can be used in the production of the cell population according to the present invention are available, for example, from R&D Systems and Fujifilm Wako Pure Chemical Industries.

Compounds well known to those skilled in the art can also be used as Wnt signaling pathway active substances. Examples of compounds having activity as Wnt signaling pathway active substances include compounds having inhibitory activity against GSK3β, compounds having inhibitory activity against Wnt deacylation enzymes, and compounds having inhibitory activity against AAK1. Examples of compounds having the above-mentioned activity include lithium chloride, SGC AAK1 1(N-(6-(3-((N,N-dimethylsulfamoyl)amino)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide, Foxy, and the like. 5 (N-formyl-L-methionyl-L-alpha-aspartyl-glycyl-L-cysteinyl-L-alpha-glutamyl-L-leucine), CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazo[ol-2-yl]pyridine) N6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-iMidazol-2-yl)-2-pyriMidinyl]aMino]ethyl]-3-nitro-2,6-pyridin ediaMine), TWS119 (3-[6-(3-amino-phenyl)-7H-pyrrolo[2,3-D]pyrimidin-4-yloxy]-phenol), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-2,5-dihydro-1H-py 1H-pyrrol-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione), BIO ((2Z,3E)-6-bromoindirubin-3-oxime), AZD2858 (3-amino-6-[4- [(4-methyl-1-piperazineyl)sulfonyl]phenyl]-N-3-pyridinyl-pyrazinecarboxamide, AZD1080 (2-hydroxy-3-(5-(morpholinomethyl)pyridin-2-yl)-1H-indole-5-carbonitrile), A R-A014418 (N-(4-Methoxybenzyl)-n-(5-nitro-1,3-thiazol-2-yl)urea), TDZD-8 (4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), LY2090314 (7-(2,5-Dihydro-4-imidazo[1,2 -a]pyridin-3-yl-2,5-dioxo-1H-pyrrol-3-yl)-9-fluoro-1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepine, IM-12 (3-(4-fluorophenethyl Indirubin ([2,3-Biindolinylidene]-2,3-dione), Bikinin (4-((5-Bromo-2-pyridinyl)amino)-4-oxobutanoic acid acid), A-1070722 (1-(7-Methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea), 3F8 (5-Ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]isoquinoline-1,3(2H)-dione), Kenpaullone (9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzozepin-6(5H)-one), 10Z-H ymenialdisine ((4Z)-4-(2-amino-1,5-dihydro-5-oxo-4H-imidazolium-4-ylidene)-2-bromo-4,5,6,7-tetrahydro-pyrrolo[2,3-c]azepin-8(1H)-one), indirubin-3'-oxime (3-[1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihydro-2H-indol-2-one), NSC 693868 (1H-pyrazolo[3,4-b]quinoxalin-3-amine), TC-G 24 (N-(3-chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine), TCS 2002 (2-methyl-5-[3-[4-(methylsulfinyl)phenyl]-5-benzofuranyl]-1,3,4-oxadiazol), TCS 21311 (3-[5-[4-(2-Hydroxy-2-Methyl-1-oxopropyl)-1-piperazinyl]-2-(trifluoromethyl)phenyl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), CP21R7 (3-(3-Aminophenyl)-4-(1-methyl-1H-indol-3-yl)pyrrole-2,5-dione), BML-284 (AMBMP Hydrochloride, N4-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)-2,4-pyrimidinediamine hydrochloride), SKL2001 (N-(3-(1H-imidazolyl-1-yl)propyl)-5-(furan-2-yl)isoxazole-3-carboxamide), WAY-262611 (1-[4-(2-Naphthalenyl)-2-pyrimidinyl]-4-piperidinemethane), IIIC3a (9-Carboxy-3-(dimethylimino)-6,7-dihydroxy-10-methyl-3H-phenoxazin-10-ium iodide), Methyl Vanillate, IQ-1 (2-[(4-Acetylphenyl)azo]-2-(3,4-dihydro-3,3-dimethyl-1(2H)-isoquinolinylidene)acetamide) and their derivatives.

In step (a), a Wnt signaling pathway inhibitor is added, and by using a Wnt signaling pathway activator and a Wnt signaling pathway inhibitor with different sites of action in combination, it is possible to activate or inhibit only a specific pathway among the multiple Wnt signaling pathways described above. The Wnt signaling pathway activator is preferably a substance that acts on a factor downstream of the Wnt signaling pathway inhibitor added in step (a). It is also preferable that the Wnt signaling pathway activator added is a substance that activates the classical Wnt pathway, and more preferably a substance that activates the Wnt signaling pathway by inhibiting and stabilizing the degradation of β-catenin, which is a Wnt signaling factor in cells. Examples of substances that inhibit and stabilize the degradation of β-catenin include GSK3 inhibitors, BML-284, SKL2001, etc. It is also preferable that the Wnt signaling pathway active substance to be added is a substance that promotes or activates β-catenin responsive transcription (CRT).

GSK3 inhibitors include CHIR99021, CHIR98014, TWS119, SB216763, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, A 1070722, 3F8, Kenpaullone, 10Z-Hymenialdisine, Indirubin-3'-oxime, NSC 693868, TC-G 24, TCS 2002, TCS 21311, CP21R7, and derivatives thereof.

A preferred combination of a Wnt signaling pathway active substance and a Wnt signaling pathway inhibitor is a GSK3 inhibitor and a PORCN inhibitor. When a GSK3 inhibitor and a PORCN inhibitor are used in combination, the classical Wnt pathway is activated and the non-classical Wnt pathway is inhibited. When a GSK3 inhibitor and a PORCN inhibitor are used in combination, the GSK3 inhibitor preferably includes at least one selected from the group consisting of CHIR99021, CHIR98014, SB216763, SB415286, and BIO, and more preferably includes CHIR99021. When a GSK3 inhibitor and a PORCN inhibitor are used in combination, the PORCN inhibitor preferably includes at least one selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, LGK-974, ETC-159, GNF-6231, and Wnt-C59, and more preferably includes IWP-2. A GSK3 inhibitor may be used in combination with KY02111 or a derivative thereof. Substances having a different mechanism of action from that of a GSK3 inhibitor and having an inhibitory or stabilizing effect on the degradation of β-catenin include, for example, BML-284 and SKL2001, and these compounds or derivatives thereof may also be used in combination with a PORCN inhibitor, KY02111 or a derivative thereof, and the like. Further preferred combinations of a Wnt signaling pathway activator and a Wnt signaling pathway inhibitor include a GSK3 inhibitor, a PORCN inhibitor, and a JNK inhibitor. When a GSK3 inhibitor is used in combination with a PORCN inhibitor and a JNK inhibitor, the JNK inhibitor preferably includes JNK-IN-8.

The concentration of the Wnt signaling pathway active substance in the medium can be appropriately set within a range that can achieve the above-mentioned effects. From the viewpoint of promoting the survival and proliferation of nervous system cells or tissues inside the cell mass, when CHIR99021 is used as the Wnt signaling pathway active substance, it is usually used at a concentration of about 10 pM to about 10 mM, preferably about 100 pM to about 1 mM, more preferably about 1 nM to about 100 μM, even more preferably about 10 nM to about 30 μM, and most preferably about 100 nM to about 3 μM. When a Wnt signaling pathway active substance other than CHIR99021 is used, it is desirable to use it at a concentration that shows the same Wnt signaling pathway promoting activity as CHIR99021 at the above concentration. The Wnt signaling pathway promoting activity can be determined, for example, by a Topflash reporter assay using HEK293 cells.

In order to improve the efficiency of producing olfactory epithelium-like tissue in step (a) and the subsequent steps, it is also preferable to add a compound that promotes differentiation into the placode region. Examples of compounds having the above-mentioned effect include BRL-54443, phenanthrolin, and parthenolide, which are described in U.S. Patent Application Publication No. 2016/0326491. When BRL-54443 is used as a compound that promotes differentiation into the placode region, it is usually used at a concentration of 10 nM to 100 μM, when phenanthrolin is used, it is usually used at a concentration of 10 nM to 100 μM, and when parthenolide is used, it is usually used at a concentration of 10 nM to 100 μM.

<Step (b)>
In step (b), the cells cultured in step (a) are cultured in the presence of a substance acting on the BMP signal transduction pathway. If the cells are cultured in suspension in step (a), the formed cell aggregates may be cultured in suspension in step (b). If the cells are cultured in adhesion in step (a), the cells may be cultured in adhesion in step (b). After the cells are cultured in suspension in step (a), they may be cultured in adhesion in step (b). After the cells are cultured in adhesion in step (a), they may be cultured in suspension in step (b).

A substance acting on the BMP signaling pathway is a substance that can enhance the signaling pathway mediated by BMP. Examples of substances acting on the BMP signaling pathway include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. These substances may be used alone or in combination. Examples of substances acting on the BMP signaling pathway from the viewpoint of biological activity include substances that have the ability to induce differentiation into osteoblast-like cells and the ability to induce alkaline phosphatase production in cells such as mouse precursor chondrocyte cell line ATDC5, mouse calvarial-derived cell line MC3T3-E1, and mouse striated muscle-derived cell line C2C12. Examples of substances having the above-mentioned activity include BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP10, BMP13/GDF6, BMP14/GDF5, GDF7, etc.

BMP2 protein and BMP4 protein are available, for example, from R&D Systems. BMP7 protein is available, for example, from Biolegend. GDF7 protein is available, for example, from Fujifilm Wako Pure Chemical Industries, Ltd. The BMP signaling pathway active substance preferably includes at least one protein selected from the group consisting of BMP2, BMP4, BMP7, BMP13, and GDF7, and more preferably includes BMP4.

The concentration of the BMP signaling pathway active substance in the medium can be appropriately set within a range that can achieve the above-mentioned effects. From the viewpoint of improving the production efficiency of cells that constitute upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, when BMP4 is used as the BMP signaling pathway active substance, it is usually used at a concentration of about 1 pM to about 100 nM, preferably about 10 pM to about 50 nM, more preferably about 25 pM to about 25 nM, even more preferably about 25 pM to about 5 nM, particularly preferably about 100 pM to about 5 nM, and most preferably about 500 pM to about 2 nM. When a BMP signaling pathway active substance other than BMP4 is used, it is desirable to use it at a concentration that shows the same BMP signaling pathway promoting activity as BMP4 at the above-mentioned concentration. When using, for example, a commercially available recombinant BMP protein as a substance acting on the BMP signaling pathway, a person skilled in the art can easily determine the concentration of the substance acting on the BMP signaling pathway to be added by comparing the activity described in the product insert, such as the ED50 value of the ability to induce alkaline phosphatase production in the mouse precursor chondrocyte cell line ATDC5, with the concentration and activity of the above-mentioned BMP4.

Compounds known to those skilled in the art can also be used as BMP signaling pathway active substances. Examples of BMP signaling pathway active substances include Smurf1 inhibitors and Chk1 inhibitors. Examples of compounds having the above-mentioned activity include A-01 ([4-[[4-Chloro-3-(trifluoromethyl)phenyl]sulfonyl]-1-piperazinyl][4-(5-methyl-1H-pyrazol-1-yl)phenyl]methaneone), PD 407824 (9-Hydroxy-4-phenyl-pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione) and derivatives thereof.

The medium used in step (b) is not particularly limited as long as it contains a substance acting on the BMP signaling pathway. Examples of the medium used in step (b) include the medium listed in step (a).

From the viewpoint of improving the efficiency of producing olfactory epithelium, the start time of step (b) is preferably 0.5 hours to 6 days, more preferably 0.5 hours to 72 hours, even more preferably 12 hours to 60 hours, and may be 18 hours to 60 hours from the start of culture in step (a). When performing suspension culture, if step (b) is started during the above period in the presence of a Wnt signal pathway inhibitor, non-neuroepithelium-like tissue is formed on the surface of the cell aggregates, and cells constituting the olfactory epithelium or their precursor cells are formed very efficiently.

When suspension culture is performed in step (a), from the viewpoint of improving the production efficiency of cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, the start time of step (b) is preferably the time when 10% to 100%, more preferably 30% to 100%, and even more preferably 50% to 100% of the cells in the surface layer of the cell aggregate formed in step (a) form tight junctions with each other. Whether tight junctions are formed in the cell aggregate can be determined by a technique such as immunostaining using an anti-ZO-1 antibody.

When starting culture in the presence of a substance acting on the BMP signaling pathway in step (b), the above-mentioned medium exchange operation (e.g., medium addition operation, half-medium exchange operation, full-medium exchange operation, etc.) may be performed using the culture vessel in which step (a) was performed, or the cells may be transferred to another culture vessel.

The period of culture in the medium containing the substance acting on the BMP signaling pathway in step (b) can be set appropriately. The culture time in step (b) is usually about 8 hours to 6 days, preferably about 10 hours to 108 hours, more preferably about 12 hours to 96 hours, even more preferably about 18 hours to 72 hours, and most preferably about 24 hours to 60 hours. In one aspect of this embodiment, the culture time in step (b) is usually about 8 hours to 6 days, preferably about 10 hours to 96 hours, more preferably about 12 hours to 72 hours, even more preferably about 14 hours to 48 hours, and most preferably about 16 hours to 36 hours.

In step (b), it is also preferable to continue adding the additives used in step (i) or step (a), such as a Wnt signaling pathway inhibitor, a Wnt signaling pathway activating substance, a TGFβ signaling pathway inhibitor, a TAK1 inhibitor, etc. The Wnt signaling pathway inhibitor, Wnt signaling pathway activating substance, or TGFβ signaling pathway inhibitor added in step (b) may be different from the substance used in the previous step, but is preferably the same. The concentration and type of additives can be appropriately adjusted. The timing of addition of these substances may be simultaneous with the start of step (b) or may be different.

<Step (c)>
In step (c), the cells cultured in step (b) are cultured in the presence of at least one selected from the group consisting of a substance acting on the FGF signaling pathway and a substance inhibiting the BMP signaling pathway. If the cells are cultured in suspension in step (b), the cell aggregates may be continued to be cultured in suspension in step (c). If the cells are cultured in adhesion in step (b), the cells may be continued to be cultured in adhesion in step (c). The cells may be cultured in suspension in step (b) and then cultured in adhesion in step (c). The cells may be cultured in adhesion in step (b) and then cultured in suspension in step (c). By carrying out the culture in step (c), a first cell population containing olfactory placode cells or olfactory epithelial stem cells is obtained.

In step (c), at least one of an FGF signaling pathway active substance and a BMP signaling pathway inhibitor is added to the medium. In one aspect of this embodiment, the cells may be cultured by adding both an FGF signaling pathway active substance and a BMP signaling pathway inhibitor.

The FGF signaling pathway active substance is not particularly limited as long as it is a substance that can enhance the signaling pathway mediated by FGF (fibroblast growth factor). Examples of FGF signaling pathway active substances include FGF proteins such as FGF1, FGF2 (sometimes referred to as "bFGF"), FGF3, FGF7, FGF8, and FGF10, anti-FGF receptor antibodies, and FGF partial peptides. These substances may be modified or fused proteins. These substances may be used alone or in combination. The FGF signaling pathway active substance used in the present invention is preferably a substance that binds to fibroblast growth factor receptor 1 (FGFR1) or fibroblast growth factor receptor 2 (FGFR2) and improves its activity as a receptor tyrosine kinase. Substances having the above activity are, for example, those described in Cancer Metastasis Rev. (2015) 34: 479-496, and are FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

The FGF2 protein and FGF8 protein are available, for example, from Fujifilm Wako Pure Chemical Industries, Ltd. The FGF signaling pathway active substance is preferably FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23, more preferably at least one selected from the group consisting of FGF2, FGF3, FGF8, and FGF10, and modified forms thereof, even more preferably FGF2, and even more preferably recombinant human FGF2.

The concentration of the FGF signaling pathway active substance in the medium can be appropriately set within a range that can achieve the above-mentioned effects. From the viewpoint of promoting differentiation into cells that constitute upper respiratory tract tissue, olfactory epithelium, or nasal epithelium, as well as cell survival and proliferation, when FGF2 is used as the FGF signaling pathway active substance, it is usually used at a concentration of about 1 pg/ml to about 100 μg/ml, preferably about 10 pg/ml to about 50 μg/ml, more preferably about 100 pg/ml to about 10 μg/ml, even more preferably about 500 pg/ml to about 1 μg/ml, and most preferably about 1 ng/ml to about 200 ng/ml. In addition, when a substance active in the FGF signaling pathway other than FGF2 is used, it is desirable to use it at a concentration that shows the same FGF signaling pathway promoting activity as FGF2 at the above-mentioned concentration. The FGF signaling pathway promoting activity of the added substance can be measured by, for example, a cell proliferation test using 3T3 cells, a kinase assay using fibroblast growth factor receptor 1, etc.

It is also preferable to add a substance that has the effect of maintaining the activity of FGF protein in the medium. Examples of substances that have the above-mentioned effect include heparin and heparan sulfate. Heparin is available as a sodium salt from, for example, Fujifilm Wako Pure Chemical Industries, Ltd. The concentration of heparin or heparan sulfate in the medium can be appropriately set within a range that can achieve the above-mentioned effects. The concentration of heparin sodium in the medium is usually about 1 ng/ml to about 100 mg/ml, preferably about 10 ng/ml to about 50 mg/ml, more preferably about 100 ng/ml to about 10 mg/ml, even more preferably about 500 ng/ml to about 1 mg/ml, and most preferably about 1 μg/ml to about 200 μg/ml. When heparan sulfate is used, it is preferable that the concentration has the same FGF protein protection activity as the above-mentioned concentration of heparin. For the purpose of maintaining the activity of FGF protein in a cell culture environment at 37°C or the like, it is also preferable to use modified FGF such as Thermostable FGF2 and FGF-1/FGF-2 chimera described in U.S. Pat. No. 8,772,460, or FGF sustained-release beads such as StemBeads FGF2 in which FGF2 is bound to a biodegradable polymer. Thermostable FGF2 is available, for example, from HumanZyme. FGF-1/FGF-2 chimera is available, for example, from Medical and Biological Laboratories. StemBeads FGF2 is available, for example, from StemCulture.

The BMP signaling pathway inhibitor is not limited as long as it can suppress signaling induced by BMP family proteins. The BMP signaling pathway inhibitor may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that inhibit BMP processing and extracellular secretion, substances that act directly on BMP (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors to BMP, and substances that inhibit physiological activity caused by signaling by BMP receptors. There are type I BMP receptors and type II BMP receptors. Known type I BMP receptors include BMPR1A, BMPR1B, and ACVR. Known type II BMP receptors include TGF-beta R-II, ActR-II, ActR-IIB, BMPR2, and MISR-II.

Proteins known as BMP signaling pathway inhibitors include, for example, Noggin, Chordin, Follistatin, Gremlin, Inhibin, Twisted Gastrulation, Coco, and secretory proteins belonging to the DAN family. These substances may be modified or fused proteins. Since a substance acting on the BMP signaling pathway is added to the culture medium in the above step (b), from the viewpoint of more effectively inhibiting the subsequent BMP signaling pathway, it is preferable that the BMP signaling pathway inhibitor in step (c) includes a substance that inhibits the signaling pathway subsequent to the secretion of BMP outside the cell, such as a substance that inhibits the binding between BMP receptor and BMP, or a substance that inhibits physiological activity resulting from signaling by the BMP receptor, and more preferably includes an inhibitor of type I BMP receptor.

Compounds well known to those skilled in the art can also be used as BMP signaling pathway inhibitors. Examples of BMP signaling pathway inhibitors include inhibitors of type I BMP receptors. Compounds having the above-mentioned activity include, for example, K02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol, 3-[6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]-phenol), Dorsomorphin ( 6-[4-[2-(1-Piperazinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidine, LDN-193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline dihydrochloride), LDN-212854 (5-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), LDN-214117 (1-(4-(6-methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine), ML347 (5-[6-(4-Methoxyphenyl l) pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), DMH2 (4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), M4K2163 dihydrochloride (1-[4-[5-(4-Fluoro-3,5-dimethoxyphenyl)-4-methyl-3-pyridinyl]phenyl]piperazine dihydrochloride) and derivatives thereof. These substances may be used alone or in combination of two or more. As the BMP signaling pathway inhibitor, for example, a substance having inhibitory activity against Alk2 described in ACS Omega 2021 6 (32), 20729-20734 can also be used. The inhibitory activity of the BMP signaling pathway inhibitor against Alk2 can be measured, for example, by measuring the inhibitory activity against Alk2 described in ACS Chem. Biol. 2013 June 21; 8(6): 1291-1302 and luciferase reporter assay using C2C12 cells.

The BMP signaling pathway inhibitor is preferably a type I BMP receptor inhibitor, more preferably at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1 and DMH2, and even more preferably K02288.

The concentration of the BMP signal transduction pathway inhibitor in the medium can be appropriately set within a range that can achieve the above-mentioned effects. From the viewpoint of the efficiency of formation of cells that constitute the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, when K02288 is used as the BMP signal transduction pathway inhibitor in step (c), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 500 nM to about 25 μM. When LDN-193189 is used as the BMP signal transduction pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 1 μM, and even more preferably about 100 nM to about 500 nM. When LDN-212854 is used as the BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When ML-347 is used as the BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 1 μM to about 25 μM. When DMH2 is used as a BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When a BMP signaling pathway inhibitor other than K02288 is used, it is desirable to use it at a concentration that shows the same BMP signaling pathway inhibitory activity as K02288 at the above concentration. The BMP signaling pathway inhibitory activity can be measured by, for example, the kinase assay, luciferase assay, etc. described in ACS Chem. Biol. 2013 Jun 21; 8(6): 1291-1302.

From the viewpoint of the efficiency of forming cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, the start time of step (c) is preferably from 0.5 hours to 240 hours after the addition of the substance acting on the BMP signaling pathway in step (b), more preferably from 6 hours to 192 hours, even more preferably from 9 hours to 120 hours, and particularly preferably from 12 hours to 72 hours. The addition times of the substance acting on the FGF signaling pathway and the substance inhibiting the BMP signaling pathway in step (c) may be simultaneous or different.

In one embodiment of step (c), a substance acting on the FGF signaling pathway may be added first, followed by the addition of a substance inhibiting the BMP signaling pathway. Specifically, for example, a substance acting on the FGF signaling pathway may be added first within 48 hours after the start of step (b), followed by the addition of a substance inhibiting the BMP signaling pathway within 96 hours. Alternatively, in another embodiment from the viewpoint of suppressing the proliferation of undifferentiated cells contaminating the cell population, a substance acting on the FGF signaling pathway and a substance inhibiting the BMP signaling pathway may be added, for example, within 120 hours after the start of step (2).

From the viewpoint of the efficiency of forming cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, it is preferable to start step (c) before non-neuroepithelial tissue is formed on the surface of the cell aggregate after the addition of the BMP signaling pathway active substance in step (b). In order to examine whether non-neuroepithelial tissue has been formed on the surface of the cell aggregate, it is possible to observe whether epithelial tissue has been formed on the surface of the cell aggregate using a microscope or the like, or to prepare slices of the cell aggregate and use techniques such as fluorescent immunostaining for markers expressed in non-neuroepithelial tissue, such as cytokeratin, E-Cadherin, and EpCAM.

When suspension culture is performed, the culture in step (c) may be performed, for example, until any one of cells of the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium differentiates in the cell aggregate. Differentiation into nasal epithelial cells can be confirmed, for example, by immunostaining of the cells. In this case, markers that can be used include cytokeratin 15, cytokeratin 18, and the marker genes of the various cells that make up the nasal epithelium described above. Alternatively, the culture in step (c) may be performed until olfactory nerve precursor cells differentiate. Differentiation into olfactory nerve precursor cells can be confirmed, for example, by immunostaining of the cells, and markers that can be used include Ebf2, NeuroD, Lhx2, Tuj1, NCAM, and Calretinin.

The medium used in step (c) is not particularly limited, but examples thereof include the medium used in step (1), as well as prepared media for culturing neural cells, such as a medium in which B27 Supplement is added to Neurobasal, a medium in which N2 Supplement is added to DMEM/F-12, and StemPro NSC SFM serum-free human neural stem cell culture medium (manufactured by Thermo Fisher Scientific).

In step (c), it is also preferable to continue to add additives used in the previous step, such as a Wnt signaling pathway inhibitor, a Wnt signaling pathway activating substance, a TGFβ signaling pathway inhibitor, a TAK1 inhibitor, etc. The Wnt signaling pathway inhibitor, Wnt signaling pathway activating substance, TGFβ signaling pathway inhibitor, and TAK1 inhibitor added in step (c) may be different from the substances used in the previous step, but are preferably the same. The concentration and type of additives can be adjusted as appropriate. The additives used in step (c) preferably include a Wnt signaling pathway inhibitor, and more preferably include a TGFβ signaling pathway inhibitor, a JNK signaling pathway inhibitor, and a TAK1 inhibitor. In one aspect of this embodiment, it is preferable to carry out step (c) in the presence of at least one selected from the group consisting of a TAK1 inhibitor, an FGF signaling pathway activating substance, and an EGF signaling pathway activating substance.

From the viewpoint of promoting the formation of the olfactory epithelium and the survival and proliferation of cells, it is also preferable that step (c) is performed in the presence of an EGF signaling pathway active substance. An EGF signaling pathway active substance is a substance that can enhance the signaling pathway mediated by epidermal growth factor (EGF). Examples of EGF signaling pathway active substances include EGFR ligand proteins such as EGF, TGF-α, AR (amphiregulin), EPG, HB-EGF (heparin-binding EGF-like growth factor), BTC (betacellulin), and EPR (epiregulin), ErbB3/4 (epidermal growth factor receptor, HRE) ligand proteins such as NRG (neuregulin) 1 to 4, and low molecular weight compounds such as alprenol and carvedilol. These substances may be used alone or in combination. EGF protein is available, for example, from Thermo Fisher Scientific and PrimeGene. TGF-α and HB-EGF proteins are available, for example, from R&D Systems. The EGF signaling pathway active agent preferably includes EGF or TGF-α.

The concentration of the EGF signaling pathway active substance in the medium can be appropriately set within a range that can achieve the above-mentioned effects. When EGF is used as an EGF signaling pathway active substance, it is usually used at a concentration of about 1 pg/ml to about 100 μg/ml, preferably about 10 pg/ml to about 50 μg/ml, more preferably about 100 pg/ml to about 10 μg/ml, even more preferably about 500 pg/ml to about 1 μg/ml, and most preferably about 1 ng/ml to about 200 ng/ml. When an EGF signaling pathway active substance other than EGF is used, it is desirable to use it at a concentration that shows the same EGF signaling pathway promoting activity as EGF at the above concentration. The activity as an EGF signaling pathway active substance can be measured by a method such as the cell proliferation assay of Balb/3T3 cells described in Science 223:1079.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, and promoting cell survival and proliferation, step (c) can also be performed in the presence of a substance acting on the Shh signaling pathway. As the substance acting on the Shh signaling pathway, those described in step (i) above can be used. The substance acting on the Shh signaling pathway in step (c) may be the same as or different from that used in step (i), but is preferably the same substance. The substance acting on the Shh signaling pathway in step (c) preferably includes at least one selected from the group consisting of SAG, Purmorphamine, and GSA-10, and more preferably includes SAG. The concentration of the substance acting on the Shh signaling pathway in the medium can be appropriately set within a range in which the above-mentioned effects can be achieved. In step (c), SAG is usually used at a concentration of about 1 nM to about 10 μM, preferably about 10 nM to about 7 μM, more preferably about 50 nM to about 5 μM, even more preferably about 100 nM to about 3 μM, particularly preferably about 300 nM to about 2 μM, and most preferably about 500 nM to about 1.5 μM. In addition, when a substance acting on the Shh signaling pathway other than SAG is used, it is desirable to use it at a concentration that exhibits Shh signaling promoting activity equivalent to that of SAG at the above concentration.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable that step (c) is carried out in the presence of a substance acting on the retinoic acid signaling pathway. Examples of substances acting on the retinoic acid signaling pathway include substances that bind to retinoic acid receptors (RAR) or retinoid X receptors (RXR) and activate downstream transcription. Examples of compounds having the above-mentioned effects include all-trans retinoic acid, isotretinoin, 9-cis retinoic acid, TTNPB (4-[(E)-2-[(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalene)-2-yl]-1-propenyl]benzoic acid), Ch55 (4-[(E)-3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid), acid), EC19 (3-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethyl]benzoic acid), EC23 (4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethyl)-benzoic acid), Phenretinide (4-hydroxyphenylretinamide), Acitetin ((all-e)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen), Trifarotene, Adapalene, AC 261066 (4-[4-(2-Butoxyethoxy-)-5-methyl-2-thiazolyl]-2-fluorobenzoic acid), AC 55649 (4-N-Octylbiphenyl-4-carboxylic acid), AM 580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid), AM80 (4-[(5,5,8,8-Tetramethyl-6,7-dihydronaphthalen-2-yl)carbamoyl]benzoic acid), BMS 753 (4-[[(2,3-Dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl)carbonyl]amino]benzoic acid), BMS 961 (3-Fluoro-4-[(r)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic acid), CD1530 (4-(6-Hydroxy-7-triclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic acid), CD2314 (5-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)-3-thiophenecarboxylic acid acid), CD437 (2-naphthalenecarboxylic acid, 6-(4-hydroxy-3-triclo(3.3.1.1(3,7))dec-1-ylphenyl), CD271 (6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthalene carboxylic acid) and derivatives thereof. These substances may be used alone or in combination.

The retinoic acid pathway acting substance in step (c) preferably includes EC23. The concentration of the retinoic acid pathway acting substance in the medium is not particularly limited as long as it is within a range in which the above-mentioned effects can be achieved. When EC23 is used as the retinoic acid pathway acting substance, the concentration of EC23 is, for example, about 10 pM to about 30 μM, preferably about 100 pM to about 20 μM, more preferably about 10 nM to about 10 μM, and even more preferably about 100 nM to about 5 μM. When a retinoic acid pathway acting substance other than EC23 is used, it is desirable to use it at a concentration that exhibits the same retinoic acid pathway acting activity as EC23 at the above concentration.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable to carry out step (c) in the presence of a Notch signaling pathway inhibitor. In the present invention, the Notch signaling pathway refers to a signaling pathway that is activated by direct interaction between the Notch protein, which is a receptor expressed on the cell membrane, and the Notch ligand (Delta, Jagged, etc.) expressed on the membrane of an adjacent cell. In cells to which the Notch signal is transmitted, the Notch protein is processed in stages, and the intracellular domain cut out on the membrane is transported into the nucleus to control the expression of downstream genes.

Examples of such substances include loss-of-function Notch receptors and their ligands, substances that inhibit Notch processing (S1 cleavage), substances that inhibit the glycosylation of Notch and Notch ligands, substances that inhibit cell membrane translocation, substances that inhibit the processing (S2 cleavage, S3 cleavage) of the intracellular domain of Notch (NICD) (gamma secretase inhibitors), substances that degrade NICD, and substances that inhibit NICD-dependent transcription.

Compounds well known to those skilled in the art can also be used as Notch signaling pathway inhibitors. Examples of compounds that have activity as Notch signaling pathway inhibitors include DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), DBZ ((2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzozepin-7-yl]propanamide), MDL28170 (benzyl N-[(2S)-3-methyl-1-oxo-1-[[(2S)-1-oxo-3-phenylpropan-2-yl]amino]butan-2-yl]carbamate, FLI-06 (cyclohexyl 2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-1,4,6,8-tetrahydroquinone-3-carboxylate), L-685,458 (tert-butyl Examples of the inhibitor include N-[6-[[1-[(1-amino-1-oxo-3-phenylpropan-2-yl)amino]-4-methyl-1-oxopentan-2-yl]amino]-5-benzyl-3-hydroxy-6-oxo-1-phenylhexan-2-yl]carbamate, CB-103 (6-(4-tert-butylphenoxy)pyridin-3-amine) and derivatives thereof, as well as substances described in Onco Targets Ther. 2013;6:943-955. The Notch signaling pathway inhibitor preferably includes DAPT.

The concentration of the Notch signaling inhibitor in the medium is not particularly limited as long as it is within a range in which the above-mentioned effects can be achieved. For example, when DAPT is used as the Notch signaling pathway inhibitor, the concentration of DAPT is, for example, about 100 pM to about 50 μM, preferably about 1 nM to about 30 μM, more preferably about 100 nM to about 20 μM, and even more preferably about 1 μM to about 10 μM. When a Notch signaling pathway inhibitor other than DAPT is used, it is desirable to use it at a concentration that exhibits Notch signaling pathway inhibitory activity equivalent to that of DAPT at the above-mentioned concentration.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable to carry out step (c) and the subsequent steps in the presence of a Hippo/YAP signaling pathway regulator. In the present invention, the Hippo/YAP signaling pathway is a signaling pathway involved in cell proliferation or apoptosis, control of self-renewal in stem cells, organ size regulation, etc., and refers to a kinase cascade composed of Mst1/2 or LATS1/2, etc., and a signaling pathway controlled by the transcription factor YAP/TAZ. For example, when attempting to proliferate cells and maintain the undifferentiated state of stem cells, a person skilled in the art can add a substance that inhibits the Hippo pathway and has the effect of promoting dephosphorylation and nuclear translocation of YAP/TAZ protein, a substance that promotes transcription induced by YAP/TAZ, etc. When promoting cell differentiation, a person skilled in the art can do so by adding a substance that activates the Hippo pathway and promotes phosphorylation of YAP/TAZ protein and degradation by the ubiquitin-proteosome system, or a substance that suppresses transcription induced by YAP/TAZ.

Hippo pathway inhibitors are not particularly limited as long as they inhibit the upstream kinase cascade or enhance the transcription or cellular response of downstream target genes induced by YAP/TAZ.

The Hippo pathway activator is not particularly limited as long as it suppresses the transcription or cellular response of downstream genes induced by YAP/TAZ. The Hippo pathway activator may be any of a nucleic acid, a protein, and a low molecular weight organic compound. Examples of the Hippo pathway activator include TEAD inhibitors, YAP-TEAD binding inhibitors, AMPK activators, YAP/TAZ transcription activity inhibitors, and pseudoactivators of adhesion stimuli between cells and to the extracellular matrix. Examples of substances having the above-mentioned effects include Verteporfin (3-[(23S,24R)-14-ethyl-22,23-bis(methoxycarbonyl)-5-(3-methoxy-3-oxopropyl)-4,10,15,24-tetramethyl-25,26,27,28-tetrazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-9-yl]propanoic acid; 3-[(23S,24R)-14-ethyl-22,23-bis(methoxycarbonyl)-9-(3-methoxy-3-oxopropyl)-4,10,15,24-tetramethyl-25,26,27,28-tetrazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-5-yl]propanoic acid), AICAR (5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide), K-975 (N-(3-(4-chlorophenoxy)-4-methylphenyl)acrylate), MYF-01-37 (1-[3-methyl-3-[3-(trifluoromethyl)anilino]pyrrolidine-1-yl]prop-2- en-1-one), TED-347 (2-chloro-1-[2-[3-(trifluoromethyl)anilino]phenyl]ethane), VT107 (N-[(1S)-1-(6-aminopyridin-2-yl)ethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide), YAP-TEAD-IN-1 ((4S)-4-[[(2S)-1-[(2S)-1-[(2S)-2-[[(3S,6S,9S,1 2S,15S,24S,27S,30S,33S)-15-[[(2S)-2-[[(2S)-1-[(2S)-2-acetamido-3-methylbutanoyl]pyrrolidine-2-carbonyl]amino]-3-(3-chlorophenyl)propanoyl]amino]-6-(4-aminobutyl)-24-benzyl-3-butyl-9-(3-carbamimidamidopropyl)-27-(hydroxymethyl)-30-methyl -12-(2-methylpropyl)-2,5,8,11,14,23,26,29,32-nonaoxo-18,19-dithia-1,4,7,10,13,22,25,28,31-nonazabicyclo[31.3.0]hexatriacontane-21-carbonyl]amino]-6-aminohexanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-5-amino-5-oxopentanoic acid), Super-TDU ((4S)-5-amino-4-[[(2S)-1-[(2S)-1-[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S,3S)-2-[2-[ (3S)-6-amino-1-[(2S)-1-[(2S)-2-[[(2S)-4-amino-2-[[(2R)-2-[[(2S,3S)-2-[[(2S)-6-amino-2-[[(2S)-1-[(2S)-4-amino-2-[[2-[[(2S)-2-[[2-[[2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[2 -[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-3-carboxy-2-[[(2S)-3-carboxy-2-[[(2S)-2-[2-[(2S)-1-hydroxy-3-oxopropan-2-yl]hydrazinyl]-3-methylbutanoyl]amino]propanoyl]amino]propane oil]amino]-3-(1H-imidazoI-5-yl)propanoyl]amino]-3-phenylpropanoyl]amino]propanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-hydroxyb utanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-4-oxobutanoyl]pyr rolidine-2-carbonyl]amino]hexanoyl]amino]-3-hydroxybutanoyl]amino]propanoyl]amino]-4-oxobutanoyl]amino]-3-methylbutanoyl]pyrrolidine-2-yl]-1,2,6-trioxohexan-3-yl]hydrazinyl]-3-hydroxybutanoyl]amino]-3-meth hylbutanoyl]pyrrolidine-2-carbonyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-4-methylpentanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-4-methylpentanoyl]pyr pyrrolidine-2-carbonyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoic acid) and derivatives thereof.

The fact that the above substances have activity as Hippo pathway inhibitors or Hippo pathway activators is described, for example, in Current Drug Targets, 2017, 18.4: 447-454, and is known to those skilled in the art. The substances described in the above literature can also be used as Hippo pathway inhibitors or Hippo pathway activators.

As described above, from the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable that step (c) is performed in the presence of a substance acting on the Wnt signaling pathway. The concentration of the substance acting on the Wnt signaling pathway in the medium can be appropriately set within a range in which the above-mentioned effects can be achieved. From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, when CHIR99021 is used as a substance acting on the Wnt signaling pathway, it is usually used at a concentration of about 10 pM to about 10 mM, preferably at a concentration of about 100 pM to about 1 mM, more preferably at a concentration of about 1 nM to about 100 μM, even more preferably at a concentration of about 10 nM to about 30 μM, and most preferably at a concentration of about 100 nM to about 3 μM. In addition, when using a substance acting on the Wnt signaling pathway other than CHIR99021, it is desirable to use it at a concentration that exhibits the same Wnt signaling pathway promoting activity as CHIR99021 at the above concentration. The Wnt signaling pathway promoting activity can be determined, for example, using a Topflash reporter assay using HEK293 cells.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable to carry out step (c) and the subsequent steps in the presence of an activin/TGF-β signaling pathway active substance. An activin/TGF-β signaling pathway active substance is a substance that can enhance the signaling pathway mediated by activin/TGF-β. Examples of activin/TGF-β signaling pathway active substances include recombinant proteins such as activin and TGF-β, and substances that activate the activin/TGF-β signaling pathway, such as alantolactone. These substances may be used alone or in combination. Examples of activin/TGF-β signaling pathway active substances from the viewpoint of biological activity include substances that have the ability to induce hemoglobin expression in cells such as human chronic myeloid leukemia-derived cell line K562. When human recombinant activin (manufactured by R&D Systems) is used as the activin/TGF-β signaling pathway active substance, it is usually used at a concentration in the range of 1 pg/ml to 1 mg/ml, preferably at a concentration in the range of 100 pg/ml to 10 μg/ml, more preferably at a concentration in the range of 1 ng/ml to 100 ng/ml. When an activin/TGF-β signaling pathway active substance other than human recombinant activin is used, it may be added at a concentration that shows the same physiological activity as the above-mentioned human recombinant activin addition concentration, which is well known to those skilled in the art. When an activin/TGF-β signaling pathway inhibitor is added in a step prior to step (c), for example, it is preferable to recover the cell aggregates from the culture vessel, wash them multiple times with a medium, buffer, etc. that does not contain an activin/TGF-β signaling pathway inhibitor, transfer the cell aggregates to a new culture vessel, and then add a medium containing an activin/TGF-β signaling pathway active substance.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable to carry out step (c) and the subsequent steps in the presence of an FGF signaling pathway inhibitor. The FGF signaling pathway inhibitor is not particularly limited as long as it is capable of suppressing signaling mediated by FGF, and may be any of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include substances that directly bind to and trap FGF, substances that inhibit the kinase activity of FGF receptors, substances that inhibit intracellular signaling activated by FGF, and substances that suppress gene expression induced by FGF stimulation. Examples of substances having the above-mentioned effects include anti-FGF antibodies, GP369 (anti-FGFR2 antibodies), SU5402 (3-[4-Methyl-2-[(2-oxo-1H-indol-3-ylidene)methyl]-1H-pyrrol-3-yl]propanoic acid, and the like. acid), BGJ398 (3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-[6-[[4-(4-ethylpiperazin-1-yl)phenyl]amino]pyrimidin-4-yl]-1-methylurea), PD173074 (1-(Tert-Butyl)-3-(2-((4-(diethylamino)butyl) amino)-6-(3,5-dimethylphenyl)pyrido[2,3-d]pyrimidin-7-yl)urea), AZD4547 (rel-N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-1H-pyrazol-3-yl]-4-[(3R,5S)-3,5-dimethyl-1-piperazinyl]benzamide), Erdafitinib (N-(3,5-dimethylphenyl)-N-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine), BYL719 ((2S)-N1-[4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimeth 2-Methyl-1-[[2-Methyl-3-(trifluoromethyl)phenyl]Methyl]-6-(4-Morpholinyl)-1H-benzimidazole-4-carboxylate acid), Taselisib (2-Methyl-2-[4-[2-(5-Methyl-2-propan-2-yl-1,2,4-triazol-3-yl)-5,6-dihydroiMidazo[1,2-d][1,4]benzoxazepin-9-yl]pyrazol-1-yl]propanamide), Capivasertib (4-Amino-N-[(1S)-1-(4-chlorophenyl)-1,2-dihydro-1,2-dihydro-1,4 ... orophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinecarboxamide, Sapanisertib (2-(((1R,4R)-4-((((4-chlorophenyl)(phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetic acid), Dabrafenib (N-[3-[5-(2-amino-4-pyrimidinyl)-2-(tert-butyl)-4-thiazolyl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide), Binimetinib (5-[(4-bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl -1H-benzimidazole-6-carboxamide), Trametinib (N-[3-[3-Cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-3,4,6,7-tetrahydro-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl]acetamide) and their derivatives.

The concentration of the FGF signaling pathway inhibitor can be appropriately set within a range that allows efficient production of cells constituting upper respiratory tract tissue, olfactory epithelium, and nasal epithelium. When SU5402 is used as the FGF signaling pathway inhibitor, the concentration of the FGF signaling pathway inhibitor in step (c) is usually 1 nM to 100 mM, preferably 10 nM to 10 mM, and more preferably 100 nM to 1 mM. When PD173074 is used as the FGF signaling pathway inhibitor, the concentration of the FGF signaling pathway inhibitor in step (c) is 1 nM to 100 mM, preferably 10 nM to 10 mM, and more preferably 100 nM to 1 mM. When an FGF signaling pathway inhibitor other than the above is used, it is preferably used at a concentration that exhibits FGF signaling pathway inhibitory activity equivalent to that of SU5402 at the above concentration. When an FGF signaling pathway active substance has been added in step (c) or an earlier step, an FGF signaling pathway inhibitor may be added directly, or the cells may be collected in another sterilized container (e.g., a 15 ml tube, a petri dish for suspension culture, etc.) and transferred after washing with medium or physiological saline. The culture container used for the culture after the transfer may be the same as that used up to step (c) (e.g., a 96-well plate), or may be a different container (e.g., a dish for suspension culture, etc.).

The timing of adding the FGF signaling pathway inhibitor in step (c) is, for example, 24 hours or more, preferably 48 hours or more, more preferably 72 hours or more, and even more preferably 96 hours or more after the addition of the FGF signaling pathway acting substance or the BMP signaling pathway inhibitor. Another embodiment of the timing of adding the FGF signaling pathway inhibitor is after an epithelial-like structure is formed on the surface of the cell mass after the addition of the FGF signaling pathway acting substance or the BMP signaling pathway inhibitor.

From the viewpoint of regulating the differentiation tendency of cells in upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue, it is also preferable to perform step (c) and the subsequent steps again in the presence of a BMP signaling pathway active substance. The type of BMP signaling pathway active substance at the time of re-addition in step (c) may be the same or different from that added in step (b), but is preferably the same. The concentration of the BMP signaling pathway active substance at the time of re-addition in step (c) can be appropriately set within a range in which the above-mentioned effect can be achieved. When BMP4 is used as the BMP signaling pathway active substance, it is usually used at a concentration of about 1 pM to about 100 nM, preferably at a concentration of about 10 pM to about 50 nM, more preferably at a concentration of about 25 pM to about 25 nM, even more preferably at a concentration of about 25 pM to about 5 nM, particularly preferably at a concentration of about 100 pM to about 5 nM, and most preferably at a concentration of about 500 pM to about 2 nM. In the case where a BMP signaling pathway inhibitor has been added in step (c), the cells may be collected in a separate sterilized container (e.g., a 15 ml tube, a petri dish for suspension culture), washed with medium or saline, and then transferred. The culture container used for the culture after the transfer may be the same as that used up to step (c) (e.g., a 96-well plate), or may be a separate container (e.g., a dish for suspension culture).

When performing suspension culture, the cell aggregates can be cultured in a viscous medium containing a thickener from the viewpoints of suppressing adhesion between cell aggregates in step (c) and subsequent steps, protecting the cells, and improving the production efficiency of upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue by simulating the physical environment of the living body.

There are no particular limitations on the means for imparting viscosity to the medium, and for example, viscosity can be imparted by adding a substance generally known as a thickening agent to the medium at an appropriate concentration. Any thickening agent can be used as long as it can impart the above-mentioned appropriate viscosity to the medium and does not adversely affect cells (is not cytotoxic) in the concentration range in which it can impart the viscosity. Examples of thickening agents include pectin, guar gum, xanthan gum, tamarind gum, carrageenan, locust bean gum, gellan gum, dextrin, diutan gum, starch, tara gum, alginic acid, curdlan, sodium caseinate, carob bean gum, chitin, chitosan, glucosamine, pullulan, dietary fiber and chemically modified substances or derivatives thereof, polysaccharides such as cellulose and agarose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl ... Examples of the thickener include polysaccharide ethers such as cellulose, hydroxypropyl ethyl cellulose, ethyl hydroxyethyl cellulose, dihydroxypropyl cellulose, and hydroxyethyl hydroxypropyl cellulose, synthetic polymers such as polyacrylamide, polyethylene oxide, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, polyethyleneimine polyvinyl methyl ether, polyvinyl alcohol, polyacrylic acid and maleic acid copolymers, and biopolymers such as collagen, gelatin, hyaluronic acid, and dextran, as well as artificial polymers that mimic them (e.g., elastin-like peptides, etc.). Preferably, these thickeners may be used alone or as a mixture of several types of thickeners. Also, copolymers of water-soluble polymers used as thickeners may be used. Preferably, methylcellulose, polyethylene glycol, polyvinylpyrrolidone, carboxymethylcellulose, or a mixture thereof, more preferably methylcellulose, may be used.

The viscous medium used is one that has a viscosity of 100 mPa·S or more, preferably 250 mPa·S or more, more preferably 500 mPa·S or more, and even more preferably 1000 mPa·S or more under 37°C conditions, which is a suitable culture environment. The upper limit of the viscosity is not particularly limited, but may be, for example, 1000 Pa·S or less. The viscosity can be measured, for example, using a capillary viscometer, a falling ball viscometer, a rotational viscometer, a vibration viscometer, or the like, based on the Japanese Industrial Standards (JIS).

The concentration of the thickener added to the medium can be adjusted as appropriate as long as it can impart viscosity and is not damaging to cells. When methylcellulose (4000 cP) is used as the thickener, it is usually used at a concentration of 0.1% to 10% by mass, preferably 0.3% to 7.5% by mass, and more preferably 0.5% to 5% by mass. "% by mass" can also be expressed as "w/w%".

When suspension culture is performed, a substance having a surfactant effect can be added from the viewpoint of suppressing adhesion or aggregation between cell aggregates in step (c) and the subsequent steps, from the viewpoint of protecting cells from physical stimuli such as shear force, and from the viewpoint of improving the production efficiency of upper respiratory tract tissue, olfactory epithelium, and nasal epithelium-like tissue by simulating the physical environment of a living body. Examples of such substances include fatty acids such as arachidonic acid, linoleic acid, linoleic acid, myristic acid, oleic acid, palmitic acid, palmitoyl acid, and stearic acid, surfactants such as Pluronic F-68 and Tween 80, and cholesterol.

When performing suspension culture, it is also desirable to take measures to prevent adhesion between the formed cell clusters from the viewpoint of making the size of the cell clusters produced in step (c) and the subsequent steps uniform and controlling the efficiency of differentiation induction. Means for achieving the above include, but are not limited to, the addition of lysophospholipids described in WO 2016/121737, the addition of a thrombin receptor agonist described in WO 2019/131941, and the addition of an inhibitor of cell adhesion described in WO 2016/121840. Another embodiment for preventing adhesion between cell clusters includes the addition of an inhibitor against the Rho signaling pathway that controls cell adhesion. The Rho signaling pathway inhibitor that prevents adhesion between cell clusters is preferably a ROCK inhibitor. When adding ROCK inhibitors Y-27632 or Chroman 1 for the purpose of preventing adhesion between cell clusters, they can be added under the same conditions as when used as a cell protective substance. When adding the ROCK inhibitor Y-27632, it is usually added to the culture environment at a concentration of 10 nM to 10 mM, preferably 100 nM to 1 mM, and more preferably 1 μM to 100 μM. When adding the ROCK inhibitor Chroman 1, it is usually added to the culture environment at a concentration of 10 pM to 1 mM, preferably 100 pM to 100 μM, and more preferably 1 nM to 10 μM.

When suspension culture is performed up to step (b), it is also preferable to perform adhesion culture of the formed cell aggregates from the viewpoint of promoting differentiation and maturation of cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium formed in step (c) and the subsequent steps. When transitioning from suspension culture to adhesion culture, the cell aggregates may be left as they are on a cell culture dish to allow them to adhere, or a cutting treatment, cell dispersion treatment, or the like may be performed to prepare a suspension of smaller cell aggregates or isolated cells, which are then allowed to adhere after seeding.

As the cultureware for adhering cells, either the flat cell culture dish described above or a thin-film cell cultureware having material permeability such as a Transwell can be used. The cultureware may be coated with a basement membrane preparation, an extracellular matrix such as laminin, or a synthetic cell adhesion molecule such as poly-D-lysine to promote cell adhesion.

In the step (c) and the following steps, from the viewpoint of promoting the survival and differentiation/maturation of cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, it is also preferable to culture the cell aggregates by the air-liquid interface culture method. The air-liquid interface culture method refers to culture under conditions in which at least one surface of the cells or tissue is exposed to the air or is located very close to the air. In order to carry out the air-liquid interface culture method, for example, a method in which the cells or tissues are cultured on a porous membrane or in a culture insert, the culture medium in the insert (inner cavity side) is removed, and the cells or tissues are cultured only in the culture medium in the well outside the insert, a method in which a hydrogel such as agarose is placed in a culture dish, and the cells or tissues are placed on it and cultured under conditions in which they are exposed to the medium in the dish but do not dry out, and a method in which the cell aggregates are embedded in agarose, etc., sliced thinly with a vibrating blade microtome, etc., and the thin slices are cultured in a cell insert, etc. can be mentioned. For the purpose of reducing the toxicity of exposure to the atmosphere, mainly due to oxygen, an oxygen-permeable membrane or plate such as PDMS can be placed on the cells or tissue to adjust the exposure conditions. The above-mentioned gas-liquid interface culture method can be carried out with reference to, for example, Respiratory research 18.1 (2017): 195, Sci Rep 6, 21472, Nat Neurosci. 2019 Apr; 22 (4): 669-679, etc.

In order to promote the survival and differentiation/maturation of cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, step (c) and the subsequent steps may include a step of embedding the cell aggregates in a gel and culturing them. Examples of gels include gels using agarose, methylcellulose, collagen, Matrigel, etc., and it is preferable to use Matrigel.

In the step (c) and the subsequent steps of embedding cells in a gel and culturing the cells, the cell aggregates may be embedded as they are, or the cells may be dispersed and isolated and then seeded in the gel. Specific cell tumors such as basal cells may be separated using a cell sorter or the like and then seeded. As a further embodiment of the culture method of embedding cells in a gel, co-culture with cells other than upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, such as fibroblasts, mesenchymal cells, and vascular cells, may also be performed. The gel-embedded culture described above can be performed with reference to, for example, Nature 501, 373-379 (2013), Nature, 499, 481-484 (2013), Nat Protoc 14, 518-540 (2019), Genes 2020, 11, 603, etc.

In step (c) and the subsequent steps, it is also preferable to carry out a culture method in which the cells are physically shaken in order to improve the supply of nutrients and oxygen to the cells, improve material exchange, and suppress fusion between cell aggregates. Such culture methods include methods other than stationary culture, such as shaking culture, rotary culture, and agitation culture. The means for carrying out shaking culture, rotary culture, agitation culture, and the like are not particularly limited, but can be carried out, for example, by placing the culture equipment in which the cells are cultured on a rotator, shaker, or the like, or by placing the cells in an environment in which a stirrer or the like is rotating. Those skilled in the art can appropriately set parameters such as the speed of shaking culture, rotary culture, and agitation culture within a range that does not cause damage to the cells. For example, when shaking culture is carried out using a 3D shaker with a wave-type shaking (e.g., Mini-Shaker 3D, manufactured by Biosan), the shaking speed range can be set, for example, to 5 to 60 rpm, preferably 5 to 40 rpm, and more preferably 5 to 20 rpm. When shaking culture is performed using a reciprocating shaker (e.g., NS-LR, manufactured by AS ONE), the shaking speed range can be set, for example, in the range of 15 to 60 rpm, preferably 15 to 50 rpm, and more preferably 15 to 45 rpm. When shaking culture is performed using a seesaw shaker (e.g., NS-S, manufactured by AS ONE), the shaking speed range can be set, for example, in the range of 5 to 50 rpm, preferably 5 to 40 rpm, and more preferably 5 to 30 rpm. When culturing cell aggregates by stirring culture or rotary culture, for example, a spinner flask (e.g., 3152, manufactured by Corning) can be placed on a magnetic stirrer, and the culture can be performed at a rotation speed at which the cell aggregates do not settle visually. Culture can also be performed using a three-dimensional rotary suspension culture device (e.g., CellPet CUBE, manufactured by J-TEC; Clinostar, manufactured by Celvivo). From the viewpoint of suppressing physical damage to cells such as friction, it is also preferable to perform shaking culture, rotary culture, or stirring culture of the cell aggregates embedded in the gel. As another embodiment for suppressing physical damage to cells such as friction, it is also preferable to perform a culture method in which the cells are physically shaken intermittently. By combining the above-mentioned shaker, stirrer, etc. with a time switch, intermittent timer, etc., shaking culture, rotary culture, stirring culture, etc. can be performed intermittently. The time conditions of intermittent operation can be set appropriately. When performing intermittent operation using an intermittent timer (e.g., FT-011, manufactured by Tokyo Kikai Glass Co., Ltd.) and a reciprocating shaker, the time conditions of intermittent operation can be set, for example, in the range of 15 seconds to 12 hours of rest and 1 second to 60 minutes of operation, preferably in the range of 30 seconds to 6 hours of rest and 1 second to 10 minutes of operation, and more preferably in the range of 60 seconds to 2 hours of rest and 2 seconds to 5 minutes of operation.

In step (c) and the subsequent steps, it is also preferable to add a further growth factor to the medium from the viewpoint of suppressing cell death and promoting cell proliferation. The type of growth factor to be added is not particularly limited as long as the above-mentioned object can be achieved. Examples of growth factors used for such purposes include insulin-like growth factor (IGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin 4/5, ciliary neurotrophic factor (CNTF), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), hepatocyte growth factor (HGF), and others. These growth factors may be added at concentrations that achieve the above objectives.

In the step (c) and the subsequent steps, it is also preferable to add a platelet-derived growth factor (PDGF) or other platelet-derived growth factor receptor agonist to the medium in order to suppress cell death and promote cell proliferation. PDGF has four types of genes, A, B, C, and D, which form homodimers of AA, AB, BB, CC, and DD or heterodimers of specific combinations to function as ligands. PDGF receptor has two types of genes, α and β, which form homodimers of αα, αβ, and ββ combinations to function as receptors. Of these, PDGFRβ is highly expressed in non-neural ectoderm including placodes. The platelet-derived growth factor receptor agonist used in the present invention preferably has an action on PDGFRββ or PDGFRαβ. The above-mentioned platelet-derived growth factor receptor agonist more preferably includes at least one selected from the group consisting of PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD, and even more preferably includes at least one selected from the group consisting of PDGF-BB and PDGF-CC. PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD are available as recombinant proteins from R&D Systems, GenScript, and the like.

In step (c) and the subsequent steps, it is also preferable to culture the cells in a high-oxygen atmosphere from the viewpoint of suppressing cell death and promoting cell proliferation. High-oxygen conditions during the culture process can be achieved, for example, by connecting an oxygen tank to the incubator in which the cells are cultured and artificially supplying oxygen. The oxygen concentration for this purpose is usually 25% to 80% by volume, more preferably 30% to 60% by volume.

In step (c) and the subsequent steps, culture equipment with high gas exchange efficiency can be used in order to increase the amount of oxygen supplied to the medium in which the cell aggregates are cultured. Examples of such culture equipment include cell culture dishes, Lumox dishes (manufactured by Sarstedt Co., Ltd.) with a gas-permeable film on the bottom of the plate, and VECELL 96-well plates (manufactured by Vecel Co., Ltd.). It is also preferable to use it in combination with the above-mentioned culture under high oxygen concentration conditions.

In step (c) and the following steps, a cell protective agent may be added to the medium from the viewpoint of maintaining the structure of the epithelial tissue in the cell aggregate. Examples of the cell protective agent used in step (c) and the following steps include the above-mentioned FGF signaling pathway active substances, heparin, ROCK inhibitors, basement membrane preparations, myosin inhibitors, polyamines, ISR inhibitors, caspase inhibitors, serum, and serum substitutes. Examples of myosin inhibitors include blebbistatin, which is an inhibitor of nonmuscle myosin II ATPase, and ML-7, ML-9, W-7, MLCK inhibitor peptide 18, and derivatives thereof, which are inhibitors of myosin light chain kinase (MLCK). The cell protective agent added may be the same as or different from the one added in step (a), but is preferably the same. A preferred cell protective agent is a ROCK inhibitor. In step (c) and the subsequent steps, when the ROCK inhibitor Y-27632 is added as a cell protective agent, it is usually added to the culture environment at a concentration of 10 nM to 10 mM, preferably at a concentration of 100 nM to 1 mM, and more preferably at a concentration of 1 μM to 100 μM. When the ROCK inhibitor Chroman 1 is added, it is usually added to the culture environment at a concentration of 10 pM to 1 mM, preferably at a concentration of 100 pM to 100 μM, and more preferably at a concentration of 1 nM to 10 μM. When the nonmuscle myosin II ATPase inhibitor Blebbistatin is added as a cell protective agent, it is usually added to the culture environment at a concentration of 10 nM to 10 mM, preferably at a concentration of 100 nM to 1 mM, and more preferably at a concentration of 1 μM to 100 μM.

In step (c) and the subsequent steps, a substance other than a cell protective agent that has the effect of maintaining the structure of epithelial tissue can also be added. Examples of such substances include substances that promote cell adhesion, substances that promote the synthesis of basement membrane components, and substances that inhibit the decomposition of basement membrane components. The substance that promotes cell adhesion may promote cell-cell adhesion, cell-basement membrane adhesion, cell-culture equipment adhesion, or the like, or may promote the production of factors involved in cell adhesion. Examples of substances that promote cell adhesion include adhesamine, adhesamine-RGDS derivatives, pyrintegrin, biotin tripeptide-1, acetyl tetrapeptide-3, RGDS peptide, and derivatives thereof. Examples of substances that promote the synthesis of basement membrane components include ascorbic acid derivatives. Examples of ascorbic acid derivatives include sodium ascorbyl phosphate, magnesium ascorbyl phosphate, ascorbyl 2-glucoside, 3-O-ethyl ascorbic acid, ascorbyl tetrahexyldecanoate, ascorbyl palmitate, ascorbyl stearate, ascorbyl 2-phosphate-6 palmitate, and glyceryl octyl ascorbic acid. Examples of substances that inhibit the decomposition of basement membrane components include inhibitors of matrix metalloproteases and serine proteases. When ascorbic acid 2-phosphate, which is a type of ascorbic acid derivative that promotes the synthesis of basement membrane components, is added, it is usually added to the culture environment at a concentration of 10 μg/ml to 1000 μg/ml, preferably 30 μg/ml to 500 μg/ml, and more preferably 50 μg/ml to 300 μg/ml. When adding other ascorbic acid or ascorbic acid derivatives, etc., add them so that the molar equivalents are approximately the same as the above concentrations.

In order to promote the survival of cells constituting the upper respiratory tract tissue, the olfactory epithelium, and the nasal epithelium, it is also preferable to add a substance that has an effect of reducing oxidative stress in step (c) and the subsequent steps. Examples of substances having such activity include antioxidants, substances with free radical scavenging action, NADPH oxidase inhibitors, cyclooxygenase inhibitors, lipoxygenase (LOX) inhibitors, superoxide dismutase (SOD)-like substances, Nrf2 activators, etc. Substances having the above-mentioned activity include, for example, ascorbic acid, N-acetyl-L-cysteine, (±)-α-tocopherol acetate, apocynin (4'-hydroxy-3'-methoxyacetophenone), nicotinamide, taurine (2-aminoethanesulfonic acid), IM-93 (1-isopropyl-3-(1-methyl-1H-indole-3-yl)-4-(N,N-dimethyl-1,3-propanediamine)-1H-pyrrole-2,5H-dione), caffeic acid (3,4-dihydroxycinnamic acid), Acid), Celastrol (3-Hydroxy-24-nor-2-oxo-1(10),3,5,7-friedelatetraen-29-oic Acid; Tripterin), Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one), (-)-Epigallocatechin Gallate ((2R,3R)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-1[2H]-benzopyran-3,5,7-triol-3-(3,4,5-trihydroxybenzoate)), EUK-8 (N,N'-Bis(salicylideneamino)ethane-manganese(i)), Edaravon (3-Methyl-1-phenyl-2-pyrazolin-5-one), MnTBAP (Mn(III) tetrakis(4-benzoic acid)porphyrin Examples of suitable ascorbic acid-reducing agents include, but are not limited to, 1,2,3,4,5-trihydroxystilbene, 1,2,4,5-trihydroxystilbene, 1,2,3 ... N-acetyl-L-cysteine can be added to the medium at a concentration of, for example, 1 nM to 1 M, preferably 10 nM to 100 mM, more preferably 100 nM to 10 mM, and even more preferably 1 μM to 5 mM.

In order to promote the survival of cells constituting the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, it is also preferable to add an inhibitor of the stress-responsive signal transduction pathway (a substance that inhibits the intracellular signal transduction mechanism in response to stress) in step (c) and the subsequent steps. The stress-responsive MAP kinase pathway (stress-activated protein kinase: SAPK) is one of the main intracellular signal transduction mechanisms in response to stress. Examples of inhibitors of the stress-responsive MAP kinase pathway include MAP3K inhibitors, MAP2K inhibitors, ASK inhibitors, MEK inhibitors, Akt inhibitors, Rho family kinase inhibitors, JNK inhibitors, p38 inhibitors, MSK inhibitors, STAT inhibitors, NF-κB inhibitors, and CAMK inhibitors.

Examples of MEK inhibitors include Selumetinib (AZD6244, 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), Mirdametinib (PD03 25901, N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, trametinib (GSK1120212, N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)benzamide oanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1-yl]phenyl]acetamide), U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadien e), PD184352 (CI-1040, 2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide), PD98059 (2-(2-amino-3-methoxyphenyl)chromen-4-one), BIX 02189 (3-[N-[3-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-N,N-dimethyl-1H-indole-6-carboxamide), Pimasertib (AS-703026, N-[(2S)-2,3-dihydroxypropyl]-3- (2-fluoro-4-iodoanilino)pyridine-4-carboxamide), Pelitinib (EKB-569, (E)-N-[4-(3-chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide), BIX 02188 (3-[N-[3-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-1H-indole-6-carboxamide), TAK-733 (3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3-d]pyrimidine-4,7-dione), AZD8330 (2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxopyridine-3-carbamide), Binimetinib (MEK162,6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carbamide), SL-327 ((Z)-3-amino-3-(4-a minophenyl)sulfanyl-2-[2-(trifluoromethyl)phenyl]prop-2-ennitrile), refametinib (RDEA119, N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2S)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide), GDC-0623 (5-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2S)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide), doanilino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide, BI-847325 (3-[3-[N-[4-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-1H-indol-6-yl]-N-ethylprop-2-ynamide), RO5126766 (CH5126766,3-[[3-fluoro-2-(m [ethylsulfamoylamino)pyridin-4-yl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one), cobimetinib (GDC-0973, [3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1-yl]methaneone) and derivatives thereof.

Examples of p38 inhibitors include SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine), Doramapimod (BIRB) 796, 1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea), SB202190 (FHPI, 4-[4-(4-fluorophenyl)-5-pyridin-4-yl-1H-imidazol-2-yl]phenol), Ralimetinib Dimesylate (5-[2-tert-butyl-4-(4-fluorophenyl)-1H-imidazo-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5-b]pyridin-2-amine; methanesulfonic acid), VX-702 (6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide), PH-797804 (3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxopyridin-1-yl]-N,4-dimethylbenzom ide), Neflamapimod (VX-745, 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenyl)sulfanylpyrimido[1,6-b]pyridazin-6-one), TAK-715 (N-[4-[2-ethyl-4-(3-methylphenyl)-1,3-thiazol-5-yl]pyridin-2-yl]benzamide), PD 169316 (4-[4-(4-fluorophenyl)-2-(4-nitrophenyl)-1H-imidazo[ol-5-yl]pyridine), TA-02 (4-[2-(2-fluorophenyl)-4-(4-fluorophenyl)-1H-imidazo[ol-5-yl]pyridine), SD 0006 (1-[4-[3-(4-chlorophenyl)-4-pyrimidin-4-yl-1H-pyrazol-5-yl]piperidin-1-yl]-2-hydroxyethane), Pamapimod (6-(2,4-difluorophenoxy)-2-(1,5-dihydroxypentan-3-ylamino)-8-methylpyrido[2,3-d]pyrimidin-7-one), BMS-582949 (4-[5-(cyclopropylcarbamoyl)-2-methylanilino]-5-methyl-N-p propylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide, SB239063 (4-[4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazol-1-yl]cyclohexane-1-o l), Skepinone-L (13-(2,4-difluoroanilino)-5-[(2R)-2,3-dihydroxypropoxy]tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,12,14-hexaen-2-one), DBM 1285 (N-cyclopropyl-4-[4-(4-fluorophenyl)-2-piperidin-4-yl-1,3-thiazol-5-yl]pyrimidin-2-amine; dihydrochloride), SB 706504 (1-cyano-2-[2-[[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxopyrido[2,3-d]pyrimidin-2-yl]amino]ethyl]guanidine), SCIO 469 (2-[6-chloro-5-[(2R,5S)-4-[(4-fluorophenyl)methyl]-2,5-dimethylpiperazine-1-carbonyl]-1-methylindol-3-yl]-N,N-dimethyl-2-oxoacetamide), Pexmetinib (1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[[5-fluoro-2-[1-( 2-hydroxyethyl)indazol-5-yl]oxyphenyl]methyl]urea), UM-164 (2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-N-[2-methyl-5-[[3-(trifluoromethyl)benzoyl]amino]phenyl]-1,3-thiazole-5-carboxamide), p38 MAPK Inhibitor (4-(2,4-difluorophenyl)-8-(2-methylphenyl)-7-oxido-1,7-naphthyridin-7-ium), p38 MAP Kinase Inhibitor
III (4-[5-(4-fluorophenyl)-2-methylsulfanyl-1H-imidazoI-4-yl]-N-(1-phenylethyl)pyridin-2-amine), p38 MAP Kinase Inhibitor IV (3,4,6-trichloro-2-(2,3,5-trichloro-6-hydroxyphenyl)sulfonylphenol), CAY105571 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]-pyridine) and derivatives thereof.

Examples of JNK inhibitors include those described in step (a). The substance that inhibits the intracellular signal transduction mechanism for stress used in the present invention is preferably one or more selected from the group consisting of MEK inhibitors, p38 inhibitors, and JNK inhibitors. When SB203580 is used as the p38 inhibitor, it can be added to the medium at a concentration of usually 1 nM to 1 mM, preferably 10 nM to 100 μM, more preferably 100 nM to 10 μM, and even more preferably 500 nM to 5 μM. When PD0325901 is used as the MEK inhibitor, it can be added to the medium at a concentration of usually 1 nM to 1 mM, preferably 10 nM to 100 μM, more preferably 100 nM to 10 μM, and even more preferably 500 nM to 5 μM. When JNK-IN-8 is used as the JNK inhibitor, it can be added to the medium at the same concentration as described in step (a). When other MEK inhibitors, p38 inhibitors, or JNK inhibitors are used, they are preferably added at a concentration that has an inhibitory activity equivalent to the concentration of the inhibitor added. The inhibitory activity of the JNK inhibitor can be measured by, for example, the DELFIA assay, kinase assay, or other methods described in Proc Natl Acad Sci U S A. 2008 Oct 28; 105(43): 16809-16813.

In one aspect of this embodiment, it is preferable to carry out one or more of steps (a) to (c) in the presence of at least one substance selected from the group consisting of a TGFβ family signaling pathway inhibitor, a ROCK inhibitor, and a JNK signaling pathway inhibitor.

<2-2. Method for producing a sheet-shaped cell structure from a first cell population>
<Step (1)>
In step (1), a first cell population containing olfactory placode cells or olfactory epithelial stem cells is dispersed to obtain single cells. The single cells may be prepared as a cell suspension. The first cell population is preferably derived from pluripotent stem cells. For convenience, the state in which the cells contained in the first cell population become single cells is also referred to as the "first cell population."

The first cell population containing olfactory placode cells or olfactory epithelial stem cells subjected to step (1) may be suspended in a medium or attached to a culture vessel or the like, but is preferably suspended in a medium. The olfactory placode cells or olfactory epithelial stem cells contained in the first cell population preferably form tissue. The first cell population subjected to step (1) may be a cell aggregate, a cell mass, an embryoid body, a sphere, a spheroid, an organoid, or the like, but is preferably an organoid containing olfactory placode cells or olfactory epithelial stem cells. The above organoid can be produced, for example, from human pluripotent stem cells by carrying out the steps described in WO2020/039732 and WO2022/026238, or steps (i), (ii), (a), (b), and (c) described in the present application.

The olfactory placode cells or olfactory epithelium stem cells contained in the first cell population express olfactory epithelium and olfactory epithelium placode marker genes. Examples of olfactory epithelium and olfactory epithelium placode marker genes include Six1, Six4, Dlx5, Eya2, Pax6, Otx2, FoxG1 (also known as Bf1), Sox2, Pou2f1, Sp8, Chd7, N-Cadherin, E-Cadherin, EpCAM, CK18, and PDGFRβ.

In one aspect of this embodiment, it is preferable that at least 20% of the first cell population is cells derived from the ectoderm, and the cells derived from the ectoderm contain cells expressing at least one olfactory placode cell marker or olfactory epithelium stem cell marker selected from the group consisting of Six1, Sp8, Otx2, cytokeratin, and E-cadherin. Examples of "cells derived from the ectoderm" other than cells expressing olfactory placode cell markers or olfactory epithelium stem cell markers include central nervous system cells, eye tissue cells, head and neck tissue cells, nasal epithelial cells, and oral epithelial cells. The content of the cells derived from the ectoderm in the first cell population is preferably 20% or more, more preferably 50% or more, and even more preferably 80% or more.

The first cell population may contain cells other than olfactory placode cells or olfactory epithelial stem cells. Examples of the "cells other than olfactory placode cells or olfactory epithelial stem cells" include central nervous system cells, ocular tissue cells, head and neck tissue cells, nasal epithelial cells, and oral epithelial cells. The cells other than olfactory placode cells or olfactory epithelial stem cells preferably form tissue.

In one aspect of this embodiment, the first cell population further comprises cells of the central nervous system,
The cells of the central nervous system
Preferably, the cells are those which 1) express at least one central nervous system marker selected from the group consisting of Pax6, Sox1, Sox2, Sox3, Dlx5, FoxG1, N-Cadherin, MAP2, RAX, and VSX2, and 2) do not express cytokeratin.

In another aspect of this embodiment, the first cell population comprises: 1) a non-neuroepithelial tissue portion comprising the olfactory placode cells or olfactory epithelial stem cells; and 2) a neural tissue portion comprising neural cells or their progenitor cells; and
A three-dimensional tissue or cell mass in which at least a portion of the surface of the nerve tissue portion is covered with the non-neuroepithelial tissue portion,
The neural cells or precursor cells thereof preferably include neural cells constituting the central nervous system or precursor cells thereof. Here, the term "three-dimensional, solid" refers to a shape having width, depth, and height, and is a concept that is distinguished from a shape that is mainly two-dimensional (e.g., a sheet shape).

In another aspect of this embodiment, the first cell population comprises: 1) a non-neuroepithelial tissue portion comprising the olfactory placode cells or olfactory epithelial stem cells; and 2) a neural tissue portion comprising neural cells or their progenitor cells; and
the non-neuroepithelial tissue portion and the neural tissue portion are attached to the same surface of the culture substrate,
The nervous system cells or their precursor cells preferably include nervous system cells that constitute the central nervous system or their precursor cells. The surface of the "same surface of the culture vessel" may be a flat surface, a wavy surface, or a surface having projections and recesses.

Methods for dispersing olfactory placode cells or olfactory epithelial stem cells contained in the first cell population include, for example, mechanical dispersion treatment, cell dispersion treatment, and cell protective agent addition treatment, and these treatments may be performed alone or in combination. The cell dispersion treatment may involve treatment with a certain cell dispersion liquid, followed by treatment with a different cell dispersion liquid. As a method for dispersing cells, it is preferable to perform a cell dispersion treatment simultaneously with a cell protective agent addition treatment, and then a mechanical dispersion treatment.

The enzyme contained in the cell dispersion liquid used in step (1) is not particularly limited as long as it is an enzyme capable of dispersing cells, and examples thereof include enzymes such as papain, EDTA, trypsin, collagenase (collagenase types I to VII), metalloprotease, hyaluronidase, elastase, dispase, and deoxyribonuclease, or mixtures thereof. As the cell dispersion liquid, for example, the solution described in step (a) above can also be used. Preferred cell dispersion liquids in step (1) are, but are not limited to, a buffer solution containing papain and a buffer solution containing DNase. The cell dispersion liquid may further contain a substance related to stabilization of enzymes or enhancement of enzyme activity, such as EDTA, L-cysteine, 2-mercaptoethanol, magnesium sulfate, etc. In order to accelerate the cell dispersion treatment, a step of physically chopping the cell population (e.g., chopping with a scalpel, scissors, etc.) or a step of physically making an incision in the cell population (e.g., making an incision with a scalpel, scissors, etc.) may be performed before the treatment. One preferred embodiment of the cell dispersion treatment in step (1) is treating the olfactory placode cells or olfactory epithelial stem cells contained in the first cell population with HBSS buffer containing papain, EDTA, L-cysteine, 2-mercaptoethanol, and Y-27632, followed by mechanical dispersion treatment, and then treatment with HBSS buffer containing DNase and magnesium sulfate.

It is also preferable to add a protease inhibitor to stop the reaction after performing the cell dispersion treatment containing a protease in step (1). The protease inhibitor to be added is not particularly limited as long as it has inhibitory activity against the protease contained in the cell dispersion. It may be any of proteins, peptides, compounds, etc. Examples of such substances include trypsin inhibitors, aprotinin, pepstatin A, chymostatin, antipain, leupeptin, ebselen, E-64, etc. When papain is used as the cell dispersion used in the cell dispersion treatment in step (1), it is preferable to use E-64 as the protease inhibitor, but this is not limited thereto.

<Step (2)>
In step (2), olfactory placode cells or olfactory epithelial stem cells are purified from the cells prepared in step (1).
In step (2), the single cells obtained in step (1) are purified to obtain a second cell population containing the olfactory placode cells or olfactory epithelial stem cells. Step (2) can also be understood as a step of purifying a first cell population containing single cells to obtain a second cell population. The content ratio of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is higher than the content ratio of the olfactory placode cells and olfactory epithelial stem cells in the first cell population. The content ratio of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is preferably 50% or more, more preferably 80% or more. The content ratio can be calculated by, for example, counting by immunostaining, image analysis, fluorescence-activated cell sorting (FACS), or single-cell analysis.

In step (2), a method for purifying olfactory placode cells or olfactory epithelial stem cells from the cell population can be, for example, a method using a marker specific to olfactory placode cells or olfactory epithelial stem cells. A reagent having specific affinity to the cell surface marker can also be used for purification. Here, the reagent having specific affinity is an antibody, an aptamer, a peptide, or a compound that specifically recognizes the marker, and is preferably an antibody or a fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody. Examples of antibody fragments include a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment "ScFv"). In order to purify olfactory placode cells or olfactory epithelial stem cells, two or more types of "reagents having specific affinity to the cell surface marker" can also be used in combination.

In order to recognize or isolate cells expressing a cell surface marker, the affinity reagent may be bound to a detectable substance, such as a fluorescent label, a radioactive label, a chemiluminescent label, an enzyme, biotin, and streptavidin, or a substance that allows for isolation and extraction, such as protein A, protein G, beads, and magnetic beads. The affinity reagent may also be indirectly labeled. An example of an indirect labeling method is the use of a pre-labeled antibody (secondary antibody) that specifically binds to the antibody. As is well known to those skilled in the art, when two or more types of "reagents having specific affinity for cell surface markers" are used in combination, it is preferable that the substance that allows for isolation and extraction is bound to or indirectly labeled by different reagents.

Methods for isolating cells include, but are not limited to, a method in which an affinity reagent is bound to particles and then precipitated, a method in which cells are sorted by magnetism using magnetic beads (e.g., MACS), a method in which a cell sorter is used with a fluorescent label (e.g., FACS), or a method in which a carrier on which an antibody or the like is immobilized (e.g., a cell concentration column) is used. That is, in step (2), the first cell population may be purified to obtain a second cell population by fluorescence-activated cell sorting (FACS) or magnetic bead cell sorting (MACS).

Purification methods using a reagent having specific affinity for the above-mentioned cell surface markers include (1) a purification method in which a reagent having specific affinity for the cell surface marker of a target cell is used to recover the target cell, and (2) a purification method in which a reagent having specific affinity for the cell surface marker of non-target cells is used to remove and recover the non-target cells. Here, "target cells" refers to the cells that are the subject of purification. "Non-target cells" refers to cells other than the "cells that are the subject of purification."

Markers that can be used in step (2) to label and purify olfactory placode cells or olfactory epithelial stem cells include, for example, EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1. That is, in one aspect of this embodiment, step (2) preferably includes purifying the first cell population to obtain the second cell population by recovering the olfactory placode cells or olfactory epithelial stem cells using a reagent having specific affinity for a cell surface marker of the target cells, and SORL1 can also be used as a cell surface marker.
The cell surface marker of the target cell preferably includes at least one selected from the group consisting of EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1.

In step (2), the target cells can be further understood to be olfactory placode cells or olfactory epithelial stem cells.

In another aspect of this embodiment, step (2) preferably further comprises purifying the first cell population by removing the non-target cells using a reagent having specific affinity for a cell surface marker of the non-target cells to obtain the second cell population,
This can also be achieved by labeling non-target cells other than olfactory placode cells or olfactory epithelial stem cells with a cell surface marker for the non-target cells, recovering a negative fraction containing olfactory placode cells or olfactory epithelial stem cells during the purification process, and removing the non-target cells from the first cell population. As a removable marker that does not label olfactory placode cells or olfactory epithelial stem cells but labels non-target cells, it is preferable to include at least one selected from the group consisting of ATP1B2, CD82, CDH6, CDON, CELSR2, CLU, CNTFR, COL11A1, CP, CRB2, DCT, DKK3, EFNB1, FBLN2, FBN2, FN1, GPM6B, ISLR2, LRP2, LRRC4B, MMRN1, PRELP, PRSS35, S1PR3, SDK2, SLC2A1, SORCS1, TFPI, THSD7A, THY1, TMEM132B, and TSPAN18. The non-target cells include mesodermal and endodermal cells, cells of the central nervous system, retinal cells, remaining pluripotent stem cells, and differentiation-resistant pluripotent stem cells. From the viewpoint of removing cells having tumorigenicity from the first cell population, a reagent that recognizes a known cell surface marker specifically expressed in undifferentiated pluripotent stem cells can also be used. The reagent that recognizes the non-target cells and is added for cell removal may be a complex with a toxin, a cytotoxic compound, or the like.

As another embodiment of purifying olfactory placode cells or olfactory epithelial stem cells from the first cell population in step (2), a compound that is specifically metabolized by olfactory placode cells or olfactory epithelial stem cells and has the effect of labeling cells can also be used. Examples of such compounds include Coumarin, gGlu-HMRG, and derivatives thereof described in iScience. 2022 May 20; 25(5): 104222.

As another method for purifying olfactory placode cells or olfactory epithelial stem cells from the cell suspension of the first cell population containing single cells prepared in step (1) and obtaining the second cell population, a label-free cell separation and analysis system that does not add a reagent that binds to cells can be used. For purification, olfactory placode cells or olfactory epithelial stem cells are purified based on non-invasively measurable parameters such as cell morphology, size, scattering of irradiated light, dielectric spectrum, and electrical properties. Examples of the label-free cell separation and analysis system and method as described above include ELESTA CROSSORTER (manufactured by SCREEN) and Ghost cytometry described in Science, 2018, 360.6394: 1246-1251.

The cells before purification or the cells after purification prepared in step (2) can also be cryopreserved. That is, in one aspect of this embodiment, step (2) may further include cryopreserving the second cell population. By carrying out cryopreservation, the intermediate product can be easily preserved, evaluated for quality, and transported. As a method for cryopreserving the purified cells, the dispersed cells are suspended in a cryopreservation medium, and a cryopreserved sample of the cells can be prepared by treating the cells at a predetermined temperature and low temperature conditions. The cryopreservation medium may contain a cryoprotectant. The freezing method for cryopreservation is not particularly limited, but either the vitrification method or the slow method can be used. The vitrification method is performed, for example, by directly immersing a tube containing cells suspended in a cryopreservation solution in liquid nitrogen. The slow method is performed, for example, by cooling a tube containing cells suspended in a cryopreservation solution to -80°C at a cooling rate of about -1°C/min in a program freezer or deep freezer. In the present invention, the slow method is preferably used. The purified cells can also be cryopreserved by the method described in WO2020/013247.

The cryopreservation medium may be any medium suitable for cryopreservation of cells, and examples of such media include physiological saline, buffer solutions such as PBS, EBSS, or HBSS, culture media such as DMEM, GMEM, or RPMI, serum, serum substitutes, or mixtures thereof, each of which contains a cryoprotectant as described below.

Cryoprotectants are added to cells during cryopreservation to maintain cell function or viability as much as possible and prevent various disorders caused by freezing. Examples of cryoprotectants include sulfoxides such as dimethyl sulfoxide (DMSO); linear polyols such as ethylene glycol, glycerol, propanediol, propylene glycol, butanediol, and polyethylene glycol. Preferably, they further contain oligosaccharides such as sucrose, trehalose, lactose, and raffinose. They may also contain amide compounds such as acetamide, Percoll, Ficoll 70, Ficoll 70000, polyvinylpyrrolidone, and the like. In the case of sulfoxides, they may be added at a final concentration of 5 to 15% (w/v), preferably 9 to 13% (w/v), and more preferably about 11% (w/v). In the case of linear polyols, they may be added at a final concentration of 4 to 15% (w/v), preferably 4.5% to 8% (w/v), and more preferably about 5.5% (w/v). In the case of oligosaccharides, they may be added at a final concentration of 5 to 20% (w/v), preferably 8 to 12% (w/v), and more preferably about 10% (w/v). The above-mentioned cell protective substances can also be used as cryoprotectants. The cryoprotectant in the present invention preferably contains a ROCK inhibitor.

As a cryopreservation medium, commercially available media such as STEM-CELLBANKER (Nihon Zenyaku Kogyo Co., Ltd.), Cellbanker 1, 1 Plus, 2, 3 (Juji Field Co., Ltd.), TC Protector (DS Pharma Biomedical Co., Ltd.), Freezing Medium for human ES/iPS Cells (ReproCell Co., Ltd.), CryoScarless DMSO Free (BioVerde Co., Ltd.), Stem Cell Keep (BioVerde Co., Ltd.), and EFS solution (NK system) can also be used.

The purified cells prepared in step (2) can also be expanded. The expansion can be either adherent or suspension culture, but is preferably adherent culture. The expansion can be performed using cells that have been awake after cryopreservation. Alternatively, the cells can be cryopreserved after expansion.

In the expansion culture by adhesion culture in step (2), from the viewpoint of promoting adhesion of olfactory placode cells or olfactory epithelial stem cells, or from the viewpoint of inducing or maintaining the properties of the cells in a state preferable for the present invention, culture equipment or the like in which the surface of a substance-permeable membrane has been artificially treated as described above can be used. One preferred embodiment of the treatment in the expansion culture by adhesion culture in step (2) is coating the culture equipment with a mixture of a synthetic peptide (Synthemax/manufactured by Corning) containing a cell adhesive domain (e.g., a domain containing an RGD sequence) and a laminin E8 fragment (iMatrix-511/manufactured by Nippi).

The medium used for the expansion culture in step (2) can be prepared using a medium normally used for the culture of the above-mentioned animal cells as the basal medium. The medium used for the expansion culture in step (2) is preferably a serum-free medium. The serum-free medium may contain the above-mentioned serum substitutes, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, etc., as appropriate. For the expansion culture in step (2), a prepared medium based on the above-mentioned basal medium, preferably a medium for culturing known epithelial cells or respiratory system cells, etc., can also be used. Examples of the above-mentioned medium include PneumaCult-Ex, Ex Plus, ALI, Airway organoid seeding medium, Airway organoid differentiation medium (manufactured by StemCell Technologies), Airway Epithelial Cell Growth Medium (manufactured by PromoCell), CnT-Prime, Epithelial Culture Medium, CnT-Prime Airway, Epithelial Culture Medium, CnT-Prime Airway Examples of media include, but are not limited to, Differentiation Medium (CELLnTEC), BEGM Bronchial Epithelial Cell Growth Medium (Lonza), etc. Known media for respiratory cell culture include, for example, the media described in Stem Cell Reports. 2018 Jan 9; 10(1): 101-119 or Nat Protoc. 2015 Mar; 10(3): 413-25.

<Step (3)>
In step (3), the cell population containing olfactory placode cells or olfactory epithelial stem cells prepared in step (2) is seeded and cultured to obtain a sheet-shaped cell structure containing upper respiratory tract cells. The cells used in step (3) may be those cryopreserved in step (2). The cells used in step (3) may be those cultured after purification in step (2). That is, in one aspect of this embodiment, the step (2) may further include cryopreserving the second cell population, and the step (3) may further include thawing the cryopreserved second cell population.

In step (3), the second cell population containing olfactory placode cells or olfactory epithelial stem cells prepared in step (2) is preferably cultured on a membrane having substance permeability. Step (3) can also be understood as culturing the second cell population on a membrane having substance permeability provided in a culture vessel. The membrane having substance permeability is, for example, a porous membrane or a membrane formed from fibers. The porous membrane can be, for example, a porous membrane made of polycarbonate, polyester, polyethylene terephthalate, polytetrafluoroethylene, or the like. The porous membrane is manufactured, for example, by a track etching method. The pore size of the porous membrane is not particularly limited as long as it has substance permeability, but is preferably a pore size that is cell impermeable. The pore size of the porous membrane as described above is, for example, about 8 μm or less, preferably about 5 μm or less, and particularly preferably about 3 μm or less. The lower limit of the pore size of the porous membrane is not particularly limited, but is, for example, 0.2 μm or more. These membranes having substance permeability are preferably made from a material having biocompatibility.

In step (3), from the viewpoint of promoting adhesion of olfactory placode cells or olfactory epithelial stem cells, or from the viewpoint of inducing or maintaining the properties of the cells in a state preferable for the present invention, culture equipment or the like in which the surface of a substance-permeable membrane has been artificially treated as described above can be used. One preferred embodiment of the treatment in step (3) is coating the culture equipment with a mixture of a synthetic peptide (Synthemax/manufactured by Corning) containing a cell adhesive domain (e.g., a domain containing an RGD sequence) and a laminin E8 fragment (iMatrix-511/manufactured by Nippi).

Examples of cultureware made of a substance-permeable membrane used in step (3) include Cell Culture Insert (manufactured by Thermo Fisher Scientific), Transwell (manufactured by Corning Incorporated), Millicell Cell Culture Insert (manufactured by Merck), and ad-MED Vitrigel (manufactured by Kanto Chemical Co., Ltd.).

In step (3), the seeding density of the olfactory placode cells or olfactory epithelial stem cells contained in the second cell population on the culture vessel is not particularly limited as long as a sheet-shaped cell structure can be formed. ^A medium in which olfactory placode cells or olfactory epithelial stem cells prepared to be usually about 1×10 ³ to about 1×10 ⁷ cells/cm ² , preferably about 2× ^{10 3} to about 5× ^{10 6 cells} /cm ² , more preferably about 5×10 ³ to about 2×10 ⁶ cells/cm ² , even more preferably about 1×10 ⁴ to about 1×10 ⁶ cells/cm 2 , and particularly preferably about 2×10 4 to about 1×10 6 cells/cm ² is suspended is added to the culture vessel, and culture is started.

The number of days of culture in step (3) is not particularly limited as long as a sheet-shaped cell structure is formed, but may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more. The upper limit of the number of days of culture in step (3) is not particularly limited, but may be, for example, 200 days or less. Those skilled in the art can determine whether the olfactory placode cells or olfactory epithelial stem cells seeded in step (3) have adhered to the culture device, whether intercellular adhesion has occurred, and whether an epithelial structure has been formed to form a sheet-shaped cell structure, for example, by observation with an optical microscope or by immunostaining using the above-mentioned cell adhesion factors and epithelial cell markers (E-Cadherin, EpCAM, ZO-1, etc.).

In step (3), the second cell population is preferably cultured in the presence of at least one selected from the group consisting of a sonic hedgehog signaling pathway agonist (a Shh signaling pathway agonist) and a BMP signaling pathway inhibitor.

The substance acting on the Shh signaling pathway used in step (3) may be the same as or different from that used in step (i), but is preferably the same. The substance acting on the Shh signaling pathway used in step (3) preferably includes at least one selected from the group consisting of SAG, purmorphamine, 20(S)-hydroxycholesterol, and GSA-10, and more preferably includes SAG. The concentration of the substance acting on the Shh signaling pathway in the medium can be appropriately set depending on the substance used within a range that can achieve the above-mentioned effects. In step (3), SAG is usually used at a concentration of about 1 nM to about 2000 nM, preferably at a concentration of about 10 nM to about 1000 nM, more preferably at a concentration of about 10 nM to about 700 nM, even more preferably at a concentration of about 50 nM to about 700 nM, particularly preferably at a concentration of about 100 nM to about 600 nM, and most preferably at a concentration of about 100 nM to about 500 nM. In addition, when a substance acting on the Shh signaling pathway other than SAG is used, it is desirable to use it at a concentration that exhibits Shh signaling promoting activity equivalent to that of SAG at the above concentration.

The BMP signaling pathway inhibitor used in step (3) may be the same as or different from that used in step (c), but is preferably the same. The BMP signaling pathway inhibitor used in step (3) is preferably a type I BMP receptor inhibitor, more preferably at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1, DMH2, Compound 1, VU5350, OD52, E6201, Saracatinib, BYL719, and derivatives thereof, and even more preferably K02288.

The concentration of the BMP signal transduction pathway inhibitor in the medium can be appropriately set within a range that can achieve the above-mentioned effects. From the viewpoint of the efficiency of formation of cells that constitute the upper respiratory tract tissue, olfactory epithelium, and nasal epithelium, when K02288 is used as the BMP signal transduction pathway inhibitor in step (3), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 500 nM to about 25 μM. When LDN-193189 is used as the BMP signal transduction pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 1 μM, and even more preferably about 100 nM to about 500 nM. When LDN-212854 is used as the BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When ML-347 is used as the BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 50 μM, more preferably about 100 nM to about 50 μM, and even more preferably about 1 μM to about 25 μM. When DMH2 is used as a BMP signaling pathway inhibitor, it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 25 nM to about 5 μM, and even more preferably about 250 nM to about 3 μM. When a BMP signaling pathway inhibitor other than K02288 is used, it is desirable to use it at a concentration that exhibits the same BMP signaling pathway inhibitory activity as K02288 at the above concentration.

In step (3), the second cell population is preferably cultured in the presence of at least one selected from the group consisting of a ROCK inhibitor, an FGF signaling pathway active substance, an EGF signaling pathway active substance, a TGFβ family signaling pathway inhibitor, a Wnt signaling pathway active substance, and a retinoic acid signaling pathway active substance.

The ROCK inhibitor, FGF signaling pathway active substance, and EGF signaling pathway active substance used in step (3) are preferably added at the same substances and final concentrations as those in step (i) and steps (a) to (c) above.

The substance acting on the Wnt signaling pathway used in step (3) may be the same as or different from the substance used in steps (i) and (a) to (c). A preferred substance acting on the Wnt signaling pathway in step (3) is a GSK3β inhibitor, more preferably CHIR99021. When CHIR99021 is used as the substance acting on the Wnt signaling pathway in step (3), it is usually used at a concentration of about 10 pM to about 10 mM, preferably about 100 pM to about 1 mM, more preferably about 1 nM to about 100 μM, even more preferably about 10 nM to about 30 μM, and most preferably about 100 nM to about 3 μM.

The TGFβ signaling pathway inhibitor used in step (3) preferably includes an Alk5/TGFβR1 inhibitor. The Alk5/TGFβR1 inhibitor is preferably SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, A77-01, RepSox, BIBF-0775, TP0427736, TGFBR1-IN-1, SM-16, TEW-7197, LY3200882, LY2109761, R268712, IN1130, Galunisertib, AZ12799734, KRCA 0008, GSK It contains at least one selected from the group consisting of 1838705, Crizotinib, Certinib, ASP 3026, TAE684, and AZD3463, and more preferably contains SB431542, RepSox, or A83-01.

The concentration of the TGFβ signaling pathway inhibitor in the medium can be set appropriately depending on the substance used within a range that can achieve the above-mentioned effects. When SB431542 is used as the TGFβ signaling pathway inhibitor in step (3), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 10 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, and particularly preferably about 1 μM to about 10 μM. When RepSox is used as the TGFβ signaling pathway inhibitor in step (3), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 10 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, and particularly preferably about 1 μM to about 10 μM. When A83-01 is used as the TGFβ signaling pathway inhibitor in step (3), it is usually used at a concentration of about 1 nM to about 100 μM, preferably about 10 nM to about 100 μM, more preferably about 20 nM to about 50 μM, even more preferably about 100 nM to about 50 μM, and particularly preferably about 200 nM to about 3 μM. A preferred embodiment of the TGFβ signaling pathway inhibitor in step (3) is a combination of SB431542, RepSox, and A83-01.

Examples of the retinoic acid pathway active substance used in step (3) include substances that bind to retinoic acid receptors (RAR) or retinoid X receptors (RXR) and activate downstream transcription. Examples of compounds having the above-mentioned effects include all-trans retinoic acid, isotretinoin, 9-cis retinoic acid, TTNPB (4-[(E)-2-[(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalene)-2-yl]-1-propenyl]benzoic acid), Ch55 (4-[(E)-3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid), EC19 (3-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethyl]benzoic acid), EC23 (4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethyl)-benzoic acid), Phenretinide (4-hydroxyphenylretinamide), Acitetin ((all-e)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen), Trifarotene, Adapalene, AC 261066 (4-[4-(2-Butoxyethoxy-)-5-methyl-2-thiazolyl]-2-fluorobenzoic acid), AC 55649 (4-N-Octylbiphenyl-4-carboxylic acid), AM 580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid), AM80 (4-[(5,5,8,8-Tetramethyl-6,7-dihydronaphthalen-2-yl)carbamoyl]benzoic acid), BMS 753 (4-[[(2,3-Dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl)carbonyl]amino]benzoic acid), BMS 961 (3-Fluoro-4-[(r)-2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-acetylamino]-benzoic acid), CD1530 (4-(6-Hydroxy-7-triclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoic acid), CD2314 (5-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)-3-thiophenecarboxylic acid acid), CD437 (2-naphthalenecarboxylic acid, 6-(4-hydroxy-3-triclo(3.3.1.1(3,7))dec-1-ylphen), CD271 (6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthalene carboxylic acid) and derivatives thereof. These substances may be used alone or in combination.

The retinoic acid pathway acting substance in step (3) preferably includes EC23. The concentration of the retinoic acid pathway acting substance in the medium is not particularly limited as long as it is within a range in which the above-mentioned effects can be achieved. When EC23 is used as the retinoic acid pathway acting substance, the concentration of EC23 is, for example, about 10 pM to about 30 μM, preferably about 100 pM to about 20 μM, more preferably about 10 nM to about 10 μM, and even more preferably about 100 nM to about 5 μM. When a retinoic acid pathway acting substance other than EC23 is used, it is desirable to use it at a concentration that exhibits the same retinoic acid pathway acting activity as EC23 at the above concentration.

From the viewpoint of regulating the differentiation tendency of cells contained in the sheet-shaped cell structure, it is also preferable to carry out step (3) in the presence of a Notch signaling pathway inhibitor. In the present invention, the Notch signaling pathway refers to a signaling pathway that is activated by direct interaction between the Notch protein, which is a receptor expressed on the cell membrane, and the Notch ligand (Delta, Jagged, etc.) expressed on the membrane of an adjacent cell. In cells to which the Notch signal is transmitted, the Notch protein is processed in stages, and the intracellular domain cut out on the membrane is transported into the nucleus to control the expression of downstream genes.

The Notch signaling pathway inhibitor is not particularly limited as long as it can suppress signaling mediated by Notch. The Notch signaling pathway inhibitor may be any of a nucleic acid, a protein, and a low molecular weight organic compound. The Notch signaling pathway inhibitor is not particularly limited as long as it can suppress signaling mediated by Notch. The Notch signaling pathway inhibitor may be any of a nucleic acid, a protein, and a low molecular weight organic compound. Examples of such substances include loss-of-function Notch receptors and ligands, substances that inhibit Notch processing (S1 cleavage), substances that inhibit glycosylation of Notch and Notch ligands, substances that inhibit cell membrane translocation, substances that inhibit processing (S2 cleavage, S3 cleavage) of the intracellular domain (NICD) of Notch (gamma secretase inhibitors), substances that degrade NICD, and substances that inhibit NICD-dependent transcription.

Compounds known to those skilled in the art can also be used as Notch signaling pathway inhibitors. Examples of compounds that have activity as Notch signaling pathway inhibitors include DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), DBZ ((2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzozepin-7-yl]propanamide), MDL28170 (benzyl N-[(2S)-3-methyl-1-oxo-1-[[(2S)-1-oxo-3-phenylpropan-2-yl]amino]butan-2-yl]carbamate, FLI-06 (cyclohexyl 2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-1,4,6,8-tetrahydroquinone-3-carboxylate), L-685,458 (tert-butyl Examples of the inhibitor include N-[6-[[1-[(1-amino-1-oxo-3-phenylpropan-2-yl)amino]-4-methyl-1-oxopentan-2-yl]amino]-5-benzyl-3-hydroxy-6-oxo-1-phenylhexan-2-yl]carbamate, CB-103 (6-(4-tert-butylphenoxy)pyridin-3-amine) and derivatives thereof, as well as substances described in Onco Targets Ther. 2013;6:943-955. The Notch signaling pathway inhibitor preferably includes DAPT.

The concentration of the Notch signaling inhibitor in the medium is not particularly limited as long as it is within a range in which the above-mentioned effects can be achieved. For example, when DAPT is used as the Notch signaling pathway inhibitor, the concentration of DAPT is, for example, about 100 pM to about 50 μM, preferably about 1 nM to about 30 μM, more preferably about 100 nM to about 20 μM, and even more preferably about 1 μM to about 10 μM. When a Notch signaling pathway inhibitor other than DAPT is used, it is desirable to use it at a concentration that exhibits Notch signaling pathway inhibitory activity equivalent to that of DAPT at the above-mentioned concentration.

From the viewpoint of regulating the differentiation tendency of cells contained in the sheet-shaped cell structure, it is also preferable to carry out step (3) and the subsequent steps in the presence of a Hippo/YAP signaling pathway regulator. In the present invention, the Hippo/YAP signaling pathway refers to a kinase cascade composed of Mst1/2, LATS1/2, etc., which is involved in the control of cell proliferation or apoptosis, the control of stem cell self-replication, and the regulation of organ size, and a signaling pathway controlled by the transcription factor YAP/TAZ. For example, when attempting to proliferate cells and maintain the undifferentiated state of stem cells, a person skilled in the art can add a substance that inhibits the Hippo pathway and has the effect of promoting the dephosphorylation and nuclear translocation of YAP/TAZ protein, a substance that promotes transcription induced by YAP/TAZ, etc. When promoting cell differentiation, a person skilled in the art can do so by adding a substance that activates the Hippo pathway and promotes phosphorylation of YAP/TAZ protein and degradation by the ubiquitin-proteosome system, or a substance that suppresses transcription induced by YAP/TAZ.

The Hippo pathway inhibitor is not particularly limited as long as it inhibits the upstream kinase cascade or enhances the transcription and cellular response of downstream target genes induced by YAP/TAZ. The Hippo pathway inhibitor may be any of a nucleic acid, a protein, and a low molecular weight organic compound. Examples of the Hippo pathway inhibitor include growth factors, cytokines, Sphingosine-1-phosphate receptor 2 (S1PR2) activators, mammalian sterile 20-like kinase (MST) 1/2 inhibitors, large tumor suppressor kinase 1 and 2 (LATS1/2) inhibitors, annexin A2 (ANXA2) binding inhibitors, and TAZ activators. Examples of substances having the above-mentioned effects include CYM-5520 (1-[2-(1-benzyl-2,5-dimethylpyrrol-3-yl)-2-oxoethyl]-6-oxopyridine-3-carbonitrile), XMU-MP-1 (4-[(2,9-dimethyl-8-oxo-6-thia-2,9,12,14-tetrazatricyclo[8.4.0.03,7]tetradeca-1(14),3(7),4,10, 12-pentaen-13-yl)amino]benzenesulfonamide), PY-60 (5-phenyl-N-(3-(thiazol-2-yl)propyl)isoxazole-3-carboxamide), GA-017 ([(E)-2-(1-(2,4-dihydroxy-3-methylphenyl)propylidene)-N-(3-hydroxybenzyl)hydrazine-1-carboxamide]), Lats-IN-1 (N-(3-benzyl-1,3-thiazol-2-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide), IBS008738 ([3-(4-methylphenyl)-5-[(E)-morpholin-4-ylmethylideneamino]imidazol-4-yl]-phenylmethaneone), TM-25659 (2-butyl-5-methyl-6- Examples of such compounds include pyridin-3-yl-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]imidazo[4,5-b]pyridine, SBP-3264 (N-[1-[5-(3-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]piperidin-4-yl]acetamide), VT02956, TDI-011536, and derivatives thereof.

The Hippo pathway inhibitor may be any of a nucleic acid, a protein, and a low molecular weight organic compound. Examples of Hippo pathway inhibitors include growth factors, cytokines, sphingosine-1-phosphate receptor 2 (S1PR2) activators, mammalian sterile 20-like kinase (MST) 1/2 inhibitors, large tumor suppressor kinase 1 and 2 (LATS1/2) inhibitors, annexin A2 (ANXA2) binding inhibitors, and TAZ activators. Examples of substances having the above-mentioned effects include CYM-5520 (1-[2-(1-benzyl-2,5-dimethylpyrrol-3-yl)-2-oxoethyl]-6-oxopyridine-3-carbonitrile), XMU-MP-1 (4-[(2,9-dimethyl-8-oxo-6-thia-2,9,12,14-tetrazatricyclo[8.4.0.03,7]tetradeca-1(14),3(7),4,10, 12-pentaen-13-yl)amino]benzenesulfonamide), PY-60 (5-phenyl-N-(3-(thiazol-2-yl)propyl)isoxazole-3-carboxamide), GA-017 ([(E)-2-(1-(2,4-dihydroxy-3-methylphenyl)propylidene)-N-(3-hydroxybenzyl)hydrazine-1-carboxamide]), Lats-IN-1 (N-(3-benzyl-1,3-thiazol-2-ylidene)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide), IBS008738 ([3-(4-methylphenyl)-5-[(E)-morpholin-4-ylmethylideneamino]imidazol-4-yl]-phenylmethaneone), TM-25659 (2-butyl-5-methyl-6- Examples of such compounds include pyridin-3-yl-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]imidazo[4,5-b]pyridine, SBP-3264 (N-[1-[5-(3-chlorophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]piperidin-4-yl]acetamide), VT02956, TDI-011536, and derivatives thereof.

The Hippo pathway activator is not particularly limited as long as it suppresses the transcription and cellular response of downstream genes induced by YAP/TAZ. The Hippo pathway activator may be any of a nucleic acid, a protein, and a low molecular weight organic compound. Examples of the Hippo pathway activator include TEAD inhibitors, YAP-TEAD binding inhibitors, AMPK activators, YAP/TAZ transcription activity inhibitors, and pseudoactivators of adhesion stimuli to intercellular and extracellular matrices. Examples of substances having the above-mentioned effects include Verteporfin (3-[(23S,24R)-14-ethyl-22,23-bis(methoxycarbonyl)-5-(3-methoxy-3-oxopropyl)-4,10,15,24-tetramethyl-25,26,27,28-tetrazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-9-yl]propanoic acid; 3-[(23S,24R)-14-ethyl-22,23-bis(methoxycarbonyl)-9-(3-methoxy-3-oxopropyl)-4,10,15,24-tetramethyl-25,26,27,28-tetrazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1,3,5,7,9,11(27),12,14,16,18(25),19,21-dodecaen-5-yl]propanoic acid), AICAR (5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide), K-975 (N-(3-(4-chlorophenoxy)-4-methylphenyl)acrylate), MYF-01-37 (1-[3-methyl-3-[3-(trifluoromethyl)anilino]pyrrolidine-1-yl]prop-2- en-1-one), TED-347 (2-chloro-1-[2-[3-(trifluoromethyl)anilino]phenyl]ethane), VT107 (N-[(1S)-1-(6-aminopyridin-2-yl)ethyl]-5-[4-(trifluoromethyl)phenyl]naphthalene-2-carboxamide), YAP-TEAD-IN-1 ((4S)-4-[[(2S)-1-[(2S)-1-[(2S)-2-[[(3S,6S,9S,1 2S,15S,24S,27S,30S,33S)-15-[[(2S)-2-[[(2S)-1-[(2S)-2-acetamido-3-methylbutanoyl]pyrrolidine-2-carbonyl]amino]-3-(3-chlorophenyl)propanoyl]amino]-6-(4-aminobutyl)-24-benzyl-3-butyl-9-(3-carbamimidamidopropyl)-27-(hydroxymethyl)-30-methyl -12-(2-methylpropyl)-2,5,8,11,14,23,26,29,32-nonaoxo-18,19-dithia-1,4,7,10,13,22,25,28,31-nonazabicyclo[31.3.0]hexatriacontane-21-carbonyl]amino]-6-aminohexanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-5-amino-5-oxopentanoic acid), Super-TDU ((4S)-5-amino-4-[[(2S)-1-[(2S)-1-[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S,3S)-2-[2-[ (3S)-6-amino-1-[(2S)-1-[(2S)-2-[[(2S)-4-amino-2-[[(2R)-2-[[(2S,3S)-2-[[(2S)-6-amino-2-[[(2S)-1-[(2S)-4-amino-2-[[2-[[(2S)-2-[[2-[[2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[2 -[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-3-carboxy-2-[[(2S)-3-carboxy-2-[[(2S)-2-[2-[(2S)-1-hydroxy-3-oxopropan-2-yl]hydrazinyl]-3-methylbutanoyl]amino]propanoyl]amino]propane oil]amino]-3-(1H-imidazoI-5-yl)propanoyl]amino]-3-phenylpropanoyl]amino]propanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-hydroxyb utanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]-4-oxobutanoyl]pyr rolidine-2-carbonyl]amino]hexanoyl]amino]-3-hydroxybutanoyl]amino]propanoyl]amino]-4-oxobutanoyl]amino]-3-methylbutanoyl]pyrrolidine-2-yl]-1,2,6-trioxohexan-3-yl]hydrazinyl]-3-hydroxybutanoyl]amino]-3-meth hylbutanoyl]pyrrolidine-2-carbonyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-4-methylpentanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]-4-methylpentanoyl]pyr pyrrolidine-2-carbonyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoic acid) and derivatives thereof.

The medium used in step (3) is not particularly limited as long as it is as described in the definition section above. The medium used in step (3) may be a serum medium or a serum-free medium. In order to avoid contamination with chemically undefined components, a serum-free medium is preferably used in the present invention. Examples of serum-free media used in step (3) include media in which N2 Supplement, B27 Supplement, and HEPES are added to DMEM/F12 medium or Advanced DMEM/F12 medium.

From the viewpoint of promoting the survival and differentiation/maturation of cells contained in the cell structure, it is also preferable to carry out step (3) under the conditions of the air-liquid interface culture method. The air-liquid interface culture method refers to culture under conditions in which at least one surface of the cells or tissue is exposed to the air or is in a very close position. In order to carry out the air-liquid interface culture method, for example, a method in which cells or tissues are cultured on a porous membrane or in a culture insert, the culture medium in the insert (inner cavity side) is removed, and cultured only with the culture medium in the well outside the insert, a method in which a hydrogel such as agarose is placed in a culture dish, cells or tissues are placed on it, and cultured under conditions in which they are exposed to the medium in the dish but do not dry out, and a method in which the cell aggregates are embedded in agarose, etc., thinly sliced with a vibrating blade microtome, etc., and the thin slices are cultured in a cell insert, etc. can be mentioned. For the purpose of reducing the toxicity of exposure to the atmosphere, mainly due to oxygen, an oxygen-permeable membrane or plate such as PDMS can be placed on the cells or tissue to adjust the exposure conditions. The above-mentioned gas-liquid interface culture method can be carried out with reference to, for example, Respiratory research 18.1 (2017): 195, Sci Rep 6, 21472, Nat Neurosci. 2019 Apr; 22 (4): 669-679, etc.

From the viewpoint of promoting survival or proliferation of cells contained in the sheet-shaped cell structure and regulating the differentiation tendency, it is also preferable to carry out step (3) in the presence of a neurotrophic factor. A neurotrophic factor is a ligand for a membrane receptor that plays an important role in maintaining the survival and function of nerves. Examples of neurotrophic factors include Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin 3 (NT-3), Neurotrophin 4/5 (NT-4/5), Neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), insulin, Insulin Like Growth Factor 1 (IGF 1), Insulin Like Growth Factor 2 (IGF 2), Insulin Like Growth Factor 3 (IGF 3), Insulin Like Growth Factor 4 (IGF 4), Insulin Like Growth Factor 5 (IGF 5), Insulin Like Growth Factor 6 (IGF 6), Insulin Like Growth Factor 7 (IGF 7), Insulin Like Growth Factor 8 (IGF 8), Insulin Like Growth Factor 9 (IGF 9), Insulin Like Growth Factor 10 (IGF 10), Insulin Like Growth Factor 11 (IGF 11), Insulin Like Growth Factor 12 (IGF 12), Insulin Like Growth Factor 13 (IGF 13), Insulin Like Growth Factor 14 (IGF 14), Insulin Like Growth Factor 15 (IGF 15), Insulin Like Growth Factor 16 (IGF 16), Insulin Like Growth Factor 17 (IGF 17), Insulin Like Growth Factor 18 (IGF 18), Insulin Like Growth Factor 19 (IGF 19), Insulin Like Growth Factor 20 (IGF 19), Insulin Like Growth Factor 21 (IGF 19), Insulin Like Growth Factor 22 (IGF 19), Insulin Like Growth Factor 23 (IGF 19), Insulin Like Growth Factor 24 (IGF 19), Insulin Like Growth Factor 25 (IGF 19), Insulin Like Growth Factor 26 (IGF 19), Insulin Like Growth Factor 27 (IGF 19), Factor 2 (IGF 2), Glia cell line-derived Neurotrophic Factor (GDNF), TGF-β2, TGF-β3, Interleukin 6 (IL-6), Ciliary Neurotrophic Factor (CNTF), and LIF. One of these may be used alone, or two or more may be used in appropriate combination.

In step (3), the obtained sheet-like cell structures may be stacked. The stacking of the sheet-like cell structures may be carried out, for example, by stacking multiple sheet-like cell structures obtained by the above-mentioned production method and incubating them for about 2 to 30 minutes. There is no particular limit to the number of sheet-like cell structures to be stacked. For example, two, three, four, or five sheet-like cell structures may be stacked. When the number of sheet-like cell structures to be stacked is three or more, three sheets may be stacked at once, or additional sheets may be stacked one by one, or stacked sheet-like cell structures may be stacked on top of each other.

In addition, any substance can be added between the sheet-like cell structures during lamination. Examples of the substance to be added include substances that promote adhesion between sheet-like cell structures (e.g., scaffolds, matrices, etc.), substances that promote cell communication between sheet-like cell structures (e.g., elongation-inducing factors, migration-inducing factors, etc.), fibronectin, growth-promoting factors, nutritional factors, differentiation-promoting or differentiation-inhibiting factors, or combinations thereof. There are no particular limitations on the method of addition. For example, addition can be performed by directly applying, spraying, or dripping onto the surface of the sheet-like cell structure, or by immersing in a medium to which the substance has been administered.

In step (3), cells other than upper respiratory tract cells can be co-cultured on the opposite side of the membrane having substance permeability on which the second cell population containing olfactory placode cells or olfactory epithelial stem cells is seeded (double-sided culture, for example, Figures 2, 3A, and 3B). Examples of cells other than upper respiratory tract cells to be co-cultured include neural crest cells, mesenchymal stem cells, mesenchymal stromal cells, vascular endothelial cells, fibroblasts, cells of the central nervous system, cells of the trachea, bronchus, and lungs, pericytes, astrocytes, microglia, choroid plexus epithelial cells, and ependymal cells. The cells other than upper respiratory tract cells to be co-cultured may be either primary cells or established cells. The cells other than upper respiratory tract cells to be co-cultured are preferably differentiated cells derived from human iPS cells.

As a specific method of double-sided culture, for example, when using an ad-MED vitrigel culture insert as a cultureware made of a substance-permeable membrane, the back side of the culture insert is turned up, and then an enclosure made of silicone or the like for holding the culture medium is placed, and the cells to be co-cultured are seeded and cultured at least until the cells adhere. After that, the medium is removed, the enclosure is removed, and the insert is placed backside down in the culture medium, and then a cell population containing olfactory placode cells or olfactory epithelial stem cells is seeded in the well inside the insert, thereby enabling double-sided culture to be performed.

In another embodiment of step (3), after seeding the second cell population onto the culture vessel, the obtained sheet-like cell structure can be isolated from the culture vessel to produce a sheet-like cell structure that does not include the culture vessel. The sheet-like cell structure as described above can be produced, for example, by using the above-mentioned temperature-responsive cell culture substrate. The obtained sheet-like cell structure that does not include the culture vessel can also be used for laminating the above-mentioned sheet-like cell structures.

In another embodiment of step (3), a sheet-shaped cell structure can be produced by seeding a second cell population containing olfactory placode cells or olfactory epithelial stem cells on a culture substrate having a micropattern. The micropattern can be composed of a cell adhesive region and a cell non-adhesive region, and it is preferable that cells are cultured in the cell adhesive region. The culture substrate having the micropattern may have substance permeability. It is preferable that the cell adhesive region has been subjected to a cell adhesive treatment (gas plasma treatment, etc.) or has been treated with a substance that promotes cell adhesion. The cell adhesive region and the cell non-adhesive region may be formed as a single region on one culture substrate, or may be formed as a plurality of regions. In this embodiment, the preferred cell adhesion promoting substance is preferably laminin, and more preferably laminin 511 to 521, but is not limited thereto. The laminin may be an E8 fragment. The cell adhesive region may be the temperature-responsive cell culture substrate or may be treated with a temperature-responsive polymer. It is preferable that the cell non-adhesive region has not been treated with cell adhesiveness or has been treated to inhibit cell adhesion. In this embodiment, preferred cell adhesion inhibition treatments include, for example, superhydrophilic treatments using coatings such as 2-methacryloyloxyethyl phosphorylcholine (MPC), poly(2-hydroxyethyl methacrylate) (Poly-HEMA), and polyethylene glycol (PEG), as well as low protein adsorption treatments.

The cell adhesive region in the culture substrate having a micropattern is preferably surrounded by a cell non-adhesive region. The shape of the cell adhesive region can be set appropriately, for example, a circle, an ellipse, a triangle, a square, a rectangle, a pentagon, a hexagon, an octagon, a star, etc. The shape of the cell adhesive region is preferably, but not limited to, a circle, a square, or a rectangle. The area of the cell adhesive region can be set appropriately, for example, 0.1 ^cm2 to ¹⁰⁰ cm2, preferably 0.3 ^cm2 to ⁸⁰ cm2, and more preferably 0.5 ^cm2 to ⁶⁰ cm2, but not limited thereto. The number of cells of the second cell population to be seeded on the culture vessel having the micropattern is, for example, 1×10 ³ cells/cm ² to 1×10 ⁷ cells/cm ² per area of the cell adhesive region, preferably 1×10 ⁴ cells/cm ² to 1×10 ⁶ cells/cm ² , but is not limited thereto. As the culture vessel having a micropattern, the above-mentioned one may be used, or it is also preferable to use a culture vessel prepared using a PDMS mold and matrix or the like.

[3. Sheet-shaped cell structure containing upper respiratory tract cells]
In this specification, the terms "cell" and "tissue" refer to the presence of cells and tissues that exist in a living body, and can be confirmed by immunostaining with markers similar to those of the corresponding cells and tissues that exist in a living body, shape observation, or other specific methods, but the functions, gene expression, structure, etc. of the "cells" and "tissues" are not necessarily the same as those of the cells and tissues that exist in a living body. In this specification, the term "cell structure" refers to an object composed of two or more elements including cells. The cell structure may include objects other than cells and living bodies, such as culture equipment. "Sheet-like" refers to a shape that has a two-dimensional surface extension and has a first main surface and a second main surface that face each other across a distance of the thickness. "Sheet-like" can also be understood as a film-like, membrane-like, or foil-like shape.

One embodiment of the present invention provides a sheet-like cell structure containing upper respiratory tract cells. The sheet-like cell structure may be produced by the above-mentioned production method. The number of cells constituting the sheet-like cell structure of the present invention is not particularly limited, but is preferably 30 or more, more preferably 500 or more. The upper limit of the number of cells constituting the sheet-like cell structure is not particularly limited, but may be, for example, 1 x 10 ⁷ or less, or 5 x 10 ⁶ or less. The size (area of the main surface) of the sheet-like cell structure is not particularly limited, but is preferably 0.4 cm ² or more, more preferably 1 cm ² or more, and even more preferably 3 cm ² or more. The upper limit of the size of the sheet-like cell structure is not particularly limited, but may be, for example, 50 cm ² or less. The thickness of the sheet-like cell structure is not particularly limited, but is preferably 10 μm or more, more preferably 15 μm or more, and even more preferably 20 μm or more. The upper limit of the thickness of the sheet-like cell structure is not particularly limited, but may be, for example, 5000 μm or less.

The cells constituting the sheet-like cell structure preferably form an epithelial structure. The epithelial structure formed by the cells may be a single layer of cells, a pseudostratified epithelium in which all cells are in contact with the basal surface and two or more cells are arranged in the apical-basal direction, or a stratified epithelium in which a plurality of cells are arranged overlapping each other, but is preferably a pseudostratified epithelium or a stratified epithelium. The sheet-like cell structure of the present invention may be a sheet-like structure in which a part is folded into pleats, or a sheet-like structure in which a part is contracted. Alternatively, a three-dimensional structure (a three-dimensional structure having convex and concave portions) that mimics the crypts and villus-like structures of the intestine, the Bowman's gland of the olfactory epithelium, etc. may be formed in a sheet shape by the method described in Science. 2022 Jan 7; 375 (6576). The height of the convex portions, the depth of the concave portions, and the diameters thereof of the above three-dimensional structures can be set as appropriate, but for example, the height of the convex portions and the depth of the concave portions are usually set in the range of 10 μm to 2 mm, preferably in the range of 30 μm to 1 mm, and more preferably in the range of 50 μm to 500 μm. The diameter of the three-dimensional structure is usually set in the range of 20 μm to 1 mm, preferably in the range of 50 μm to 800 μm, and more preferably in the range of 100 μm to 500 μm.

In one embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells includes a membrane that is preferably permeable to substances other than cells and the living body.

In one embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells contains at least three types of upper respiratory tract cells: 1) basal cells or their precursor cells, 2) olfactory placode cells or olfactory epithelial stem cells, and 3) olfactory nerve cells or their precursor cells.

Basal cells or their precursor cells refer to cells that express at least one basal cell marker selected from the group consisting of p63, cytokeratin 5, and p75, and preferably further express Wt1 and Lgr5.

Olfactory placode cells or olfactory epithelial stem cells refer to cells that express at least one or more markers of olfactory placode cells or olfactory epithelial stem cells selected from the group consisting of Six1, Sp8, Otx2, cytokeratin, and E-Cadherin, and preferably further express Sox2, Sox3, Dlx5, Emx2, Pax6, EpCAM, and N-Cadherin.

Olfactory nerve cells or their precursor cells refer to cells expressing at least one or more markers of olfactory nerve cells or their precursor cells selected from the group consisting of βIII tubulin, Calretinin, Ebf2, and Lhx2, and preferably further express NCAM, N-Cadherin, Ebf1, Ebf3, NeuroD, and Ascl1. Furthermore, the olfactory nerve cells contained in the sheet-shaped cell structure of the present invention preferably express olfactory receptors, and more preferably can receive olfactory information and transmit information to the central nervous system in the same way as olfactory nerve cells localized in the olfactory epithelium in the living body. Olfactory nerve cell precursor cells refer to cells that become olfactory nerve cells when matured, and also include cells that have the ability to differentiate into olfactory nerve cells. Olfactory nerve cell precursor cells preferably exhibit properties similar to those of nerve cells present in the olfactory epithelium placode.

That is, the sheet-shaped cell structure of the present embodiment is a sheet-shaped cell structure containing upper respiratory tract cells,
The upper respiratory tract cells are
1) a basal cell or a precursor cell thereof expressing at least one basal cell marker selected from the group consisting of p63, cytokeratin 5, and p75;
2) Olfactory placode cells or olfactory epithelial stem cells expressing at least one olfactory epithelial stem cell marker selected from the group consisting of Six1, Sp8, Otx2, Sox2, Dlx5, cytokeratin, and E-Cadherin; and
3) olfactory nerve cells or precursor cells thereof expressing at least one olfactory nerve cell marker selected from the group consisting of βIII tubulin, Calretinin, Ebf1, Ebf2, Ebf3, NeuroD1, and Lhx2;
(e.g., FIG. 1A and FIG. 2A).

In one embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells may further contain cells that constitute the olfactory epithelium or their precursor cells. Examples of such cells include supporting cells, Bowman's gland cells, and olfactory ensheathing cells.

Supportive cells or their precursor cells refer to cells expressing at least one selected from the group consisting of HIF2α, Sox2, Hes1, Hes5, and Six1.

Bowman's gland cells or their precursor cells refer to cells that express at least one selected from the group consisting of cytokeratin and Ascl3.

Olfactory ensheathing cells or their precursor cells refer to cells expressing at least one selected from the group consisting of S100, Sox10, and vimentin. Markers for olfactory ensheathing cells or their precursor cells can also be, for example, those described in Brain Structure and Function 222.4 (2017): 1877-1895.

The supporting cells, Bowman's gland cells, or olfactory ensheathing cells, or their precursor cells, preferably exhibit properties similar to those of supporting cells, Bowman's gland cells, or olfactory ensheathing cells, or their precursor cells, present in the olfactory epithelium or olfactory epithelium placode in vivo.

In another embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells contains at least two types of upper respiratory tract cells: 1) ciliated cells and 2) secretory cells.

Ciliated cells refer to cells that express at least one or more ciliated epithelial cell markers selected from the group consisting of FoxJ1, FoxN4, p73, acetylated tubulin, and β4-tubulin.

Secretory cells refer to cells expressing at least one secretory cell marker selected from the group consisting of mucin 1, mucin 2, mucin 3, mucin 4, mucin 5AC, mucin 5B, FoxA1, FoxA2, Nkx2.1/TTF-1, and SPDEF.

That is, the sheet-shaped cell structure of the present embodiment is a sheet-shaped cell structure containing upper respiratory tract cells,
The upper respiratory tract cells are
1) ciliated cells expressing at least one or more ciliated epithelial cell markers selected from the group consisting of FoxJ1, FoxN4, p73, MYB, acetylated tubulin, and β4-tubulin; and
2) secretory cells expressing at least one secretory cell marker selected from the group consisting of mucin 1, mucin 2, mucin 3, mucin 4, mucin 5AC, mucin 5B, FoxA1, FoxA2, Nkx2.1/TTF-1, and SPDEF;
(e.g., FIG. 1B and FIG. 2B).

In another aspect of this embodiment, the sheet-shaped cell structure comprises:
and an apical surface expressing at least one selected from the group consisting of Ezrin, Ezrin, and PKCζ;
the upper airway cells form an epithelial structure between the apical surface and a culture substrate;
The upper airway cells have epithelial cell polarity;
At least 70% of the cells constituting the sheet-shaped cell structure are localized in the epithelial structure,
The epithelial structure preferably includes basal cells in contact with the culture substrate (eg, FIG. 1 and FIG. 2).

In another embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells may further contain cells constituting the nasal epithelium or their precursor cells. Examples of such cells include club cells, basal cells, salt cells, neuroendocrine cells, and isolated chemosensory epithelial cells. The cells constituting the nasal epithelium contain at least one type of these cells, and preferably multiple types of cells.

Since the nasal epithelium is mainly derived from ectodermal non-neural epithelium, the cells that make up the nasal epithelium or their precursor cells preferably express ectodermal markers. Precursor cells of cells that make up the nasal epithelium refer to cells that become cells that make up the nasal epithelium when matured, and also include cells that have the ability to differentiate into cells that make up the nasal epithelium.

The cells constituting the nasal epithelium preferably include club cells. Club cells or their precursor cells can be defined as cells expressing at least one selected from the group consisting of SCGB1A1, SCGB3A2, Cyp2f2, uroplakin3a, Cldn10, Aox3, Pon1, and Cckar. Club cells may include Hillock cells expressing at least one selected from cytokeratin 4 and cytokeratin 13.

The cells constituting the nasal epithelium preferably include basal cells. Basal cells are located on the basal surface of non-neuroepithelial tissue and are preferably aligned on the basement membrane structure. Basal cells or their precursor cells can be defined as cells expressing at least one selected from the group consisting of cytokeratin 5, cytokeratin 14, cytokeratin 15, p63, ΔNp63, and p75.

The cells constituting the nasal epithelium preferably include salt cells. Salt cells or their precursor cells can be defined as cells that express CFTR and preferably express at least one selected from the group consisting of Foxi1, vacuolar H+-ATPase (vacuolar H-ATPase, V-ATPase), Ascl3, Tfcp2l1, and DMRT2.

The cells constituting the nasal epithelium preferably include neuroendocrine cells. Neuroendocrine cells or their precursor cells can be defined as cells expressing at least one selected from the group consisting of Ascl1, NeuroD1, Chromogranin A, Synaptophisin, and neuron-specific enolase (NSE).

The cells constituting the nasal epithelium preferably include isolated chemosensory epithelial cells. Isolated chemosensory epithelial cells or their precursor cells can be defined as cells expressing at least one selected from the group consisting of ChAT/Trpm5, Pou2f3/Skn-1a, Tas2r, Gnat3, and Plcb2.

In one embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells is a cell structure containing upper respiratory tract cells on the first main surface of the sheet-shaped cell structure and cells other than upper respiratory tract cells on the second main surface (for example, (A) and (B) in FIG. 2). The cells other than upper respiratory tract cells contained in the sheet-shaped cell structure are not particularly limited, but cells that have the effect of promoting the survival, proliferation, differentiation/maturation, and functional expression of upper respiratory tract cells on the second main surface, as well as the engraftment of the sheet-shaped cell structure into the recipient tissue, etc. are preferred.

It is also preferable that the cells other than upper respiratory tract cells contained in the another sheet-shaped cell structure are cells that interact with upper respiratory tract cells on the second main surface and transmit viruses, pathogens, etc. Examples of the cells other than upper respiratory tract cells contained in the sheet-shaped cell structure include neural crest cells, mesenchymal stem cells, mesenchymal stromal cells, vascular endothelial cells, fibroblasts, cells of the central nervous system, cells of the trachea, bronchus, and lungs, pericytes, astrocytes, microglia, choroid plexus epithelial cells, and ependymal cells. The cells other than upper respiratory tract cells contained in the sheet-shaped cell structure may be either primary cells or established cells. The cells other than upper respiratory tract cells contained in the sheet-shaped cell structure are preferably cells derived from human iPS cells.

The upper respiratory tract cells contained in the sheet-like cell structure include cells constituting the olfactory epithelium or cells constituting the nasal epithelium. The sheet-like cell structure further includes a substance-permeable membrane, and the upper respiratory tract cells adhere to one surface of the substance-permeable membrane (the surface on the first main surface side), and cells other than the upper respiratory tract cells adhere to the opposite surface (the surface on the second main surface side).

In one embodiment of the present invention, the sheet-shaped cell structure containing upper respiratory tract cells further contains a membrane, preferably permeable to substances, other than the cells and the living body. In one aspect of this embodiment, the membrane to which the cells are attached may be placed on the surface of a scaffold material, and a complex between the cell sheet and the scaffold material may be formed.

From the viewpoint of transplantation into a living body, the scaffold material is preferably a biocompatible material. Examples of such materials include hydroxyapatite, calcium phosphate such as β-TCP, inorganic compounds such as calcium carbonate, metals such as titanium, and polymeric materials. The polymers are divided into two types: synthetic polymers and natural polymers. Examples of synthetic polymers include biodegradable polyurethane, poly-L-lactic acid (PLLA), polyglycolic acid (PGA), copolymers of lactic acid and glycolic acid (PLGA), and polycaprolactone (PCL). Examples of natural polymers include collagen, gelatin, glycosaminoglycan, chitin, chitosan, hyaluronic acid, and polypeptides. The polymers are preferably biodegradable or bioabsorbable materials.

The polymer is preferably formed as a porous hydrogel having material permeability. The polymer can be processed into materials such as fibers, nonwoven fabrics, textiles, and sponges, and can be molded into ropes, sheets, tubes, blocks, and other shapes to match the shape of the target tissue, and used as a scaffolding material.

The cells contained in the sheet-shaped cell structure containing upper respiratory tract cells of the present invention preferably express factors related to infectious diseases, such as virus receptors, virus infection-related factors, and pattern recognition receptors. The virus receptors and virus infection-related factors may be receptors of viruses that infect the upper respiratory tract or virus infection-related factors. Examples of the virus receptors and virus infection-related factors include ACE2, Tmprss2, NRP1, CTSB, CTSL, BSG, HSPA5, DPP4, FURIN, ANPEP, Tmprss11D, ST6GAL1, ST3GAL4, CEACAM1, sialic acid α2,6Gal, and sialic acid α2,3Gal.

The cells contained in the sheet-shaped cell structure containing upper respiratory tract cells of the present invention may be deficient or mutated in a viral infection-related factor or a disease-related gene. That is, the upper respiratory tract cells may be deficient or mutated in one or more genes selected from the group consisting of genes related to genetic diseases or infectious diseases, and genes encoding viral receptors or viral infection-related factors. A cell population containing such cells can be used, for example, for transplant cells (transplant compositions) for regenerative medicine applications described below, disease models (e.g., upper respiratory tract disease models), and screening of compounds (e.g., disease treatment compounds). Viral infection-related factors include, for example, viral receptors and ACE2. It is expected that the deletion or mutation of viral infection-related factors will provide resistance to viral infection.

The disease-related gene is, for example, a gene associated with a genetic disease or a gene associated with an infectious disease. Cells constituting the olfactory epithelium or nasal epithelium with a deleted or mutated gene, or their precursor cells, can be obtained by differentiation from pluripotent stem cells established or genome-edited from patients with genetic diseases. Examples of diseases include cystic fibrosis, ciliary dyskinesia syndrome, Kallmann syndrome, Kartagener syndrome, mucopolysaccharidoses, asthma, etc. Another embodiment of the disease-related gene is a gene involved in cancer and tumor development of the nasal cavity or upper respiratory tract. Examples of such disease-related genes include ABL1, BRAF, DDR, EGFR, EPHA2, ERBB2, ERBB4, FGFR1, FGFR2, FLT1, FLT4, FRK, KDR, KIT, MAPK11, PDGFRA, PDGFRB, RAF1, RET, TEK, TRNAK2, VEGFA, VEGFB, VEGFC, TP53, CDKN2A, P Examples of such genes include, but are not limited to, IK3CA, PTEN, KMT2D, FBXW7, SMARCB1, PREX2, ARID1A, FANCA, IDH2, PRKDC, HRAS, NOTCH2, NOTCH1, NF1, TSC2, EPHA7, CDKN2B, ATRX, ARID2, AKT3, BRCA2, BCL6, ASXL1, AR, and ALK. Cells can also be collected from normal and diseased areas of the nasal cavity or upper respiratory tract tissue, iPS cells can be established, and the cells contained in the sheet-shaped cell structure described in the present application can be prepared.

There are no particular limitations on the method for detecting the cells and tissues contained in the sheet-like cell structure containing the produced upper respiratory tract cells, but a method that is highly reliable, reproducible, and easy to implement is preferable. Examples include optical observation under a microscope, immunostaining using antibodies against each cell or tissue marker, gene expression analysis such as real-time PCR for marker genes, single-cell RNA-Seq analysis, observation of cell type-specific microstructures under an electron microscope, transplantation into mice, rats, etc. Preferred is immunostaining using antibodies against the markers described above or single-cell RNA-Seq analysis.

The results of immunostaining can be interpreted by methods well known to those skilled in the art, such as visual inspection or quantitative analysis of the captured images using image analysis software. When interpreting the results of immunostaining, it is necessary to refer to, for example, Protein Nucleic Acid Enzyme Vol. 54 No. 2 (2009) pp. 185-192, and to eliminate false positives caused by autofluorescence, nonspecific adsorption of secondary antibodies, and leakage of fluorescence during multiple staining.

The results of single-cell RNA-Seq analysis can be used to infer the cells and tissues contained in the produced cell population, for example, by comparing the expression profile of the obtained cell population with the profiles of biological tissues in public databases, etc., and based on the similarities in gene expression and the constituent cell populations.

The microfluidic chip or biomimetic system according to this embodiment includes:
1) the sheet-shaped cell structure, and 2) at least one of the cells and cell populations other than the upper respiratory tract cells,
(For example, FIG. 3A and FIG. 3B ). The above-mentioned microfluidic chip or biomimetic system can be used in a screening method and a screening kit for a compound for treating a disease, use as a disease model of the upper respiratory tract, a method and an evaluation kit for evaluating the efficacy or toxicity of a target substance, a method for searching for a disease-specific biomarker, a composition for transplantation, isolation and culture of a virus or a microorganism, and an upper respiratory tract tissue-bearing model animal or a human tissue-bearing model animal.

One embodiment of the present invention provides an in vitro disease model of the upper respiratory tract using the above-mentioned "sheet-shaped cell structure containing upper respiratory tract cells". More specifically, the sheet-shaped cell structure containing upper respiratory tract cells can be used as a disease model of infectious diseases caused by pathogens such as viruses and microorganisms. A method for using the sheet-shaped cell structure containing upper respiratory tract cells as an in vitro disease model includes, for example, a step of contacting the structure with a pathogen, and a step of confirming the infection of the pathogen in the structure, such as detecting the proliferation of the pathogen. Furthermore, by analyzing the infected cells, it is also possible to identify target cells of the pathogen and analyze host factors. Another method for using the sheet-shaped cell structure containing upper respiratory tract cells as an in vitro disease model includes, for example, a step of contacting the sheet-shaped cell structure with a pathogen, a step of contacting a substance to be evaluated, and a step of detecting the proliferation of the pathogen, and can be used as a drug efficacy evaluation system for the substance to be evaluated. An in vitro disease model of the upper respiratory tract may be performed, for example, by the method described in WO2022/026238.

One embodiment of the present invention provides an evaluation system for gene therapy of the upper respiratory tract using the above-mentioned "sheet-shaped cell structure containing upper respiratory tract cells". More specifically, the sheet-shaped cell structure containing upper respiratory tract cells can be used as a disease model for evaluating the efficiency of introducing a target gene of a vector used in gene therapy into cells, and for infectious diseases caused by pathogens such as viruses and microorganisms. A method for using the sheet-shaped cell structure containing upper respiratory tract cells as an evaluation system for gene therapy includes, for example, a step of contacting the sheet-shaped cell structure with a vector, and a step of confirming infection of the pathogen in the structure, such as detecting the proliferation of the pathogen. Furthermore, by analyzing the infected cells, it is also possible to identify target cells of the pathogen and analyze host factors. Another method for using the sheet-shaped cell structure containing upper respiratory tract cells as an in vitro disease model includes, for example, a step of contacting the sheet-shaped cell structure with a pathogen, a step of contacting a substance to be evaluated, and a step of detecting the proliferation of the pathogen, and can be used as a drug efficacy evaluation system for the substance to be evaluated. An in vitro disease model of the upper respiratory tract may be performed, for example, by the method described in WO2022/026238.

The present invention will be described in more detail below with reference to examples, but these do not limit the scope of the present invention. In addition, the reagents and materials used are commercially available unless otherwise specified. In this specification, the criteria for judging the results of immunostaining of cells are the same as those in Preliminary Experiment 1 of WO 2020/039732. The criteria for judging the results of immunostaining can be set appropriately by a person skilled in the art, but for example, a negative control is prepared using serial sections of the target sample, in which no primary antibody is added during staining and only a fluorescently labeled secondary antibody is used, and a result showing at least three times the brightness and fluorescence intensity as the staining result of the adjacent area of the tissue on the negative control can be considered as positive.

[Experiment 1: Method for producing a cell mass containing olfactory placode cells or olfactory epithelial stem cells]
According to the culture process shown in the upper part of FIG. 4, a cell mass containing non-neuroepithelial tissue including olfactory epithelium-like tissue (a first cell population containing olfactory placode cells or olfactory epithelial stem cells) was produced from pluripotent stem cells. Human iPS cells (1231A3 strain, obtained from Riken BRC and β-actin GFP strain, purchased from Merck) were used as pluripotent stem cells. As described in International Publication No. 2020/039732, the pluripotent stem cells were maintained and cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK02N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (manufactured by Nippi Co., Ltd.) was used as the feeder-free scaffold. StemFit medium is a medium containing bFGF as an undifferentiated maintenance factor.

As a specific maintenance culture operation, first, the subconfluent human iPS cells were washed twice with 5 mM EDTA/PBS, and then further incubated with 5 mM EDTA/PBS at 37°C for 10 minutes. After removing 5 mM EDTA/PBS from the culture equipment (culture dish) after incubation, StemFit medium was added and pipetting was performed to detach the cells from the surface of the culture dish and disperse them into single cells. Thereafter, the human iPS cells dispersed into single cells were seeded on a plastic culture dish coated with Laminin511-E8, and cultured feeder-free in StemFit medium in the presence of Y27632 (ROCK inhibitor, Fujifilm Wako Pure Chemical Industries, Ltd., 10 μM). When a 6-well plate (manufactured by Corning, for cell culture, culture area 9.5 cm ² ) was used as the plastic culture dish, the seeding cell number of human iPS cells dispersed into single cells was 5×10 ³ cells/well, and when a 10 cm dish (manufactured by Corning, for cell culture, culture area 58 cm ² ) was used, the seeding cell number was 3×10 ⁴ cells/dish. One day after seeding, the entire medium was replaced with StemFit medium not containing Y27632. Thereafter, the entire medium was replaced with StemFit medium not containing Y27632 once every 1 to 2 days. The cells were cultured for 7 days after seeding until they became subconfluent (about 60% of the culture area was covered with cells).

When the cultured human iPS cells were used for differentiation induction, SB431542 (TGF-β signaling pathway inhibitor, Fujifilm Wako Pure Chemical Industries, Ltd., final concentration 5 μM, indicated as "SB431" in Figure 4, etc.) and SAG (Shh signaling pathway activator, Enzo Life Sciences, Inc., final concentration 300 nM) were added 6 days after seeding, and the cells were cultured for 24 hours (start of step (i)).

The prepared subconfluent human iPS cells were incubated in 5 mM EDTA/PBS in the same manner as in the above subculture. Serum-free medium for differentiation induction was added, and the cells were detached from the surface of the culture dish by pipetting and dispersed into single cells.

Thereafter, the human iPS cells dispersed into single cells were suspended in 100 μl of serum-free medium in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, MS-9096V, manufactured by Sumitomo Bakelite Co., Ltd.) to give 1.35 × 10 ⁴ cells per well, and cultured in suspension at 37°C and 5% CO ₂ . The serum-free medium used in this experiment was a mixture (volume ratio 1:1) of F-12+Glutamax medium (Thermo Fisher Scientific) and IMDM+Glutamax medium (Thermo Fisher Scientific), supplemented with 5% Knockout Serum Replacement (Thermo Fisher Scientific), 450 μM 1-monothioglycerol (Fujifilm Wako Pure Chemical Industries, Ltd.), 1x chemically defined lipid concentrate (Thermo Fisher Scientific), and 1x Glutamax medium (Thermo Fisher Scientific). A serum-free medium supplemented with 5% KSR gfCDM (manufactured by Nacalai Tesque, Inc.), 50 units/ml penicillin-50 μg/ml streptomycin (manufactured by Nacalai Tesque, Inc.) was used (start of step (a)). Hereinafter, this serum-free medium is also referred to as 5% KSR gfCDM.

At the start of suspension culture (day 0 after the start of suspension culture, start of step (a)), Y-27632 (final concentration 20 μM), IWP-2 (Wnt signaling pathway inhibitor, manufactured by Tocris Bioscience, final concentration 1 μM), SB431542 (TGF signaling pathway inhibitor, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., final concentration 1 μM), RepSox (TGF signaling pathway inhibitor, manufactured by Cayman Chemicals, final concentration 1 μM), and JNK-IN-8 (JNK signaling pathway inhibitor, manufactured by Merck, final concentration 1 μM) were added to the serum-free medium.

One day after the start of suspension culture, 50 μl of serum-free medium containing Y-27632, IWP-2, SB431542, RepSox, JNK-IN-8, and BMP4 (BMP signaling pathway active substance, R&D Systems, final concentration 0.5 nM) was added per well (start of step (b)).

Furthermore, on the third day after the start of suspension culture, 50 μl of serum-free medium was added per well, which did not contain BMP4 but contained Y-27632, IWP-2, SB431542, RepSox, JNK-IN-8, 5Z-7-Oxo Zeaenol (TAK1 inhibitor, manufactured by Cayman Chemicals, final concentration 2 μM), and K02288 (BMP signaling pathway inhibitor, manufactured by Cayman Chemicals, final concentration 300 nM) (start of step (c)). Thereafter, half of the medium was replaced in the same manner on the 6th, 10th, 13th, 17th, and 20th days of culture. When changing the medium after the 10th day of culture, 1 mM N-acetyl-L-cysteine (NAC), 250 μM ascorbic acid 2-phosphate (AA2P), and 500 μM nicotinamide (NAD) were added, and when changing the medium after the 13th day of culture, human recombinant EGF (PrimeGene, final concentration 20 ng/ml) was added.

On the 23rd day after the start of suspension culture, an inverted microscope (Keyence Corporation, BIOREVO) was used to perform bright-field observation and fluorescence observation of the cell aggregates derived from the β-actin GFP strain by oblique illumination (Figure 4A and B). The above differentiation induction method formed spherical cell aggregates (first cell population) with a diameter of about 400 μm to 600 μm. A thick epithelial-like structure was formed on the surface of the spherical cell aggregates, which appeared bright when observed, and showed the characteristics of a placode, which is a thickened epithelium. Fluorescence observation of the fluorescent protein GFP, which is constitutively expressed under the β-actin promoter in the β-actin GFP strain, showed that the cell aggregates had a double structure consisting of a cell aggregate portion inside the cell aggregate and a thick epithelial-like structure on the surface of the cell aggregate. The cell aggregates derived from the 1231A3 strain also had a thick epithelial-like structure formed on the outside of the cell aggregates, and had the same morphology as the cell aggregates derived from the β-actin GFP strain.

The cell aggregates derived from the 1231A3 strain on the 23rd day after the start of the suspension culture were collected in a culture tube using a 200 μl tip with a wide tip diameter, washed twice with PBS, and then fixed in 4% paraformaldehyde-phosphate buffer (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) at room temperature for 15 minutes. The fixed cell aggregates were washed three times with PBS, and then immersed in 20% sucrose/PBS at 4°C for at least one night for cryoprotection. The cryoprotected aggregates were transferred to a cryomold <No. 2> (manufactured by Sakura Finetek), and the excess 20% sucrose/PBS around the cell aggregates was removed with a micropipette, after which they were embedded in O.C.T compound (manufactured by Sakura Finetek), and rapidly frozen on an aluminum heat block cooled with dry ice to prepare blocks for frozen sections. A 10 μm-thick frozen section was prepared from the block in which the aggregates were embedded using a Leica CM1950 cryostat (Leica) and attached to a Crest slide glass or MAS-coated slide glass (Matsunami Glass Industry Co., Ltd.). The frozen section on the slide glass was surrounded by an immunohistochemistry pap pen (Daido Sangyo Co., Ltd.), and then permeabilized with 0.2% Triton-X100/TBS at room temperature for 10 minutes, followed by blocking with a mixture of blocking reagent N-102 (NOF Corporation) and SuperBlock (TBS) Blocking Buffer (Thermo Fisher Scientific) (volume ratio 1:4) at room temperature for 30 minutes.

For frozen sections after blocking, 250 μl of primary antibody diluted in SuperBlock (TBS) Blocking Buffer was added per slide and allowed to react overnight at 4°C in a humidified box. After washing the frozen sections after primary antibody treatment with 0.05% Tween-20/TBS for 10 minutes at room temperature three times, the secondary antibody was diluted with SuperBlock (TBS) Blocking Buffer, 250 μl of the diluted solution was added per slide and allowed to react for 1 hour in the dark at room temperature. After the secondary antibody treatment, the frozen sections were washed three times with 0.05% Tween-20/TBS for 10 minutes at room temperature, and then washed once with pure water. The frozen sections were then mounted using NEO cover glass (Matsunami Glass Industry Co., Ltd.) and 30 μl of Prolong Glass mounting medium (Thermo Fisher Scientific) per slide glass. The mounted specimen was left overnight in a dark place at room temperature to solidify the Prolong Glass mounting medium, and the periphery of the cover glass was coated with transparent nail polish and air-dried in a dark place at room temperature, followed by observation under a fluorescent microscope (FIG. 5). An upright fluorescent microscope Axio Imager M2 (Carl Zeiss) and its accompanying software Axio Vision were used to observe the specimens and acquire images.

The antibodies listed in Table 1 were used as primary antibodies. The fluorescently labeled antibodies listed in Table 2 were used as secondary antibodies. Multiple staining was performed using these antibodies. For nuclear counterstaining, 1 μg/ml Hoechst 33342 (Sigma Aldrich) was added to the secondary antibody dilution solution.

As a result, it was shown that an epithelial-like structure containing cells positive for Six1, Sp8, cytokeratin 18, Pax6, Sox2, EpCAM, E-Cadherin, and P-Cadherin was formed on the surface of the cell aggregates derived from the 1231A3 strain on the 23rd day of culture. In addition, the surface of the cell aggregates was Ezrin positive and was on the apical side. The inside of the cell aggregates contained many dead cells with collapsed nuclear structures. At least 80% of the live cells localized mainly on the surface of the cell aggregates were Otx2-positive ectodermal cells. In addition, proliferative Ki67-positive cells were also contained in the live cells. The results of the gene expression showed that at least 80% of the cell aggregates were ectodermal cells, and that the ectodermal cells contained proliferative olfactory placode cells or olfactory epithelial stem cells. The cell aggregates correspond to the first cell population of the present application, and it was shown that the first cell population can be produced by the differentiation induction method.

[Experiment 2: Single-cell analysis of human iPS cell-derived olfactory epithelium and nasal epithelium organoids]
Cells prepared from olfactory epithelium/nasal epithelium organoids corresponding to the first cell population derived from the human iPS cell line 1231A3 strain on the 20th day of culture prepared by the method described in Experiment 1 and the upper part of Figure 6 were used in the experiment. As a specific method, on the 20th day after the start of suspension culture, bright field observation was performed using an inverted microscope (Keyence Corporation, BIOREVO), and then single cell analysis was performed (Figure 5A). As conditions for sample preparation for single cell analysis, 9 organoids per condition were collected in a low-adsorption 15 ml tube (Sumitomo Bakelite Co., Ltd., Stemful) and washed with PBS. Then, the cells were incubated at 37 ° C. for 20 minutes with 10 U / mL papain (Nacalai Tesque) and 100 μg / mL DNase I (Worthington Biochemical Co., DP). The reaction solution used was HBSS(-) (Nacalai Tesque) supplemented with 5 mM L-cysteine (Fujifilm Wako Pure Chemical Industries, Ltd.), 6 μM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM EDTA (Nacalai Tesque, Inc.), and 10 μM Y-27632.

After the tissue dispersion treatment with papain, the cells were dispersed into single cells using a P1000 micropipette, and 4 ml of 5% KSR gfCDM was added, and impurities were removed using a cell strainer with a pore size of 40 μm. The cell suspension was centrifuged with a swing rotor (1000 rpm, 5 minutes), and after removing the supernatant, the cell pellet was suspended in 5% KSR gfCDM containing 10 μM Y-27632 and 100 μg/mL DNase I, and the number of cells was counted after incubation at 37 ° C. for 5 minutes. The suspension containing about 3 x 10 ⁵ cells was centrifuged again with a swing rotor (120 G, 5 minutes), and after removing the supernatant, 600 μl of ice-cold Sample Buffer in BD Rhapsody Cartridge Reagent Kit was added, and the cells were suspended again. The subsequent steps were performed according to the instructions included with the kit, and a cDNA library for single cell analysis was prepared using the BD Rhapsody Targeted mRNA and AbSeq Reagent Kit. The cDNA library of each sample prepared was sequenced using a large-scale sequencer HiSeqX_Ten (manufactured by Illumina), and the obtained data was subjected to primary analysis using a platform from Seven Bridge Genomics to calculate a gene expression matrix. Then, secondary analysis was performed using single cell data analysis software SeqGeq v1.8 (manufactured by FlowJo).

The obtained gene expression matrix was subjected to dimensionality reduction using principal component analysis (PCA) and uniform manifold approximation and projection (UMAP), and the plot obtained by clustering using the PhenoGraph algorithm based on the k-nearest neighbor method is shown in Figure 7. As a result of clustering, the cells contained in the organoids were classified into a cell population of non-neuroepithelium and upper respiratory tract cells including olfactory epithelium and nasal epithelium (OE/NE) and a cell population of the central nervous system (CNS). Each cell population can be further classified into subclusters, and the non-neuroepithelial/upper airway cell population contains a cluster of olfactory nerve stem cells with high expression of Dlx5, Pax6, Sp8, Six1, Otx2, Sox2, and Ki67, a cluster of olfactory nerve progenitor cells with high expression of Ebf2, NeuroD1, and Lhx2, and a cluster of immature olfactory nerve cells with high expression of Ebf2, NeuroD1, Lhx2, and Calretinin and low expression of Ki67. The central nervous system cell population contains Sox1, Pax6, and Ki67-positive central nervous nerve progenitor cells, clusters of Rax and Vsx2-positive retinal cells, other nerve nuclei, and region-specific nerve clusters (Figures 8, 9, 10, and 11).

[Experiment 3: Identification of cell surface markers of olfactory epithelium and nasal epithelium derived from human iPS cells by single cell analysis]
Using the single cell analysis data obtained by the method described in Experiment 2, we attempted to identify surface markers expressed in olfactory epithelium/nasal epithelium cell populations containing human iPS cell-derived olfactory epithelium stem cells. Specifically, we extracted genes with expression levels that differ between cell populations of non-neuroepithelium/upper respiratory tract cells, including olfactory epithelium/nasal epithelium, and other cell populations from the genes encoding 1,492 types of human cell surface proteins described in PLoS One 10: e0121314. Tables of gene expression levels obtained by the above method and plots of single cell analysis are shown in Figures 12 to 16.

As a result, it was shown that among the genes encoding human cell surface proteins, EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1 are highly expressed in cell populations of non-neuroepithelial and upper respiratory tract cells, including olfactory epithelium and nasal epithelium, and that reagents such as antibodies that specifically recognize these proteins can be applied to identify and purify olfactory epithelial and nasal epithelial cells, including olfactory epithelial stem cells, from cell clusters (Figures 12, 13, 14, 15, 16). In addition, ATP1B2, CD82, CDH6, CDON, CELSR2, CLU, CNTFR, COL11A1, CP, CRB2, DCT, DKK3, EFNB1, FBLN2, FBN2, FN1, GPM6B, ISLR2, LRP2, LRRC4B, MMRN1, PRELP, PRSS35, S1PR3, SDK2, SLC2A1, SORCS1, TFPI, THSD 7A, THY1, TMEM132B, and TSPAN18 are highly expressed in cell populations of the central nervous system, including neural progenitor cells and retinal cells, and it has been shown that reagents such as antibodies that specifically recognize these proteins can be used to remove by-products of unintended cells when identifying and purifying olfactory epithelium and nasal epithelial cells, including olfactory epithelial stem cells, from cell clusters (Figure 12).

[Experiment 4: Preparation of cell aggregates from human iPS cells and purification of EpCAM-positive olfactory placode cells or olfactory epithelial stem cells]
EpCAM-positive olfactory placode cells or olfactory epithelial stem cells were purified from cell masses containing olfactory placode cells or olfactory epithelial stem cells produced according to the culture steps described in the upper part of Fig. 17 and in Experiment 1. As a specific production method, as described in the upper part of Fig. 17, cell masses (first cell population) derived from human iPS cell line 1231A3 on the 23rd day of culture were collected in a low-adsorption 15 ml tube (Stemful, manufactured by Sumitomo Bakelite Co., Ltd.), washed with PBS, and then incubated with 10 U/mL papain (manufactured by Nacalai Tesque, Inc.) (final concentration) at 37°C for 20 minutes. The reaction solution used was HBSS(-) (Nacalai Tesque) to which 5 mM L-cysteine (Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration), 6 μM 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) (final concentration), 1 mM EDTA (Nacalai Tesque) (final concentration), and 10 μM Y-27632 (final concentration) were added, and the pH was adjusted to 7.2. The precipitated cell mass was mixed by tapping the tube about once every 5 minutes. After the above incubation treatment, the cells were dispersed into single cells by pipetting using a P1000 micropipette and a low-adsorption tip. After dispersion, an equal volume of a reaction solution containing HBSS(-) containing ₅ mM MgSO (final concentration), 200 μg/ml DNAse I (final concentration), 20 μM E-64 (cysteine protease inhibitor, Cayman Chemicals) (final concentration), and 10 μM Y-27632 (final concentration) was added to the cell suspension and incubated at room temperature for 5 minutes to inactivate papain and reduce viscosity by cleaving DNA (step (1)).

The number of viable cells in the cell suspension obtained in the step (1) was counted by trypan blue staining. The cell suspension was then centrifuged in a swing rotor (120G, 5 minutes), and the supernatant was removed. The cell pellet was then resuspended in cell separation buffer to a concentration of ^2x107 cells/ml. The cell separation buffer used was HBSS(-) to which 2% Knockout Serum Replacement (final concentration), 1 mM EDTA (final concentration), and 10 μM Y-27632 (final concentration) had been added. A portion of the cell suspension was collected in a 1.5 ml tube for analysis by FACS, and then BD Fc Block Reagent for Human (BD Biosciences) was added to the cell suspension in an amount (volume ratio) of 1/100 of the cell suspension, and the mixture was allowed to react on ice for 15 minutes. MicroBeads, human (Miltenyi Biotech, 130-061-101) that specifically recognizes CD326 (EpCAM, one of the cell surface proteins identified in Experiment 3) was added to the cell suspension after the reaction in an amount (volume ratio) of 1/10 of the reaction solution, and the mixture was allowed to react on ice for 30 minutes. During this time, the precipitated cells were resuspended by tapping about every 10 minutes. The cell suspension after the reaction was washed by adding the cell separation buffer, centrifuged with a swing rotor (120G, 5 minutes), and the supernatant was removed. The cell pellet was then resuspended in cell separation buffer to ^2x107 cells/ml. The obtained cell suspension was collected from the MACS positive fraction (second cell population) using QuadroMACS Separator and LS Columns (Miltenyi Biotech, 130-091-051 and 130-042-401) according to the instructions for the instrument (step (2)). A portion of the positive fraction was collected in a 1.5 ml tube for analysis by FACS.

APC-labeled anti-EpCAM antibody (Miltenyi Biotech, 130-111-117) was added to each of the cell population before MACS purification (first cell population) and the cell population after purification (second cell population) separated during step (2), and reacted on ice for 30 minutes. The cell separation buffer was added to each of the cell suspensions after the reaction, and the cells were washed and centrifuged (120 G, 5 minutes) with an angle rotor. After centrifugation, the supernatant was removed and the cell pellet was resuspended in cell separation buffer to 1 x 10 ⁶ cells/ml. At that time, propidium iodide (Dojindo Laboratories, Cellstain PI solution, P378) was added at a final concentration of 1 μg/ml to distinguish dead cells. The stained cell suspension was analyzed using a cell sorter (SONY, MA900) to determine the percentage of EpCAM-positive cells before and after purification ( FIG. 17 ). At this time, the unstained cell suspension was used as a negative control. As a result, about 11.5% of the cell suspension before purification (first cell population) was EpCAM-positive. Furthermore, about 88% of the cell suspension after purification (second cell population) was EpCAM-positive ( FIG. 17 , lower left), indicating that purification increases the percentage of olfactory system cells, including olfactory placode cells or olfactory epithelial stem cells, compared to the first cell population. In addition, more than 99% of the MACS-negative flow-through fraction was EpCAM-negative ( FIG. 17 , lower right).

[Experiment 5: Preparation of cell aggregates from human iPS cells using cultureware with divided microwells, and purification of EpCAM-positive olfactory placode cells or olfactory epithelial stem cells]
EpCAM-positive olfactory placode cells or olfactory epithelial stem cells were purified from cell masses containing olfactory placode cells or olfactory epithelial stem cells produced according to the culture process described in the upper part of FIG. 18. The specific method is as follows. First, 2 ml of Anti-Adherence Rinsing Solution (07010, STEMCELL Technologies) was added to the wells of AggreWell 800, 6-well plate (34821, STEMCELL Technologies). The 6-well plate was centrifuged using a swing rotor centrifuge to remove air bubbles in the solution. After centrifugation, the Rinsing Solution was removed, and each well was washed with a serum-free medium for differentiation induction described below. Thereafter, 5 ml of fresh serum-free medium was added to each well, and the wells were allowed to stand in a CO ₂ incubator for about 1 hour for equilibration.

Thereafter, step (i) was carried out, and the prepared subconfluent human iPS cell line β-actin GFP line was treated with 5 mM EDTA/PBS in the same manner as in the above-mentioned passage. When the cells were collected, serum-free medium for differentiation induction was added, and the cells were detached from the surface of the culture dish by pipetting and dispersed into single cells. The single cells were suspended in 5 ml of serum-free medium equilibrated to 5.4 x 10 ⁶ cells per well in a previously prepared AggreWell 800, 6-well plate. The single cells were then cultured in suspension under conditions of 37°C and 5% CO _2. 5% KSR gfCDM was used as the serum-free medium at that time (start of step (a)).

At the start of suspension culture (day 0 after the start of suspension culture, start of step (a)), Y27632 (final concentration 20 μM), IWP-2 (final concentration 1 μM), SB431542 (final concentration 1 μM), and JNK-IN-8 (final concentration 1 μM) were added to the serum-free medium.

After confirming the formation of cell aggregates in the microwells under a microscope one day after the start of suspension culture, 1 ml of serum-free medium containing Y27632, IWP-2, SB431542, JNK-IN-8, and BMP4 (final concentration of each, 1 nM) was added per well (start of step (b)).

Furthermore, on the third day after the start of suspension culture, cell aggregates were collected into a 37 μm Reversible Strainer Large (27250, STEMCELL Technologies) using a wide-bore 1000 μl pipette tip, and after removing dead cells and debris, they were transferred to a 10 cm suspension culture dish. The medium used at that time was 12 ml of 5% KSR gfCDM containing no BMP4, Y27632, IWP-2, SB431542, JNK-IN-8, K02288 (final concentration 1 μM), and 5Z-7-Oxo Zeaenol (final concentration of each was 2 μM) (start of step (c)). Thereafter, on days 6, 10, 13, and 17 after the start of culture, 6 ml of medium was collected from the dish while being careful not to aspirate the cell aggregates, and 6 ml of new medium was added for half-volume medium exchange. When changing the medium on day 6 after the start of culture, medium without Y27632 was used. When changing the medium on day 10 after the start of culture, N-acetyl-L-cysteine (NAC) (final concentration 1 mM), ascorbic acid 2-phosphate (AA2P) (final concentration 250 μM), and nicotinamide (NAD) (final concentration 500 μM) were added. When changing the medium on day 13 after the start of culture, EGF (final concentration 20 ng/ml) was added.

The cell mass (first cell population) on the 20th day of culture prepared by the above method was purified by MACS using the method described in Experiment 4 to obtain a second cell population (steps (1) and (2)). The first cell population and the second cell population were then analyzed by FACS (Figure 18). As a result, approximately 41.9% of the cell suspension before purification (first cell population) was EpCAM positive. Furthermore, approximately 92.8% of the cell suspension after purification (second cell population) was EpCAM positive (Figure 18, lower left), indicating that it is possible to prepare an EpCAM-positive cell population containing olfactory placode cells and olfactory epithelial stem cells by using a cultureware with divided microwells. In addition, more than 99% of the MACS-negative passing fraction was EpCAM negative (Figure 18, lower right).

[Experiment 6: Production of a sheet-shaped cell structure containing olfactory epithelium-like upper respiratory tract cells and olfactory epithelium tissue from a cell mass containing olfactory placode cells or olfactory epithelium stem cells]
The purified cells (second cell population) obtained in step (2) of the method described in Experiment 4 and Fig. 17 were suspended in a medium. The obtained cell suspension was seeded on a culture vessel to carry out step (3) described in Fig. 19, thereby producing a sheet-shaped cell structure containing upper respiratory tract cells. The medium used was Advanced DMEM/F-12 supplemented with 1/100 N2 Supplement, 1/50 B27 Supplement w/o VA, HEPES (Thermo Fisher Scientific, 12634010, 17502048, 12587010) (final concentration 10 mM), L-alanyl-L-glutamine (final concentration 2 mM), N-acetyl-L-cysteine (NAC) (final concentration 1 mM), ascorbic acid 2-phosphate (AA2P) (final concentration 250 μM), nicotinamide (NAD) (Nacalai Tesque, Inc.) (final concentration 500 μM), and Y27632 (final concentration 10 μM). The culture equipment used was a cell culture insert (Kanto Chemical Co., Ltd., ad-MED Vitrigel 2 24well, 08364-96) to which 200 μl of 20 μg/ml Synthemax-2 and 0.5 μg/ml Laminin 511E8 diluted with PBS was added, and the resulting mixture was coated at 37° C. for 1 hour or more. The seeding conditions were as follows: approximately 2×10 ⁵ purified cells were suspended in 200 μl of medium and added to the inner well of the culture insert. 800 μl of medium of the same composition but without cells was added to the outer well. The entire medium was replaced every 3 or 4 days from the start of culture.

Furthermore, to examine the medium conditions, the addition of FGF, EGF, Wnt3a+R-Spondin, Noggin, BMP4, and TGFβ signaling pathway inhibitors was examined. As a result, cell proliferation was observed with the addition of either FGF, EGF, or TGFβ signaling pathway inhibitors, and proliferation was further promoted by using these in combination (Figure 19).

Based on the above-mentioned investigation, which showed excellent cell proliferation under the conditions of adding FGF, EGF, and TGFβ signaling pathway inhibitors, further investigation of medium conditions was carried out. Specifically, the addition of Shh signaling pathway active substances, BMP4, BMP inhibitors, Wnt/porcupine inhibitors, Wnt agonists, and retinoic acid analogs was investigated. As a result, further cell proliferation was confirmed under the conditions where a combination of Shh signaling pathway active substances and BMP inhibitors was added (Figure 19). Under the above-mentioned conditions, a dense epithelial sheet-like structure with cell-to-cell adhesion was formed on the culture insert.

The sheet-like cell structures produced under conditions in which the above-mentioned FGF, EGF, and TGFβ signaling pathway inhibitors, Shh signaling pathway active substances, and BMP inhibitors were added in combination were analyzed by immunostaining on the 28th and 39th days after the start of culture (Figures 20 and 21).

As a result, the formed sheet-like cell structure was shown to contain olfactory placode cells and olfactory epithelial stem cells positive for Dlx5, Six1, Sp8, Pax6, Sox2, E-Cadherin, EpCAM, and cytokeratin at 28 days after the start of culture. In addition, it was shown to contain basal cells positive for cytokeratin 5, p63, and p75, and olfactory nerve precursor cells and immature olfactory nerve cells positive for NeuroD1, Lhx2, Calretinin, Ebf2, and Tuj1. Furthermore, the sheet-like cell structure on day 39 after the start of culture had further thickened while retaining olfactory placode cells and olfactory epithelial stem cells compared to day 28 after the start of culture, indicating that the sheet-like cell structure can be maintained in vitro for at least 15 days. The above results demonstrate that the method described in the present application can be used to produce a sheet-like cell structure containing olfactory epithelial stem cells, nerve cells, and basal cells.

[Experiment 7: Production of a sheet-shaped cell structure containing olfactory epithelium-like upper airway cells from cryopreserved purified olfactory placode cells or olfactory epithelium stem cells]
The second cell population containing EpCAM-positive olfactory placode cells or olfactory epithelial stem cells purified by the method described in Experiment 4 was frozen and then thawed and put to sleep to produce a sheet-shaped cell structure. As a specific method, the second cell population obtained by MACS-purifying cell aggregates derived from the 1231A3 strain 24 days after the start of culture was centrifuged after cell counting, and resuspended in STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) to a concentration of 4 x 10 ⁶ cells/ml. Thereafter, the cell suspension was dispensed into cryotubes (manufactured by Thermo Fisher Scientific) to a cell density of 8 x 10 ⁵ cells/200 μl/vial, and stored in a cryopreservation container (manufactured by Nippon Freezer Co., Ltd., BICELL) precooled at 4 ° C., and then left to stand at -80 ° C. for 3 hours or more, and cell cryopreservation was performed by the slow freezing method.

Then, the cryotube containing the frozen second cell population was thawed quickly while shaking in a 37°C water bath until small pieces of ice remained in the liquid. Immediately after thawing, the cell suspension containing the second cell population was mixed with about 10 ml of medium dispensed in a 15 ml tube, centrifuged (120 G, 5 minutes), and the supernatant was removed with an aspirator. The cell pellet after centrifugation was suspended in Advanced DMEM/F-12+N2B27 medium to which FGF, EGF, TGFβ signaling pathway inhibitors, Shh signaling pathway active substances, and BMP inhibitors described in Experiment 6 were added in combination, and the number of live cells was measured by cell counting. Thereafter, step (3) was carried out under the same conditions as in Experiment 6, and the cells were cultured until the 40th day after the start of culture, to produce a sheet-like cell structure. The produced sheet-like cell structure was analyzed by immunostaining in the same manner as in Experiment 6 (Figure 22).

As a result, the sheet-like cell structure formed contained olfactory placode cells and olfactory epithelium stem cells that were positive for Dlx5, Six1, Sp8, Pax6, Sox2, E-Cadherin, P-Cadherin, EpCAM, and cytokeratin, basal cells that were positive for cytokeratin 5, p63, and p75, and olfactory nerve precursor cells and immature olfactory nerve cells that were positive for NeuroD1, Lhx2, Calretinin, Ebf2, and Tuj1. From the above results, it was found that the method described in the present application can be used to produce an olfactory epithelium-like sheet-like cell structure that contains olfactory epithelium stem cells, neurons, and basal cells from the second cell population after cryopreservation and thawing through step (3).

[Experiment 8: Production of a sheet-shaped cell structure containing nasal epithelium-like upper airway cells from cryopreserved purified olfactory placode cells or olfactory epithelial stem cells]
The purified cells (second cell population) obtained in step (2) of the method described in Experiment 5, Experiment 6, and FIG. 23 were suspended in a medium and seeded on a culture vessel in step (3), to produce a sheet-shaped cell structure containing upper respiratory tract cells. As a specific method, cell aggregates derived from the β-actin GFP strain produced using a microwell plate on the 23rd day after the start of culture were purified by MACS. The second cell population obtained by purification was centrifuged after cell counting, and cryopreserved under the condition of 2x10 ⁶ cells/1 ml/vial according to the method of Experiment 6. The thawed cells were seeded at 1.8x10 ⁶ cells in a T75 cell culture flask for cell culture, and cultured for 4 days. The medium used was 20 ml of PneumaCult Ex-plus medium (manufactured by StemCell Technologies) supplemented with Y27632 (final concentration 10 μM). The cell culture flask was treated at 37° C. for at least 1 hour with 10 ml of PBS diluted with Synthemax II (manufactured by Corning) (final concentration 0.01 mg/ml) and Laminin 511-E8 (final concentration 0.005 μg/ml) to perform coating treatment.

After the cells were washed twice with 5 mM EDTA/PBS on the fourth day after the start of culture, 5 ml of TrypLE Express (Thermo Fisher) was added and treated at 37 ° C for 10 minutes. 5 ml of PneumaCult Ex-plus medium was added to the treated cells to recover the cells, and the cells were centrifuged (120 G, 5 minutes) and the supernatant was removed. The obtained cells were suspended again in PneumaCult Ex-plus medium containing Y27632 (final concentration 10 μM), and seeded in the upper well of a 24-well Transwell cell culture insert (Corning, # 3470) at 6.6 x 10 ⁴ cells / 200 μl / well and cultured for 3 days. 500 μl of medium of the same composition was added to the outer well of the culture insert. 10 ml of PBS in which Synthemax II (manufactured by Corning) (final concentration 0.01 mg/ml) and Laminin 511-E8 (final concentration 0.005 μg/ml) were diluted was added to the cell culture insert, and the insert was treated at 37° C. for at least 1 hour to perform coating treatment.

Three days after the start of culture, the entire medium in the inner well of the cells was removed, and the entire medium in the outer well was replaced with 500 μl of PneumaCult ALI medium (StemCell Technologies), and air-liquid interface culture was performed. The entire medium in the outer well was replaced every 3 or 4 days from the start of culture.

Bright-field observation was performed using an inverted microscope on the 27th day after the start of culture in a culture flask, on the 30th day after the start of culture at the start of air-liquid interface culture, and on the 37th, 44th, and 74th days after the start of culture (Figure 23). As a result, the cells, which were a single layer of epithelium like paving stones on the 27th day after the start of culture, had formed a sheet-like tissue on the 30th day after the start of culture. Furthermore, the cells matured through air-liquid interface culture, and three-dimensional protuberances were formed on the 37th, 44th, and 74th days after the start of culture. At the same time, ciliary movement was observed in cells from the 37th day after the start of culture by observation at a high magnification. Furthermore, cells on the 44th and 74th days after the start of culture were fixed and immunostained (Figure 24).

As a result, the sheet-like cell structure formed 44 days after the start of culture contained CK5, p75, p63, and ICAM-1 positive basal cells, Pax6, Sox2, and Six1 positive olfactory epithelial stem cells, and MUC5B and MUC5AC positive secretory cells. Furthermore, the sheet-like cell structure formed 74 days after the start of culture contained ciliated cells with an acetylated tubulin positive structure, as confirmed by ciliary movement through bright field observation. From the above results, it was found that the production method described in this application caused partial transdifferentiation of olfactory epithelial stem cells into nasal epithelium, and it is possible to produce a sheet-like cell structure resembling nasal epithelium that contains olfactory epithelial stem cells, ciliated cells, basal cells, and secretory cells.

[Experiment 9: Electron microscopic observation of sheet-like cell structures containing upper airway cells derived from cryopreserved purified olfactory placode cells or olfactory epithelial stem cells]
A second cell population containing EpCAM-positive olfactory placode cells or olfactory epithelial stem cells purified by the method described in Experiment 4 was cryopreserved, thawed, and put to sleep to produce a sheet-shaped cell structure. The produced sheet-shaped cell structure was then analyzed by electron microscopy. As a specific method, a first cell population, which is a cell aggregate on the 27th day after the start of culture, was prepared from a human iPS cell line β-actin GFP line by the production method using a microwell plate described in Experiment 5. The prepared first cell population was then purified by the method described in Experiment 4 to obtain a second cell population. The obtained second cell population was seeded in a 10 cm dish, cultured for 3 days, and then collected and cryopreserved.

The above-mentioned cryopreserved cells were seeded in a 12-well size vitrigel culture insert at 1.3 x 10 ⁵ cells/well and cultured for 14 days in PneumaCult Ex Plus medium. The cultured cells were then fixed to prepare samples for electron microscopy. The sample was prepared as follows. First, the cultured cells were treated overnight with 4% paraformaldehyde/2.5% glutaraldehyde, and then the cells were fixed with osmium, followed by dehydration, t-butanol freeze-drying, and platinum-palladium coating. The samples were observed using a scanning electron microscope (JSM-7900F, manufactured by JEOL Ltd.). The electron microscope images taken are shown in FIG. 25.

As a result, it was shown that the sheet-like cell structure produced had a smooth surface and a structure in which the cells were tightly bound together. Furthermore, some of the cells had cilia on their surface, a structure characteristic of respiratory system cells. The above results demonstrate that the cell structure formed by the production method described in this application is sheet-like and contains cells with characteristics of respiratory system tissue.

[Experiment 10: Method for producing a cell mass containing olfactory placode cells or olfactory epithelial stem cells]
A cell mass containing non-neuroepithelial tissue including olfactory epithelium-like tissue (a first cell population containing olfactory placode cells or olfactory epithelium stem cells) was produced from pluripotent stem cells according to the culture process shown in the upper part of Figure 26. The maintenance culture operations of the human iPS cell line and human pluripotent stem cells used were performed in the same manner as in Experiment 1.

When the cultured pluripotent stem cells were used for differentiation induction, the medium was replaced with StemFit medium 5 days after seeding and PD173074 (FGF signal pathway inhibitor, manufactured by Cayman Chemicals, final concentration 100 nM) was added at the same time, and SB431542 and SAG were added 6 days after seeding, followed by culture for approximately 48 hours and 24 hours (start of step (i)).

The subconfluent human iPS cells prepared by carrying out the above step (i) were induced to differentiate by the method described in the upper part of Figure 26 and in Experiment 1. On the 23rd day after the start of suspension culture, bright-field observation was performed using an inverted microscope (BIOREVO, manufactured by Keyence Corporation) (Figure 26A). The above differentiation induction method resulted in the formation of spherical cell aggregates (first cell population) with a diameter of about 400 μm to 600 μm. On the surface of the spherical cell aggregates, a thick epithelial-like structure was formed that appeared brightly when observed, and showed the characteristics of a placode, which is a thickened epithelium. When fluorescence observation was performed on the fluorescent protein GFP, which is constitutively expressed by the β-actin GFP strain under the β-actin promoter, it was shown that the cell aggregates had a double structure consisting of a cell aggregate portion inside the cell aggregates and a thick epithelial-like structure on the surface of the cell aggregates. The cell aggregates derived from the 1231A3 strain also had a thick epithelial-like structure formed on the outside of the cell aggregates, and had a morphology similar to that of the cell aggregates derived from the β-actin GFP strain.

Furthermore, under the same conditions as in Experiment 1, analysis was performed by immunostaining of cell aggregates derived from the 1231A3 strain. As a result, as in Experiment 1, under the conditions of Experiment 10, an epithelial structure containing cells positive for Six1, Sp8, cytokeratin 18, Pax6, Sox2, EpCAM, E-Cadherin, and P-Cadherin was formed on the surface of the cell aggregates derived from the 1231A3 strain on the 23rd day of culture (Figure 27). In addition, the surface of the cell aggregate was Ezrin positive and on the apical side. The inside of the cell aggregate contained many dead cells with collapsed nuclear structures, and at least 80% of the live cells localized mainly on the surface of the cell aggregate were Otx2 positive ectodermal cells, and Ki67 positive proliferative cells were also contained. The results of the immunostaining analysis showed that the first cell population could be produced under the conditions of Experiment 10, in which treatment with an FGF signaling pathway inhibitor was further performed in step (i).

[Experiment 11: Transplantation of a sheet-shaped cell structure containing upper respiratory tract cells into animals and creation of a model animal bearing human upper respiratory tract tissue]
The second cell population containing EpCAM-positive olfactory placode cells or olfactory epithelial stem cells derived from human iPS cell 1231A3 strain purified by the method described in Experiment 4 was cryopreserved and then thawed and put to sleep by the method described in Experiment 7 to produce a sheet-like cell structure. Thereafter, Tissue Eng Part C Methods. 2023 Nov; 29 (11): 526-534. , Japanese Patent No. 4496425, Japanese Patent No. 7066162, and Japanese Patent Publication No. 2021-24825 were referred to, and the produced sheet-like cell structure was transplanted into an animal and a human upper respiratory tract tissue model animal was produced. An overview of the test is shown in the upper part of FIG.

A collagen sponge was prepared as a scaffold material for transplantation of the sheet-shaped cell structure. The specific method for preparing the collagen sponge and the transplantation formulation, i.e., the cell sheet-scaffold material complex, is as follows.
1) 18 g of NMP collagen PS (manufactured by Nippon Ham Co., Ltd.) was added to 3 L of ultrapure water (4° C.) and stirred at 4° C. for 3 days to dissolve.
2) The collagen solution was placed on ice, 1N NaOH was added, and the solution was vigorously stirred with a glass rod until the viscosity decreased, and then further stirred at 4°C for 5 minutes using a stirrer.
3) The collagen solution was placed on ice again and 1N NaOH was added with stirring until the pH was 7.0-7.3.
4) The above collagen solution was stirred at 4° C. for 2 hours using a stirrer.
5) The collagen solution was reconfirmed to be in the pH range of 7.0-7.3. If the pH was less than 7.0, steps 3) and 4) were repeated.
6) The collagen solution was centrifuged at 3,500 g for 30 minutes at 4° C. to obtain a collagen precipitate.
7) The obtained collagen precipitate was placed in a plastic case and left to stand at -80°C overnight or longer to freeze.
8) The collagen precipitate was placed in a freeze-dryer whose trap temperature had been cooled to -80°C in advance, and freeze-dried for 4 days. If the dried collagen was not to be used immediately, it was stored in a desiccator.
9) 2.5 g of dried collagen was weighed out and suspended in 230 mL of ultrapure water.
10) The resulting suspension was stirred with a stirrer for 2 minutes, and then 14 mL of 0.1 N hydrochloric acid was added.
11) The above suspension was briefly stirred with a glass rod and then stirred every hour with a mechanical stirrer.
12) For three days, the suspension was stirred with a stirrer for 1 hour about 3 to 7 times per day, while adding 0.1 N hydrochloric acid appropriately to adjust the pH to about 3.0 to 3.3.
13) After all the collagen in the suspension was dissolved, it was used as a 1% collagen solution for preparing a collagen sponge. If not used immediately, it was stored at 4°C.
14) A polypropylene mesh (Bard mesh, Becton Dickinson Japan) coated with 17.5 mL of 1% collagen solution (4°C) was placed in a plastic container, and 17.5 mL of 1% collagen solution (4°C) was layered on top of it, followed by allowing to stand overnight at -80°C.
15) The plastic container of the collagen solution was placed in a freeze-dryer whose trap temperature had been cooled to -80°C in advance, and freeze-dried for one week.
16) The freeze-dried collagen was placed in a vacuum drying oven that had been preheated to 140° C., and heated at 140° C. while being degassed (thermal dehydration crosslinking).
17) After thermal dehydration crosslinking, the collagen was sterilized with ethylene oxide gas.
18) The upper respiratory tract cell sheet during culture, produced by the method described in Experiment 7, was cut from the culture insert together with the scaffolding vitrigel membrane, and attached so as to cover the surface of the collagen sponge prepared through the above process, to prepare a cell sheet-scaffolding material composite for transplantation.

For animal transplantation, immunodeficient rats (strain: F344-Il2rgem1IexasRag2em1Iexas, 16-20 weeks old) were anesthetized with intraperitoneal administration of medetomidine hydrochloride (0.14 mg/kg), midazolam (1.9 mg/kg), and butorphanol tartrate (2.4 mg/kg). A midline skin incision was made along the internal nasal suture of the nasal bone to expose the nasal bone. In addition, the right side of the internal nasal suture of the exposed nasal bone was incised with a drill to create a nasal defect measuring 2 mm in length and 1 mm in width. A cell sheet-scaffold material composite made of a collagen sponge covered with a vitrigel membrane on which upper airway cells had been engrafted was inserted into the nasal defect with the apex of the sheet facing the nasal cavity, and the incised skin was sutured.

One week after transplantation, the head of the transplanted rat was removed. To prepare slice samples for immunostaining, the skin, cerebrum, eyeballs, and mandible were removed after perfusion fixation. The transplanted rat head was then immersed overnight in 4% PFA and then immersed in 10% EDTA decalcification solution for 10 days for decalcification. The decalcified transplanted rat head was then immersed in 10%, 20%, and 30% sucrose solutions for one day each for dehydration. The cryoprotected sample was placed in a mold, embedded in O.C.T. compound, and frozen to create a block for sectioning. A 10 μm-thick coronal section of the transplanted rat nasal cavity was created from the created block using a cryostat.

Fluorescent immunostaining was performed on the above sections using the method described in Experiment 1. An upright fluorescent microscope Axio Imager M2 (Carl Zeiss) and its accompanying software Axio Vision, and an inverted fluorescent microscope BZ-X800 (Keyence) and its accompanying software BZ-X Viewer were used to observe the specimens and obtain images. The primary antibodies and fluorescently labeled secondary antibodies used were those listed in Tables 1, 2, and 3.

The results showed that the transplanted tissue survived in the nasal tissue of the transplanted rats. The transplanted upper respiratory tract sheet was detectable by bright-field observation using oblique illumination (Figure 28A). Furthermore, fluorescence microscopy showed that the transplanted tissue was positive for anti-human nuclear antibodies that specifically detect human cells, human Lamin B1-specific antibodies, and human NCAM-specific antibodies, and was olfactory epithelium- and nasal epithelium-like tissue that was positive for NCAM, Tuj1, EpCAM, EGFR, and cytokeratin (Figures 28B-M). The above results demonstrated that the produced upper respiratory tract cell sheet can be transplanted and engrafted in the living body, and that it is possible to create a model animal bearing human upper respiratory tract tissue.

Claims (40)

A method for producing a sheet-shaped cell structure containing upper respiratory tract cells, comprising the steps of:
(1) dispersing a first cell population containing olfactory placode cells or olfactory epithelial stem cells to obtain single cells;
(2) purifying the single cells obtained in the step (1) to obtain a second cell population containing the olfactory placode cells or olfactory epithelial stem cells, wherein the content of the olfactory placode cells and olfactory epithelial stem cells in the second cell population is higher than the content of the olfactory placode cells and olfactory epithelial stem cells in the first cell population; and
(3) seeding the second cell population obtained in the step (2) on a culture vessel, culturing the second cell population, and obtaining the sheet-shaped cell structure;
A manufacturing method comprising: At least 20% of the first cell population in step (1) are cells of ectodermal origin,
The method according to claim 1, wherein the ectoderm-derived cells contain cells expressing at least one olfactory placode cell marker or olfactory epithelium stem cell marker selected from the group consisting of Six1, Sp8, Otx2, cytokeratin, and E-cadherin. The first cell population in step (1) further comprises cells of the central nervous system,
The cells of the central nervous system
The method according to claim 1 or 2, wherein the cells 1) express at least one central nervous system marker selected from the group consisting of Pax6, Sox1, Sox2, Sox3, Dlx5, FoxG1, N-Cadherin, MAP2, RAX, and VSX2, and 2) do not express cytokeratin. The first cell population in the step (1) includes 1) a non-neuroepithelial tissue portion including the olfactory placode cells or olfactory epithelial stem cells, and 2) a neural tissue portion including neural cells or their precursor cells,
A three-dimensional tissue or cell mass in which at least a portion of the surface of the nerve tissue section is covered with the non-neuroepithelial tissue section,
The method according to claim 1 or 2, wherein the nervous system cells or their precursor cells include nervous system cells constituting the central nervous system or their precursor cells. The first cell population in the step (1) comprises: 1) a non-neuroepithelial tissue portion containing the olfactory placode cells or olfactory epithelial stem cells; and 2) a neural tissue portion containing neural cells or their precursor cells; and
the non-neuroepithelial tissue portion and the neural tissue portion are attached to the same surface of the culture substrate,
The method according to claim 1 or 2, wherein the nervous system cells or their precursor cells include nervous system cells constituting the central nervous system or their precursor cells. The method according to claim 1 or 2, wherein the first cell population in step (1) is derived from pluripotent stem cells. The first cell population in the step (1)
(a) culturing pluripotent stem cells in the presence of a Wnt signaling pathway inhibitor;
(b) culturing the cells cultured in step (a) in the presence of a BMP signaling pathway agent; and
(c) culturing the cells cultured in the step (b) in the presence of at least one substance selected from the group consisting of an FGF signaling pathway agonist and a BMP signaling pathway inhibitor, thereby producing the first cell population containing the olfactory placode cells or olfactory epithelial stem cells;
The method according to claim 1 or 2, wherein the composition is produced by a method comprising the steps of: Prior to the step (a), the pluripotent stem cells are cultured in the absence of feeder cells,
1) at least one selected from the group consisting of a TGFβ family signaling pathway inhibitor, a sonic hedgehog signaling pathway agonist, and an FGF signaling pathway inhibitor;
2) Undifferentiated cell maintenance factor;
The method of claim 7, further comprising carrying out step (i) of culturing in a medium comprising: The step (2) includes purifying the first cell population to obtain the second cell population by recovering the olfactory placode cells or olfactory epithelial stem cells using a reagent having specific affinity for a cell surface marker of a target cell,
The method according to claim 1 or 2, wherein the cell surface marker of the target cell comprises at least one selected from the group consisting of EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1. The step (2) further comprises purifying the first cell population by removing the non-target cells using a reagent having specific affinity for a cell surface marker of the non-target cells to obtain the second cell population;
The method of claim 9, wherein the cell surface marker of the non-target cells includes at least one selected from the group consisting of ATP1B2, CD82, CDH6, CDON, CELSR2, CLU, CNTFR, COL11A1, CP, CRB2, DCT, DKK3, EFNB1, FBLN2, FBN2, FN1, GPM6B, ISLR2, LRP2, LRRC4B, MMRN1, PRELP, PRSS35, S1PR3, SDK2, SLC2A1, SORCS1, TFPI, THSD7A, THY1, TMEM132B, and TSPAN18. The method according to claim 9, wherein the step (2) comprises purifying the first cell population to obtain the second cell population by fluorescence activated cell sorting (FACS) or magnetic bead cell sorting (MACS). The step (2) further comprises cryopreserving the second cell population;
The method of claim 1 or 2, wherein the step (3) further comprises thawing the cryopreserved second cell population. The method according to claim 1 or 2, wherein the step (3) comprises culturing the second cell population in the presence of at least one selected from the group consisting of a sonic hedgehog signaling pathway activator and a BMP signaling pathway inhibitor. The method of claim 13, wherein the Sonic Hedgehog signaling pathway active substance comprises at least one selected from the group consisting of Sonic Hedgehog protein, SAG, Purmorphamine, 20(S)-hydroxycholesterol, and GSA-10. The BMP signaling pathway inhibitor is a type I BMP receptor inhibitor,
The method according to claim 13, wherein the type I BMP receptor inhibitor comprises at least one selected from the group consisting of K02288, Dorsomorphin, LDN-193189, LDN-212854, LDN-214117, ML347, DMH1, DMH2, Compound 1, VU5350, OD52, E6201, Saracatinib, BYL719 and derivatives thereof. The method according to claim 1 or 2, wherein the step (3) comprises culturing the second cell population in the presence of at least one selected from the group consisting of a ROCK inhibitor, an FGF signaling pathway active substance, an EGF signaling pathway active substance, a TGFβ family signaling pathway inhibitor, a Wnt signaling pathway active substance, and a retinoic acid signaling pathway active substance. The TGFβ family signaling pathway inhibitor is an Alk5/TGFβR1 inhibitor,
The Alk5/TGFβR1 inhibitors include SB431542, SB505124, SB525334, LY2157299, GW788388, LY364947, SD-208, EW-7197, A83-01, A77-01, RepSox, BIBF-0775, TP0427736, TGFBR1-IN-1, SM-16, TEW-7197, LY3200882, LY2109761, R268712, IN1130, Galunisertib, AZ12799734, KRCA 0008, GSK The method according to claim 16, comprising at least one selected from the group consisting of 1838705, Crizotinib, Certinib, ASP 3026, TAE684, AZD3463 and derivatives thereof. The method according to claim 1 or 2, wherein the step (3) comprises culturing the second cell population on a substance-permeable membrane provided on the culture vessel. The method according to claim 7, wherein the Wnt signaling pathway inhibitor has at least one activity selected from the group consisting of 1) inhibitory activity against the non-canonical Wnt pathway, 2) inhibitory activity against the extracellular secretion of Wnt family proteins, 3) inhibitory activity against porcupine, and 4) inhibitory activity against ROR. the Wnt signaling pathway inhibitor 1) has an inhibitory activity against a non-canonical Wnt pathway, the inhibitory activity being an inhibitory activity against acylation or palmitoylation of a target protein by porcupine;
The method according to claim 19, wherein the substance having inhibitory activity comprises at least one selected from the group consisting of IWP-2, IWP-3, IWP-4, IWP-L6, IWP-12, IWP-O1, LGK-974, Wnt-C59, ETC-131, ETC-159, GNF-1331, GNF-6231, Porcn-IN-1, RXC004, CGX1321, AZD5055, and derivatives thereof. The method according to claim 19, wherein the Wnt signaling pathway inhibitor comprises at least one selected from the group consisting of XAV-939, KY02111, KY03-I, and derivatives thereof. The method according to claim 7, wherein one or more of steps (a) to (c) are carried out in the presence of at least one selected from the group consisting of a TGFβ family signaling pathway inhibitor, a ROCK inhibitor, and a JNK signaling pathway inhibitor. The method according to claim 7, wherein step (c) is carried out in the presence of at least one selected from the group consisting of a TAK1 inhibitor, an FGF signaling pathway active substance, and an EGF signaling pathway active substance. A sheet-shaped cell structure containing upper respiratory tract cells, produced by the production method described in claim 1 or 2. A sheet-shaped cell structure comprising upper respiratory tract cells,
The upper respiratory tract cells are
1) a basal cell or a precursor cell thereof expressing at least one basal cell marker selected from the group consisting of p63, cytokeratin 5, and p75;
2) Olfactory placode cells or olfactory epithelial stem cells expressing at least one olfactory epithelial stem cell marker selected from the group consisting of Six1, Sp8, Otx2, Sox2, Dlx5, cytokeratin, and E-Cadherin; and
3) olfactory nerve cells or precursor cells thereof expressing at least one olfactory nerve cell marker selected from the group consisting of βIII tubulin, Calretinin, Ebf1, Ebf2, Ebf3, NeuroD1, and Lhx2;
A sheet-like cell structure comprising: A sheet-shaped cell structure comprising upper respiratory tract cells,
The upper respiratory tract cells are
1) ciliated cells expressing at least one or more ciliated epithelial cell markers selected from the group consisting of FoxJ1, FoxN4, p73, MYB, acetylated tubulin, and β4-tubulin; and
2) secretory cells expressing at least one secretory cell marker selected from the group consisting of mucin 1, mucin 2, mucin 3, mucin 4, mucin 5AC, mucin 5B, FoxA1, FoxA2, Nkx2.1/TTF-1, and SPDEF;
A sheet-like cell structure comprising: The sheet-shaped cell structure is
and an apical surface expressing at least one selected from the group consisting of Ezrin, Ezrin, and PKCζ;
the upper airway cells form an epithelial structure between the apical surface and a culture substrate;
the upper airway cells have epithelial cell polarity;
At least 70% of the cells constituting the sheet-shaped cell structure are localized in the epithelial structure,
The sheet-shaped cell structure according to claim 25 or 26, wherein the epithelial structure comprises basal cells in contact with the culture substrate. The sheet-shaped cell structure according to claim 25 or 26, wherein the sheet-shaped cell structure contains upper respiratory tract cells expressing at least one virus receptor or virus infection-related factor selected from the group consisting of ACE2, Tmprss2, NRP1, CTSB, CTSL, BSG, HSPA5, DPP4, FURIN, ANPEP, Tmprss11D, ST6GAL1, ST3GAL4, CEACAM1, sialic acid α2,6Gal, and sialic acid α2,3Gal. The sheet-shaped cell structure according to claim 25 or 26, wherein the upper respiratory tract cells are deficient or mutated in one or more genes selected from the group consisting of genes associated with genetic diseases or infectious diseases, and genes encoding viral receptors or viral infection-related factors. 1) The sheet-shaped cell structure according to claim 25 or 26, and 2) at least one of the cells and cell populations other than upper respiratory tract cells,
A microfluidic chip or biomimetic system comprising: A method for screening a compound for treating a disease using an apparatus, chip, system, or equipment containing the sheet-shaped cell structure according to claim 25 or 26. A screening kit for a compound for treating a disease, comprising an apparatus, chip, system, or equipment containing the sheet-shaped cell structure according to claim 25 or 26. A method for using a device, chip, system, or equipment containing the sheet-shaped cell structure according to claim 25 or 26 as a disease model for the upper respiratory tract. A method for evaluating the efficacy or toxicity of a target substance using an instrument, chip, system, or device containing the sheet-shaped cell structure according to claim 25 or 26. A kit for evaluating the efficacy or toxicity of a target substance, comprising an instrument, chip, system, or device containing the sheet-shaped cell structure according to claim 25 or 26. A method for searching for disease-specific biomarkers using an instrument, chip, system, or device containing the sheet-shaped cell structure according to claim 25 or 26. A transplant composition comprising an instrument, chip, system, or device containing the sheet-shaped cell structure according to claim 25 or 26. Use of an instrument, chip, system, or device containing the sheet-shaped cell structure according to claim 25 or 26 in the isolation and culture of viruses or microorganisms. An upper airway tissue model animal or human tissue model animal to which the sheet-shaped cell structure according to claim 25 or 26 has been transplanted. A method for purifying olfactory placode cells or olfactory epithelial stem cells contained in a sample, comprising the steps of:
and recovering the olfactory placode cells or olfactory epithelial stem cells from the sample using a reagent having a specific affinity for a cell surface marker of the olfactory placode cells or olfactory epithelial stem cells;
The cell surface marker of the olfactory placode cell or olfactory epithelial stem cell comprises at least one selected from the group consisting of EpCAM, E-Cadherin, P-Cadherin, Cldn4, Cldn6, Cldn7, LAM1B, FREM2, TACSTD2, IGSF1, F11R, FRAS1, and SORL1.

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