Method for producing nail matrix cells

Abstract

The present invention provides a method for producing nail matrix cells, the method including a step for performing shaking culturing of nail stem cells under suspension culture conditions in the presence of a Wnt signal transduction promoting substance, an FGF signal transduction promoting substance, and an adrenocortical hormone.

Description

Nail matrix production method

[0001] The present invention relates to a method for producing nail matrix cells, nail matrix cells produced by the method, and a therapeutic agent for fingertip disorders containing the cells. The present invention also relates to nail matrix cells containing a reporter gene and a method for screening for a preventive or therapeutic agent for fingertip disorders using the cells.

[0002] Nails are important tissues that are evolutionarily conserved in all known terrestrial mammals. Human nails are particularly important for digit function, sensation, protection, and aesthetics. Congenital malformations, fingertip injuries, and anti-cancer chemotherapy can cause intractable nail abnormalities, but there are no effective treatments for severe cases.

[0003] The nail matrix, a population of nail stem or progenitor cells, generates nails throughout life. The nail matrix actively divides asymmetrically, and its progeny gradually keratinize to form mature nails. Embryologically, a digit rudiment (initially called the limb bud) emerges from the lateral body wall on day 24 of gestation. The limb bud, which has a mesenchymal core and an epithelial cap, elongates to form arms and legs, stimulated by FGF8 and Wnt proteins secreted by the apical ectodermal ridge (AER), similar to the amphibian blastocyst, and then segments. Segmentation ensues with segment-specific expression of homeobox genes, and the final, most distal segment, expressing Hoxd13, forms the digit. The nail rudiment appears as an epidermal thickening near the distal end of the dorsal digit at approximately 10 weeks of gestation. The nail matrix forms from the proximal nail fold at approximately 16 weeks.

[0004] It has previously been reported that limb buds that can become forelimbs and hindlimbs were successfully differentiated from mouse pluripotent stem cells (Non-Patent Document 1). However, to date, there have been no reports of nail matrix cells being produced from pluripotent stem cells.

[0005] Mori S, et al., Nat Commun. 2019;10: 3802

[0006] Therefore, an objective of the present invention is to provide a method for producing nail matrix cells, and also to provide nail matrix cells produced by the method, a therapeutic agent for fingertip disorders such as nail disorders containing the cells, and a method for screening for substances for preventing or treating fingertip disorders such as nail disorders using the cells.

[0007] The present inventors conceived the idea that organoids containing nail matrix cells could be obtained by further inducing differentiation of the limb bud organoids described in Non-Patent Document 1. Therefore, when they attempted to induce differentiation of pluripotent stem cells into limb bud organoids using the method described in Non-Patent Document 1, they unexpectedly observed a high level of cell death. Therefore, to prevent cell death, they changed the method to allow cells to grow to a certain extent in the early stages of differentiation, and then milder environmental changes to allow a soft landing. This resulted in efficient induction of limb bud organoids. Furthermore, after repeated trial and error to further induce differentiation of the limb bud organoids induced in this way, they successfully differentiated the organoids into finger organoids by shaking culture under suspension culture conditions in the presence of a Wnt signaling promoter, an FGF signaling promoter, and adrenocortical hormones. They concluded that the finger organoids produced in this way contained nail matrix cells and primitive nails. To the inventors' knowledge, there had been no reports of nail matrix cell induction in vitro, so this success was surprising.

[0008] Furthermore, we found that in finger organoids, Lgr5 protein-expressing cells are present near Lgr6 protein-expressing cells, and that there are also cells co-expressing both. From these results, the inventors suggested a strong relationship between Lgr5-positive cells and Lgr6-positive cells, and concluded that the Lgr6-positive nail matrix cells contained in finger organoids are likely derived from Lgr5-positive nail stem cells. Furthermore, the inventors designed a reporter gene under the control of the transcriptional regulatory region of the Lgr6 gene and differentiated finger organoids containing nail matrix cells from pluripotent stem cells into which the gene had been introduced. It was confirmed that the reporter protein was also expressed in Lgr6-expressing cells. Based on these findings, the inventors conducted further research and completed the present invention.

[0009] That is, the present invention provides the following: [1] A method for producing nail matrix cells, comprising a step of shaking culture of nail stem cells under suspension culture conditions in the presence of a Wnt signaling promoter, an FGF signaling promoter, and adrenocortical hormone. [2] The method according to [1], wherein the nail stem cells are contained in limb bud organoids. [3] The method according to [1] or [2], wherein the nail matrix cells are contained in digital organoids. [4] The method according to any one of [1] to [3], wherein the Wnt signaling promoter is CHIR98014. [5] The method according to any one of [1] to [4], wherein the FGF signaling promoter is bFGF and / or FGF8. [6] The method according to any one of [1] to [5], wherein the adrenocortical hormone is hydrocortisone. [7] The method according to any one of [1] to [6], wherein the nail stem cells are derived from pluripotent stem cells. [8-1] The method according to [7], wherein the pluripotent stem cells are derived from humans. [8-2] The method according to any one of [1] to [8-1], wherein the nail stem cells are of human origin. [9] A nail matrix cell or a finger organoid comprising said cell, produced by the method according to any one of [1] to [8-2].

[10] A therapeutic agent for fingertip disorders, comprising the nail matrix cell or finger organoid according to [9].

[11] The agent according to

[10] , wherein the fingertip disorder is a nail disorder.

[12] A nail matrix cell having a nucleic acid comprising a reporter gene under the control of a transcriptional regulatory region of the Lgr6 gene.

[13] The nail matrix cell according to

[12] , produced by the method according to any one of [1] to [8-2].

[14] The nail matrix cell according to

[12] or

[13] , wherein the reporter gene is a gene encoding a fluorescent protein.

[15] A method for screening for a preventive or therapeutic agent for fingertip disorders, comprising the following steps (1) to (3): (1) contacting the nail matrix cells according to any one of

[12] to

[14] with a test substance; (2) measuring the expression level of a reporter protein in the cells; and (3) selecting the test substance as a candidate for a preventive or therapeutic agent for fingertip disorders when the expression level measured in step (2) is higher than the expression level in a control group not contacted with the test substance or the expression level measured in the absence of the test substance.

[16] The method according to

[15] , wherein the fingertip disorder is a nail disorder.

[17] A method for treating a fingertip disorder, comprising transplanting an effective amount of the nail matrix cell or digital organoid according to [9] into a subject.

[18] The nail matrix cell or digital organoid according to [9] for use in treating a fingertip disorder.

[19] Use of the nail matrix cell or digital organoid according to [9] in the manufacture of a therapeutic agent for a fingertip disorder.

[0010] The present invention makes it possible to provide nail matrix cells or finger organoids in vitro, enabling the development of regenerative medicine by transplanting the nail matrix cells or finger organoids into patients with fingertip disorders such as nail disorders. Nail regeneration, in particular, can be extremely important for the quality of life (QOL) of patients with nail disorders. It also makes it possible to provide finger organoids with a low risk of tumorigenesis due to the contamination of undifferentiated cells when transplanted into a living organism. Furthermore, the present invention provides nail matrix cells carrying nucleic acids containing a reporter gene under the control of the transcriptional regulatory region of the Lgr6 gene, enabling the use of the nail matrix cells to screen for preventive or therapeutic drugs for fingertip disorders.

[0011] The differentiation scheme of digital organoids and the early expression of limb bud-related proteins are shown. (A) Differentiation scheme; (B) Immunofluorescent staining of early spheres. Scale bar = 200 μm. The time course of gene expression from pluripotent stem cells through limb bud formation to digital organoids is shown. Gene expression levels were normalized to the expression level of internal ribosomal protein S18. **p < 0.01, *p < 0.05. Data are shown as mean ± SD. Immunohistochemical detection of Lgr6 and sclerokeratin is shown. Immunohistochemical staining detected the expression of (A) Lgr6, (B) keratin 17, and (C) keratin 81 proteins. Scale bar = 200 μm. Immunohistochemical detection of Lgr5 and Lgr6 is shown. Immunohistochemical staining detected the presence of Lgr5 protein-expressing cells in close proximity to Lgr6 protein-expressing cells, as well as the presence of cells co-expressing both proteins. Scale bar = 50 μm. Functionality of Lgr6-GFP reporter iPSCs is demonstrated. (A) Design of the Lgr6-GFP reporter gene in the piggyback system. (B) Expression of reporter GFP in organoids on day 44. (C) Immunohistochemical detection of Lgr6 protein and reporter GFP. Scale bar = 200 μm. The sequences in the figure are listed in the sequence listing as SEQ ID NOs: 20 and 21, from top to bottom. Overall view of the comprehensive comparative gene expression analysis between undifferentiated iPS cells and digital organoids. The mRNA expression level of each gene was quantified using RNA sequencing analysis, and the expression ratio (horizontal axis) and significance (P value) are shown. On the horizontal axis: negative log2 (fold change) indicates higher expression in iPS cells, while positive log2 indicates higher expression in digital organoids. The upward movement of the vertical axis (larger -log10 value) indicates a smaller P value, indicating a higher probability of a statistical difference. The ratio of the number of gene sequences per given number of sequences analyzed by RNA sequencing: FPKM (fragments per kilobase of exon per million reads mapped). The table below shows the ratio of the number of sequence bases present for each gene relative to the total number of bases that could be sequenced from the RNA. Transcriptional regulation-related genes involved in the development of limbs and digits are shown in A, extracellular trophic factors, etc. in B, and keratin groups in C.RNA sequencing analysis shows the results of a comparison of specific gene expression levels between undifferentiated iPS cells and finger organoids. For the genes shown in Figure 7, the expression levels of finger organoids were compared to those of iPS cells, and the bar graph shows the log2 (fold change) plot. Purification of Lgr6-expressing cells, preparation of cell clusters, and subcutaneous dorsal transplantation and establishment. Figure A shows the Lgr6 reporter iPS cells prepared in Example 5 differentiated into finger organoids as described in Examples 1 and 2. Fluorescent cells (GFP-positive cells) were collected using a cell sorter (FACS AriaIIIBD). The cell clusters were prepared in a non-adhesive 96-well round-bottom flask (SUMILON MS-9096U, M) (scale bar = 100 μm). Figure B shows a section of the cell clusters formed 20 days after subcutaneous transplantation of the cells shown in Figure 9A, which were mass-produced using an EZsphere 10 cm dish (AGC Technoglass). The host mouse erythrocytes (stained for mouse-specific Ter119 antigen with Thermo Scientific rat anti-mouse Ter-119 antibody (model number: 12-5921-82)) were surrounded by transplanted human cells (stained for human-specific nuclear antigen (hNA) with Chemicon mouse antibody (model number: MAB1281)), demonstrating successful engraftment through the host's circulation (scale bar, left: 50 μm, right: 10 μm). Figure C shows, from left to right, the presence of Lgr6 reporter GFP in the transplanted human cells (hNA-positive cells) (scale bar = 100 μm), along with the coexistence of nail-forming sclerotins KRT17 and KRT81 (scale bar = 100 μm). The Lgr6-expressing cell cluster was transplanted subcutaneously into the fingertip and was present in the nail matrix region. Histological images are shown 20 days after transplantation of approximately 300 Lgr6-positive cell clusters, prepared as in Figure 9, into the fingertip of an immunodeficient mouse. Cells immunostained for Lgr6 reporter GFP using Santacruz antibody SC-393010 are present in the fingertip tissue and nail matrix region (NM, indicated by white arrows) (scale bar = 200 μm). The nail is indicated by NP (nail plate).

[0012] 1. Method for Producing Nail Matrix Cells The present invention provides a method for producing nail matrix cells from nail stem cells (hereinafter, sometimes referred to as the "production method of the present invention"). Specifically, the present invention provides a method for producing nail matrix cells, which includes a step of shaking culture of nail stem cells under suspension culture conditions in the presence of a Wnt signaling promoter, an FGF signaling promoter, and adrenocortical hormone.

[0013] In this specification, unless otherwise specified, "cell" includes "cell population". Furthermore, "cell population" includes "cell aggregate" and "organoid". Furthermore, unless otherwise specified, "cell" refers to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

[0014] Nail matrix cells are cells derived from nail stem cells in vivo, actively dividing asymmetrically, and their progeny cells gradually keratinize to form mature nails. As used herein, "nail matrix cells" refer to cells that express Lgr6 and have the potential to differentiate into nail cells that express sclerokeratins (e.g., keratin 17 (Krt-17), keratin 81 (Krt-81), etc.). Furthermore, as used herein, "nail stem cells" refer to stem cells that express Lgr5 and have the potential to differentiate into nail matrix cells.

[0015] The nail stem cells used in the present invention may be, for example, an already established cell line, or may be selected from biological tissue (e.g., finger tissue) or the limb bud organoids described below using a cell marker such as Lgr5 as an indicator, using methods such as flow cytometry or mass cytometry, magnetic cell separation, or an affinity column on which a desired antigen is immobilized.

[0016] In addition, in the present invention, nail stem cells can be used in any form: single cell, cell population containing said cells, and cell aggregates or organoids containing said cells (such as limb bud organoids), but preferably in the form of limb bud organoids.In addition, nail matrix cells obtained by the production method of the present invention can be used in any form: single cell, cell population containing said cells, and cell aggregates or organoids containing said cells (such as finger organoids), but preferably in the form of limb bud organoids.Therefore, in another embodiment, a method for producing nail matrix cells or finger organoids containing said cells is provided, which comprises the step of shaking culture of limb bud organoids under suspension culture conditions in the presence of a Wnt signaling promoter, an FGF signaling promoter and adrenocortical hormones.

[0017] In this specification, "organoid" refers to a structure that contains multiple types of cells, and typically has a structure and / or function similar to that of tissue in vivo.Whether a certain structure is organoid can be determined, for example, by carrying out staining (e.g., immunostaining, hematoxylin-eosin (HE) staining, etc.) on a sample as needed, and then observing the localization of cells and the presence or absence of layer structure formation under a microscope.

[0018] As used herein, "finger organoid" refers to an organoid comprising nail matrix cells, and typically, the organoid further comprises nail cells. In one embodiment, the "finger organoid" of the present invention has nail cells expressing Krt-81 and nail cells expressing Krt-17 in the vicinity of nail matrix cells expressing Lgr6. In addition, in addition to nail matrix cells expressing Lgr6, nail cells expressing Krt-81 and nail cells expressing Krt-17, finger organoids may also contain at least one (preferably all) of the following: cells expressing Hoxd13 gene, which is essential for the development of fingers and wrists; cells expressing forelimb-specific Tbx5 gene; cells expressing hindlimb-specific Pitx1 gene; and cells expressing Runx2 gene, which is involved in bone formation; and cells expressing Lgr5. The finger organoids may express at least one (preferably all) of the Hox genes selected from the group consisting of Lgr6 and / or Lgr5, and / or Hoxa11, Hoxb7, Hoxd12 and Hoxd13. The finger organoids may also express at least one (preferably all) of the genes associated with morphology selected from the group consisting of BMP2, BMP4, BMP7, FGF8, FGF18, FGF19, TGFB1 and WNT5A, and / or at least one (preferably all) of the keratin genes selected from the group consisting of Krt-6A, Krt-6C, Krt-16, Krt-23 and Krt-31, and may not express at least one (preferably all) of the genes associated with an undifferentiated state selected from the group consisting of LIN28A, POU5F1 and ESRG. In one embodiment, the finger organoids of the present invention exhibit the gene expression pattern shown in Figure 8, but the finger organoids of the present invention are not limited to those exhibiting these gene expression patterns.

[0019] Limb buds are the primordium of vertebrate limbs, originating from a portion of the lateral plate mesoderm during early embryogenesis. A single layer of epithelial cells bulges outward, covering the mesenchymal tissue. The apical ectodermal ridge (AER) is located at the dorsoventral boundary of the distal end of the limb bud, while the zone of polarizing activity (ZPA) is located posteriorly. These play an important role as signaling centers for limb bud outgrowth. As used herein, "limb bud organoid" refers to an organoid containing a region containing lateral plate mesoderm cells and E-cadherin-expressing epithelial tissue surrounding the region. Examples of such lateral plate mesoderm cells include cells expressing Hand2 and / or Pitx1. Limb bud organoids also typically contain nail stem cells expressing Lgr5.

[0020] As used herein, the term "Wnt signaling promoter" refers to a substance that activates a signaling pathway that utilizes Wnt family proteins as ligands and primarily Frizzled as a receptor. Examples of such signaling pathways include the canonical Wnt pathway and the non-canonical Wnt pathway. The canonical Wnt pathway is mediated by β-catenin. Non-canonical Wnt pathways include the planar cell polarity (PCP) pathway, Wnt / JNK pathway, Wnt / Calcium pathway, Wnt-RAP1 pathway, Wnt-Ror2 pathway, Wnt-PKA pathway, Wnt-GSK3MT pathway, Wnt-aPKC pathway, Wnt-RYK pathway, and Wnt-mTOR pathway. In the non-canonical Wnt pathway, there are common signaling factors that are also activated in signaling pathways other than Wnt. Activators of these factors are also included in Wnt signaling pathway inhibitors. Examples of Wnt signaling promoters include GSK3 inhibitors and Wnt proteins. These substances may be used alone or in combination.

[0021] Examples of GSK3 inhibitors include CHIR98014 (N-6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), 1-Azakenpaullone (9-bromo-7,12-dihydro-pyrido[3',2':2,3]azepino[4,5-b]indol-6(5H)-one), and AZD 2858 (3-amino-6-[4-[(4-methyl-1- piperazinyl)sulfonyl]phenyl]-N-3-pyridinyl-2-pyrazinecarboxamide), BIO(6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3- dihydro-(3Z)-2H-indol-2-one), CP21R7 (3-(3-aminophenyl)-4-(1-methyl-1H-indol3-yl)-1H-pyrrole-2,5-dione), Sotrastaurin (3-(1H-indol-3-yl)-4-[2-(4-methyl1-piperazinyl)-4-quinazolinyl]-1Hpyrrole-2,5-dione), TWS119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol), Valproic Acid (2-propyl-pentanoic acid), and the like are included, and CHIR98014 is preferred.Examples of Wnt proteins include Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16, with Wnt3 being preferred.

[0022] The concentration of the Wnt signaling promoter in the medium can be appropriately set depending on the substance used. For example, when CHIR98014, a GSK3 inhibitor, is used as the Wnt signaling promoter, its concentration is usually 0.1 μM to 100 μM, preferably 1 μM to 20 μM, and more preferably 4 μM to 10 μM (7 μM in one embodiment). When a Wnt signaling promoter other than CHIR98014 is used, the concentration of the Wnt signaling promoter in the medium is appropriately selected.

[0023] The FGF signaling promoter 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 promoters include FGF proteins such as FGF1, FGF2 (also known as bFGF), FGF3, FGF8, and FGF10, anti-FGF receptor antibodies, and FGF partial peptides. These substances may be used alone or in combination.

[0024] The FGF signaling promoter is preferably at least one selected from the group consisting of FGF2, FGF3, FGF8, FGF10, and modified forms thereof, and more preferably at least one of FGF2 and FGF8 (preferably both).

[0025] The concentration of the FGF signaling promoter in the medium can be appropriately set depending on the substance used. For example, when FGF2 is used as the FGF signaling promoter, the concentration is usually 10 pg / ml to 1 μg / ml, preferably 100 pg / ml to 100 ng / ml, and more preferably 1 ng / ml to 20 ng / ml (in one embodiment, 10 ng / ml). When FGF8 is used, the concentration is usually 10 pg / ml to 1 μg / ml, preferably 100 pg / ml to 100 ng / ml, and more preferably 1 ng / ml to 20 ng / ml (in one embodiment, 10 ng / ml). When an FGF other than FGF2 and FGF8 is used, the concentration of FGF in the medium is appropriately selected.

[0026] Adrenal cortical hormones are hormones produced in the adrenal cortex, and examples of the adrenal cortical hormones used in the present invention include natural glucocorticoids such as hydrocortisone, cortisone acetate, and fludrocortisone acetate, and artificially synthesized glucocorticoids such as dexamethasone, betamethasone, prednisolone, methylprednisolone, and triamcinolone, with hydrocortisone being preferred. These substances may be used alone or in combination.

[0027] The concentration of the adrenal cortical hormone in the medium can be set appropriately depending on the substance used. For example, in the case of hydrocortisone, the concentration is usually 100 ng / ml to 1000 μg / ml, preferably 500 ng / ml to 100 μg / ml, and more preferably 1 μg / ml to 10 μg / ml (4 μg / ml in one embodiment).

[0028] In the production method of the present invention, nail stem cells or limb bud organoids are cultured under suspension culture conditions. As used herein, "suspension culture" refers to culture performed under conditions that maintain cells or cell clumps suspended in a culture medium, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell clumps and the culture vessel.

[0029] The culture vessel used for suspension culture is not particularly limited, but examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, etc., but for the shaking culture described below, sealed culture vessels are preferred. Examples of such culture vessels include tissue culture flasks, culture bags, roller bottles, etc. Furthermore, to enable culture under non-adhesive conditions, the culture vessel is preferably non-adhesive. Examples of non-adhesive culture vessels include those whose surfaces have been artificially treated to make them non-adhesive, and those that have not been artificially treated (e.g., coated with an extracellular matrix) to improve cell adhesion.

[0030] As shown in the examples below, finger organoids could be induced by orbital culture of limb bud organoids. Without being bound by any theory, it is speculated that the physical shaking of the medium caused by orbital culture allows for the supply of oxygen and nutrients and the efficient exchange of excreted waste products, thereby inducing nail matrix cells and finger organoids. Therefore, as long as the medium can be physically shaken, the shaking culture used in the present invention is not limited to orbital culture, and may be, for example, agitation culture. Shaking culture can be performed, for example, by placing the cultureware in which nail stem cells or limb bud organoids are cultured on a shaker or the like. Orbital culture can be performed, for example, by placing the cultureware in which nail stem cells or limb bud organoids are cultured on a rotator or the like. Orbital culture can be performed, for example, by placing the limb bud organoids in an environment where a stirrer or the like is rotating.

[0031] Those skilled in the art can appropriately set parameters such as the speed of shaking culture within a range that does not cause damage to nail stem cells or limb bud organoids. For example, when shaking culture is performed using a wave-type 3D shaker (e.g., Mini-Shaker 3D, Biosan), the shaking speed can be set within a range of, for example, 5 to 60 rpm. When shaking culture is performed using a reciprocating shaker (e.g., NS-LR, AS ONE), the shaking speed can be set within a range of, for example, 15 to 60 rpm. When shaking culture is performed using a seesaw shaker (e.g., NS-S, AS ONE), the shaking speed can be set within a range of, for example, 5 to 50 rpm. When performing agitation culture, for example, a spinner flask (e.g., 3152, Corning) can be placed on a magnetic stirrer and culture can be performed at a rotation speed that does not cause nail stem cells or limb bud organoids to settle visually. When performing rotational culture, a three-dimensional rotary suspension culture device (e.g., CellPet CUBE, manufactured by J-Tech Corporation; Clinostar, manufactured by Cellvivo) can also be used to perform the culture. When performing rotational culture using a three-dimensional rotary suspension culture device, the rotation speed range can be set, for example, between 15 and 60 rpm. Furthermore, when performing rotational culture using a three-dimensional rotary suspension culture device, the cells may be rotated perpendicular to the direction of gravity (horizontal rotation) or parallel to the direction of gravity (vertical rotation), with horizontal rotation being preferred. From the viewpoint of suppressing physical damage to the cells, such as friction, it is also preferable to culture nail stem cells or limb bud organoids embedded in gel with shaking.

[0032] The period for shaking culture of nail stem cells or limb bud organoids under suspension culture conditions is not particularly limited as long as it is the period during which Lgr6-positive cells appear, but is usually 10 days or more, preferably 20 days or more, more preferably 30 days or more, even more preferably 40 days or more, most preferably 50 days or more, and may be cultured for 60 days or more. Furthermore, since nail matrix cells or finger organoids can be maintained by culturing under culture conditions, the upper limit of the culture period is not particularly limited, but is usually 100 days or less, preferably 90 days or less, more preferably 80 days or less, and most preferably 70 days or less.

[0033] The medium used in the present invention can be prepared using a medium used for culturing mammalian cells as a basal medium. Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, Ham's F-12 medium, RPMI1640 medium, Fischer's medium, Neurobasal medium, Essential 8 (manufactured by Thermo Fisher Scientific), Essential 6 (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), and StemFit. Examples of suitable medium include AK02N (Ajinomoto Co., Inc.), StemFit AK03N (Ajinomoto Co., Inc.), ReproMed iPSC Medium (ReproCELL, Inc.), NutriStem XF (Biological Industries, Inc.), NutriStem V9 (Biological Industries, Inc.), Cellartis DEF-CS Xeno-Free Culture Medium (Takara Bio Inc.), Stem-Partner SF (Kyokuto Pharmaceutical Co., Ltd.), PluriSTEM Human ES / iPS Cell Medium (Merck & Co., Inc.), StemSurehPSCMediumΔ (Fujifilm Wako Pure Chemical Industries, Ltd.), and mixed media thereof (e.g., DMEM / F-12 medium (a 1:1 mixture of DMEM medium and Ham's F-12 medium)). In one embodiment, a mixed medium of DMEM / F-12 medium and Neurobasal medium is used. The mixing ratio, by volume, is, for example, DMEM / F-12:Neurobasal=0.8-1.2:1.2-0.8 (preferably 1:1).

[0034] The medium used in the present invention may be either serum-containing or serum-free. A serum-free medium refers to a medium that does not contain unprepared or unpurified serum. A medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) is considered to be serum-free. The medium used in the present invention may also contain a serum substitute. The serum substitute may contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such a serum substitute can be prepared, for example, by the method described in WO98 / 30679. To simplify the production method of the present invention, commercially available serum substitutes can be used. Examples of commercially available serum substitutes include KSR (knockout serum replacement) (Invitrogen), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0035] The medium may contain other additives, such as, but not limited to, insulin, an iron source (e.g., transferrin), minerals (e.g., sodium selenate), sugars (e.g., glucose), organic acids (e.g., pyruvic acid, lactic acid), serum proteins (e.g., albumin), amino acids (e.g., L-glutamine), reducing agents (e.g., 2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin), antibiotics (e.g., streptomycin, penicillin, gentamicin), and buffers (e.g., HEPES).

[0036] The culture temperature is not particularly limited, but is usually 30 to 40°C, preferably 37°C, and culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being 2 to 5%.

[0037] The limb bud organoids used in the present invention can be produced, for example, by inducing differentiation from pluripotent stem cells. Examples of such methods include the following methods (A) to (D). Therefore, the production method of the present invention may include at least one of steps (A) to (D). (A) A step of suspension culture of pluripotent stem cells to form cellular aggregates of pluripotent stem cells. (B) A step of suspension culture of the cellular aggregates obtained in step (A) in the presence of a BMP signaling promoter to induce cellular aggregates. (C) A step of suspension culture of the cellular aggregates obtained in step (B) in the presence of a BMP signaling promoter and a retinoic acid signaling promoter to induce cellular aggregates. (D) A step of suspension culture of the cellular aggregates obtained in step (C) in the presence of a BMP signaling inhibitor to induce limb bud organoids.

[0038] The pluripotent stem cells used in the present invention may be any undifferentiated cells that possess both the "self-renewal ability" (ability to proliferate while maintaining an undifferentiated state) and the "pluripotency" (ability to differentiate into all three primary germ layers). Examples of such pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (nuclear transfer embryonic stem cells (ntES cells)), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, these are iPS cells (more preferably, human iPS cells). When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of embryos. However, from an ethical standpoint, they are preferably produced without the destruction of embryos.

[0039] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (Takahashi K. and Yamanaka S. (2006) Cell, 126:663-676; Takahashi K. et al. (2007), Cell, 131:861-872; Yu J. et al. (2007), Science, 318:1917-1920; Nakagawa M. et al., Nat. Biotechnol.26:101-106 (2008); WO 2007 / 069666). When iPS cells are used, the iPS cells may be produced from somatic cells by a method known per se, or iPS cells that have already been established and stored may be used. The reprogramming factors may be composed of genes that are specifically expressed in ES cells, their gene products, or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products, or non-coding RNAs, or low-molecular-weight compounds. Examples of genes contained in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO 2007 / 069666, WO 2008 / 118820, WO 2009 / 007852, WO 2009 / 032194, WO 2009 / 058413, WO 2009 / 057831, WO 2009 / 075119, WO 2009 / 079007, WO 2009 / 091659, WO 2009 / 101084, WO 2009 / 101407, WO 2009 / 102983, WO 2009 / 114949, WO 2009 / 117439, WO 2009 / 126250, WO 2009 / 126251, WO 2009 / 126655, WO 2009 / 157593, WO 2010 / 009015, WO 2010 / 033906, WO 2010 / 033920, WO 2010 / 042800, WO 2010 / 050626, WO 2010 / 056831, WO 2010 / 068955, WO 2010 / 098419, WO 2010 / 102267, WO 2010 / 111409, WO 2010 / 111422, WO 2010 / 115050, WO 2010 / 124290, WO 2010 / 147395, WO 2010 / 147612, Huangfu D, et al. (2008), Nat.The combinations described in Biotechnol., 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9 are exemplified.

[0040] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; Suemori H. et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; Ueno M. et al. (2006), Proc. Natl. Acad. Sci. USA 103:9554-9559; Suemori H. et al. (2001), Dev. Dyn., 222:273-279; Kawasaki H. et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I. et al. (2006), Nature. 444:481-485, etc.Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0041] ntES cells are ES cells derived from cloned embryos by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). Specifically, ntES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate ntES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (see above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0042] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under culture conditions similar to those for ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0043] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0044] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0045] In step (A), there are no particular limitations on the method for preparing pluripotent stem cell aggregates, and in a preferred embodiment, pluripotent stem cell aggregates can be obtained by culturing dispersed pluripotent stem cells under suspension culture conditions and allowing multiple pluripotent stem cells to aggregate to form aggregates.

[0046] The formation of pluripotent stem cell aggregates in step (A) is typically carried out by the following method. First, pluripotent stem cells are recovered from subculture and dispersed into single cells or a state close to single cells. Dispersion of pluripotent stem cells is carried out using an appropriate cell dissociation solution. Examples of cell dissociation solutions include EDTA; proteolytic enzymes such as trypsin, collagenase IV, and metalloproteases, which can be used alone or in appropriate combinations. Among these, those with minimal cytotoxicity are preferred, and commercially available cell dissociation solutions include, for example, Dispase (Eidea), TrypLE (Invitrogen), and Accutase (MILLIPORE). The dispersed pluripotent stem cells are suspended in the above-mentioned medium.

[0047] To suppress cell death of pluripotent stem cells (especially human pluripotent stem cells) induced by dissociation, it is preferable to add a Rho-associated coiled-coil kinase (ROCK) inhibitor from the start of culture (JP 2008-99662 A). Examples of ROCK inhibitors include Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), fasudil / HA1077, SR3677, GSK269962, GSK429286A, H1152, Wf-536, thiazovivin, and salts or derivatives thereof. The concentration of ROCK inhibitor used for suspension culture is sufficient to suppress cell death of pluripotent stem cells induced by dissociation. For example, for Y-27632, such a concentration is usually 0.1 to 200 μM, preferably 2 to 50 μM (10 μM in one embodiment). Treatment with a ROCK inhibitor is typically carried out for one day, after which it is preferable to continue culturing in a medium containing no ROCK inhibitor.

[0048] When a medium exchange operation is performed in step (A) or any of the subsequent steps, examples include an operation in which new medium is added without discarding the original medium (medium addition operation), an operation in which about half of the original medium (about 30 to 90%, for example, about 40 to 60% of the volume of the original medium) is discarded and about half of the new medium (about 30 to 90%, for example, about 40 to 60% of the volume of the original medium) is added (half medium exchange operation), and an operation in which about the entire volume of the original medium (90% or more of the volume of the original medium) is discarded and about the entire volume of new medium (90% or more of the volume of the original medium) is added (full medium exchange operation).

[0049] The basal medium used in step (A) includes the above-mentioned basal medium that can be used in the production method of the present invention, but StemFit AK02N medium and Essential 8 medium are preferred. Essential 8 medium is a DMEM / F12 medium containing the following additives: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO (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)). It is also preferable to change the basal medium during the culture. For example, StemFit AK02N medium can be used at the start of the culture, and Essential 8 medium can be used for the last day of the culture. The medium used in step (A) may contain the above-mentioned medium additives that can be used in the production method of the present invention.

[0050] The period of step (A) can be appropriately determined by those skilled in the art, and is usually 1 to 10 days, preferably 2 to 8 days, and more preferably 3 to 7 days (5 days in one embodiment).

[0051] In step (B), the cell aggregates obtained in step (A) are cultured in suspension in the presence of a BMP signaling promoter to induce cell aggregates. In one embodiment, the cell aggregates obtained in step (B) are cell aggregates containing posterior primitive streak (PPS) cells.

[0052] The BMP signaling promoter used in step (B) is a substance capable of enhancing the signaling pathway mediated by bone morphogenetic protein (BMP). Examples of substances capable of enhancing the signaling pathway mediated by BMP include substances that stabilize BMP ligands in the culture environment and increase their potency, substances that bind to type I BMP receptors ALK-1, ALK-2, ALK-3, and ALK-6 and activate or induce intracellular signaling downstream of the receptor, substances that induce phosphorylation of Smad-1, Smad-5, Smad-8, and Smad-9 involved in intracellular BMP signaling, and substances that induce or enhance functions such as activation or repression of gene transcription by Smad-1 / 5 / 8 / 9. Examples of BMP signaling promoters include proteins such as BMP2, BMP4, BMP7, BMP13, and GDF7 (among which BMP2, BMP4, and BMP7 are preferred, with BMP4 being particularly preferred), GDF proteins such as GDF5, 6, and 7, anti-BMP receptor antibodies, and BMP partial peptides. These substances may be used alone or in combination.

[0053] The concentration of the BMP signaling promoter in the medium can be appropriately set depending on the substance used. For example, when BMP4 is used as the BMP signaling promoter, it is usually used at a concentration of 1 ng / ml to 200 ng / ml, preferably 5 ng / ml to 40 ng / ml, and more preferably 10 ng / ml to 20 ng / ml. When a BMP signaling promoter other than BMP4 is used, the concentration of the BMP signaling promoter in the medium is appropriately selected.

[0054] Compounds known to those skilled in the art can also be used as BMP signaling promoters. Examples of BMP signaling promoters include Smurf1 inhibitors, Chk1 inhibitors, and phosphorylated Smad stabilizers. Examples of compounds exhibiting the above-described activity include A-01 ([4-[[4-Chloro-3-(trifluoromethyl)phenyl]sulfonyl]-1-piperazinyl][4-(5-methyl-1H-pyrazol-1-yl)phenyl]methanone), PD407824 (9-Hydroxy-4-phenyl-pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione), SB4 (2-[[(4-Bromophenyl)methyl]thio]benzoxazole), SJ000291942 (2-(4-Ethylphenoxy)-N-(4-fluoro-3-nitrophenyl)-acetamide), and derivatives thereof.

[0055] The basal medium used in step (B) may be the same as the medium described above that can be used in the production method of the present invention. Essential 6 medium and DMEM medium are preferred as basal media. It is also preferable to change the basal medium during the culture; for example, Essential 6 medium can be used at the start of culture, and DMEM medium can be used for the last day of culture. The medium used in step (B) may contain the medium additives described above that can be used in the production method of the present invention.

[0056] The culture period in step (B) can be appropriately determined by those skilled in the art, and is usually 4 to 14 days, preferably 5 to 13 days, and more preferably 6 to 12 days (9 days in one embodiment).

[0057] In one embodiment, the medium used in step (B) (particularly for the culture in the latter half of step (B)) may further contain at least one (preferably all) selected from the group consisting of insulin, transferrin, and sodium selenite. The concentration of insulin in the medium is typically 0.1 to 100 μg / mL, preferably 1 to 50 μg / mL, more preferably 3 to 20 μg / mL (particularly 10 μg / mL). The concentration of transferrin in the medium is typically 0.1 to 100 μg / mL, preferably 1 to 50 μg / mL, more preferably 2 to 10 μg / mL (particularly 5.5 μg / mL). The concentration of sodium selenite in the medium is typically 0.1 to 100 pg / mL, preferably 1 to 50 pg / mL, more preferably 2 to 10 pg / mL (particularly 6.7 pg / mL).

[0058] In step (C), the cell aggregates obtained in step (B) are cultured in suspension in the presence of a BMP signaling promoter and a retinoic acid signaling promoter to induce cell aggregates. The BMP signaling pathway active substance may be the same as that used in step (B). In one embodiment, the cell aggregates obtained in step (C) are cell aggregates containing lateral plate mesoderm (LPM) cells.

[0059] Examples of retinoic acid signal transduction promoters include substances that bind to retinoic acid receptors (RAR) or retinoid X receptors (RXR) and activate downstream transcription. Examples of compounds that have 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), and EC19 (3-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl]benzoic acid). acid), EC23(4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethynyl)-benzoicacid), Fenretinide(4-hydroxyphenylre tinamide), Acitretin((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-fluorobenzoicacid), 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]benzoicacid), 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-tricyclo[3.3.1.13,7]dec-1-yl-2-naphthalenyl)benzoicacid), CD2314(5-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)-3-thiophenecarboxylic acid), CD437 (2-naphthalenecarboxylic acid, 6-(4-hydroxy-3-tricyclo(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.

[0060] The retinoic acid signaling promoter in step (C) is preferably all-trans retinoic acid. The concentration of the retinoic acid signaling promoter in the medium can be appropriately set depending on the substance used. When all-trans retinoic acid is used as the retinoic acid signaling promoter, the concentration of all-trans retinoic acid is, for example, 200 nM to 20 μM, preferably 500 nM to 5 μM (in one embodiment, 2 μM). When a retinoic acid signaling promoter other than all-trans retinoic acid is used, the concentration of the retinoic acid signaling promoter in the medium is appropriately selected.

[0061] The basal medium used in step (C) may be, for example, the same medium as the medium that can be used in the production method of the present invention. DMEM medium is preferred as the basal medium. The medium used in step (C) may contain the medium additives that can be used in the production method of the present invention.

[0062] The culture period in step (C) can be appropriately determined by those skilled in the art, and is usually 1 to 7 days, preferably 2 to 6 days, and more preferably 3 to 5 days (4 days in one embodiment).

[0063] In one embodiment, the medium used in step (C) may further contain at least one (preferably all) selected from the group consisting of insulin, transferrin, and sodium selenite, similar to the medium in step (B). The concentrations of insulin, transferrin, and sodium selenite in the medium may be the same as those in the medium in step (B).

[0064] In step (D), the cell aggregates obtained in step (C) are cultured in suspension in the presence of a BMP signaling inhibitor to induce limb bud organoids.

[0065] The BMP signaling inhibitor used in step (D) is not limited as long as it can inhibit signaling induced by BMP family proteins. It may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such inhibitors include substances that inhibit BMP processing and extracellular secretion, substances that directly act on BMP (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of BMP-encoding genes (e.g., antisense oligonucleotides, siRNAs, etc.), substances that inhibit the binding of BMP receptors to BMP, and substances that inhibit physiological activities resulting from signaling mediated by BMP receptors. BMP receptors include type I BMP receptors and type II BMP receptors. Known type I BMP receptors include BMPR1A, BMPR1B, and ACVR, while known type II BMP receptors include TGF-beta R-II, ActR-II, ActR-IIB, BMPR2, and MISR-II.

[0066] Proteins known as BMP signaling inhibitors include, for example, Noggin, Chordin, Follistatin, Gremlin, Inhibin, Twisted Gastrulation, Coco, and secreted proteins belonging to the DAN family. Preferred BMP signaling inhibitors include substances that inhibit the binding of BMP receptors to BMPs, and substances that inhibit physiological activities resulting from signal transduction by BMP receptors, and more preferably inhibitors of type I BMP receptors.

[0067] Compounds well known to those skilled in the art can also be used as BMP signaling inhibitors. Examples of BMP signaling 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-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-a]pyrimidine), LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), and the like. dihydrochloride), LDN212854(5-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyriMidin-3-yl]quinoline), LDN214 117(1-(4-(6-methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine), ML347(5-[6-(4-Methoxyphenyl) )pyrazolo[1,5-a]pyrimidin-3-yl]quinoline)), DMH1(4-(6-(4-Isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)q uinoline), DMH2(4-[6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), Compound 1(3-(1,2,3-benzothiadiazol-6-yl)-1-[2-(cyclohex-1-en-1-yl)ethyl]urea), VU0465350(7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine), VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone), OD36(4-chloro-7,10-dioxa-13,17,18,21-tetrazatetracyclo[12.5.2.12,6.017,20]docosa-1( 20),2(22),3,5,14(21),15,18-heptaene), OD52, E6201((3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-1H-benzo[c] [1]oxacyclotetradecine-1,7(8H)-dione), Saracatinib (N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(oxan-4-yloxy)quinazolin-4-amine), BYL719 ((2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide), etc. These substances may be used alone or in combination.

[0068] The concentration of the BMP signaling inhibitor in the medium can be appropriately set depending on the substance used. For example, when LDN193189 is used as the BMP signaling inhibitor in step (D), it is typically used at a concentration of 20 nM to 100 μM, preferably 100 nM to 50 μM, more preferably 200 nM to 10 μM, and even more preferably 500 nM to 5 μM (in one embodiment, 2 μM). Furthermore, when a BMP signaling inhibitor other than LDN193189 is used, it is desirably used at a concentration that exhibits BMP signaling inhibitory activity equivalent to that of LDN193189 at the above-mentioned concentrations. When a BMP signaling inhibitor other than LDN193189 is used, the concentration of the BMP signaling inhibitor in the medium is appropriately selected.

[0069] Examples of the medium used in step (D) include the same medium as the medium described above that can be used in the production method of the present invention. The medium used in step (D) may contain the medium additives described above that can be used in the production method of the present invention.

[0070] The culture period in step (D) can be appropriately determined by those skilled in the art, and is, for example, usually 6 hours to 2 days, preferably 12 hours to 36 hours (in one embodiment, 24 hours).

[0071] In one embodiment, the medium used in step (D) may further contain at least one (preferably all) selected from the group consisting of insulin, transferrin, and sodium selenite, similar to the medium in step (B). The concentrations of insulin, transferrin, and sodium selenite in the medium may be the same as those in step (B).

[0072] The culture temperature in the above steps (A) to (D) is not particularly limited, but is usually 30 to 40°C, preferably 37°C, and culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration preferably being 2% to 5%.

[0073] The limb bud organoids used in the production method of the present invention may be obtained by methods other than those described above. Examples of such methods include the method described in Non-Patent Document 1. Specifically, in Non-Patent Document 1, limb bud organoids are produced by the following procedure. Mouse embryonic stem cells maintained in maintenance medium (G-MEM supplemented with 1% FBS, 10% KSR, 0.1 mM non-essential amino acids, 1 mM pyruvate, 0.1 mM 2-mercaptoethanol, 2000 U / ml LIF, 1 μM PD0325901, and 3 μM CHIR99021) are suspended in differentiation medium (G-MEM supplemented with 1.5% KSR, 0.1 mM non-essential amino acids, 1 mM pyruvate, and 0.1 mM 2-mercaptoethanol) and seeded onto a non-adhesive plate at a concentration of 3000 cells per well to produce pluripotent stem cell aggregates (SFEBq method) (culture day 0). From day 1 to day 5 of culture, differentiation into PPS (posterior primitive streak) aggregates is induced by adding human BMP4 and Matrigel to the culture medium to final concentrations of 10 ng / ml and 4% (v / v), respectively. On day 5 of culture, the aggregates are transferred to a plastic petri dish, and the amniotic membrane is removed from the surface of the PPS aggregates with tweezers. Different differentiation-inducing factors are then added depending on whether the PPS aggregates are to differentiate into mouse hind limbs or forelimbs. For mouse hind limb differentiation, PPS aggregates are cultured in 2.5% KSR differentiation medium from day 5 to day 6 of culture. From day 6, the medium is changed to high-glucose Dulbecco's modified Eagle's medium (LB medium) supplemented with ITS+1 and 0.1 mM 2-mercaptoethanol. From day 6 to day 6.5, 1 μM AGN193109 (a retinoic acid antagonist (RAA)) is added to induce differentiation into LPM (lateral plate mesoderm) aggregates for hindlimb formation. For mouse forelimb development, PPS aggregates are cultured in 2.5% KSR differentiation medium containing 1 μM all-trans retinoic acid (RA) from day 5 to day 6, and then switched to LB medium from day 6 to day 6.5 to induce differentiation into LPM aggregates for forelimb formation.From day 6.5, both aggregates were cultured in LB medium supplemented with 4 μg / ml hydrocortisone and 0.8% methylcellulose 40, and 0.1 μM LDN193189 was added from day 6.5 to day 7 to induce differentiation into limb bud organoids.

[0074] 2. Nail matrix cells produced by the production method of the present invention As described above, nail matrix cells or finger organoids containing said cells can be produced from nail stem cells or limb bud organoids. Therefore, in another aspect of the present invention, nail matrix cells or finger organoids containing said cells produced by the production method of the present invention (hereinafter, sometimes referred to as "cells or organoids of the present invention") are provided. Furthermore, nail matrix cells can be isolated by known means from cell populations, cell aggregates, or finger organoids containing nail matrix cells produced by the production method of the present invention. Therefore, the present invention also provides a method for isolating nail matrix cells from cell populations, cell aggregates, or finger organoids containing nail matrix cells produced by the production method of the present invention, as well as nail matrix cells isolated by said method.

[0075] Isolation of nail matrix cells from cell populations, cell aggregates, or finger organoids containing nail matrix cells can be performed, for example, by the following method. Cell aggregates, such as organoids, are dispersed into a single-cell population, and then cells expressing marker proteins present on the cell surface of nail matrix cells, such as Lgr6, are isolated. The pretreated cell population is then dispersed by enzyme treatment. Specifically, the pretreated cell population is first transferred to an incubator containing PBS and washed with the same medium. The enzyme used for dispersion is not particularly limited as long as it can disperse cells, but examples include enzymes such as papain, EDTA, trypsin, collagenase (collagenase types I to VII), metalloprotease, hyaluronidase, elastase, dispase, and deoxyribonuclease, as well as mixtures thereof. A preferred enzyme is papain. The conditions for the enzyme treatment (temperature, time, etc.) can be appropriately determined depending on the enzyme used.

[0076] 3. Treatment for Fingertip Disorders Comprising Nail Matrix Cells or Finger Organoids Nail matrix cells undergo continuous cell division in vivo, and the divided cells keratinize to form mature nails. However, injury to the fingertip or chemotherapy with anticancer drugs can cause the loss of nail matrix cells, resulting in serious impairment of nail formation. As shown in the Examples below, in one embodiment, the finger organoids of the present invention expressed the Hoxd13 gene, a transcription factor involved in finger formation, and the Runx2 gene, which is involved in skeletal formation. Sarcomas derived from nail matrix cells or nail stem cells have been reported to recapitulate finger-like structures (e.g., Godse R. et al., J. CutanPathol. 2023 Sep;50(9):779-782; Lee S., Kim BJ and Lee W., Australas J. Dermatol. 2019 Feb;60(1):69-70, etc.). Furthermore, as shown in the Examples below, nail organoids have been demonstrated to engraft in vivo when transplanted into mice. Therefore, by transplanting the cells or organoids of the present invention into patients with fingertip disorders, including nail disorders, it is expected that fingertip disorders can be treated by regenerating fingertips and nails. Therefore, the present invention provides a therapeutic agent for fingertip disorders (hereinafter, sometimes referred to as the "therapeutic agent of the present invention") containing the cells or organoids of the present invention. In one embodiment, the therapeutic agent of the present invention is a therapeutic agent for nail disorders. The nail matrix cells contained in the therapeutic agent of the present invention may be isolated from a cell population such as a finger organoid. As used herein, "fingertip" refers to the region from the second joint of the finger to the distal end of the finger, but typically refers to the region from the first joint of the finger to the distal end of the finger. The present invention also encompasses a method for treating fingertip disorders, in which an effective amount of the nail matrix cells or finger organoids of the present invention is transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) to be treated.

[0077] The cells or organoids of the present invention can be used as a therapeutic agent for fingertip disorders, or as a therapeutic agent for nail disorders caused by damage to the nail matrix, or to replenish the corresponding damaged area when the nail matrix is ​​damaged. Fingertip disorders may be caused by congenital factors or acquired factors. Causes of fingertip disorders include, for example, congenital limb or finger malformation or hypoplasia, loss or damage to the fingertip due to an accident, necrosis of fingertip tissue or fingertip loss due to frostbite, infection, internal organ disease, tumor, and side effects of drugs such as anticancer drugs. Nail disorders include, for example, congenital anonychia, total or partial loss of the nail due to injury, green nail syndrome, onychomycosis, acute paronychia, and nail tumors.

[0078] The therapeutic agent of the present invention contains a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include physiological saline, phosphate-buffered saline (PBS), culture media, etc., which are used to stably maintain cells. Examples of culture media include, but are not limited to, RPMI, AIM-V, and X-VIVO10. Furthermore, commonly used preservatives, stabilizers, reducing agents, isotonicity agents, etc. may also be added to the pharmaceutically acceptable carrier. If necessary, a cryopreservative may be added, the cells may be cryopreserved, thawed at the time of use, washed with a buffer solution, and then used in transplantation therapy.

[0079] The effective amount of nail matrix cells or finger organoids contained in the therapeutic agent of the present invention is appropriately selected depending on the condition, weight, age, etc. of the subject. Generally, the number of cells is 1 × 10 per administration. 2 ~1×10 5 It is contained so that there are only one.

[0080] The therapeutic agent of the present invention can be in a known form suitable for parenteral administration, such as an injection or infusion. When used as an infusion, it may be implanted once or multiple times into the base of the nail of a subject requiring treatment.

[0081] 4. Nail matrix cells containing a nucleic acid encoding a reporter protein The present invention also provides nail matrix cells (hereinafter sometimes referred to as "reporter cells of the present invention") containing a nucleic acid (hereinafter referred to as "nucleic acid of the present invention") comprising a reporter gene under the control of the transcriptional regulatory region of the Lgr6 gene.

[0082] As used herein, the term "transcriptional regulatory region" generally refers to a region ranging from several kb to several tens of kb upstream of a gene, and can be identified by a method including, for example, (i) determining the 5' end by a conventional method such as the 5'-RACE method (which can be performed using, for example, a 5'-full Race Core Kit (manufactured by Takara Bio Inc.)), the oligo-capping method, or S1 primer mapping; and (ii) obtaining the 5'-upstream region using a Genome Walker Kit (manufactured by Clontech) or the like, and measuring the promoter activity of the obtained upstream region.

[0083] Human Lgr6 has three alternative splicing variants, and the transcriptional regulatory regions of any of these variants can be used in the present invention, although the transcriptional regulatory region of variant 2 is preferred. Examples of the transcriptional regulatory region of the Lgr6 gene contained in the nucleic acid of the present invention include, but are not limited to, the nucleotide sequence set forth in SEQ ID NO: 1 and sequences having high identity to said sequence (e.g., 80% or more, 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, or 99% or more identity).

[0084] Examples of reporter genes used in the present invention include, but are not limited to, fluorescent protein genes, luciferase genes, and chromogenic enzyme genes. Examples of luciferases encoded by reporter genes include firefly luciferase, bacterial luciferase, synthetic Renilla luciferase, Oplophorus gracilirostris luciferase (e.g., NanoLuc (registered trademark)), and secreted luciferases. Examples of fluorescent proteins encoded by reporter genes include green fluorescent proteins such as GFP and EGFP, blue fluorescent proteins such as BFP and TagBFP, and red fluorescent proteins such as RFP and DsRed. Examples of chromogenic enzyme genes encoded by reporter genes include β-galactosidase, β-glucuronidase, and alkaline phosphatase. These marker genes can be used alone or in combination.

[0085] The nucleic acid of the present invention preferably has a sequence (polyadenylation (poly A) signal, also called a terminator) downstream of the reporter gene that terminates transcription of mRNA from the gene. For example, a terminator sequence derived from a viral gene or from various mammalian or avian genes can be used. Preferably, a terminator derived from SV40 is used.

[0086] The nucleic acids of the present invention may further contain a drug resistance gene or another reporter gene, and cells into which the nucleic acids have been introduced can be selected using drug resistance or reporter activity as an indicator. Examples of drug resistance genes include the neomycin phosphotransferase II (nptII) gene, the hygromycin phosphotransferase (hpt) gene, and the puromycin-N-acetyltransferase (Pac) gene, and examples of other reporter genes include, but are not limited to, the β-galactosidase (lacZ) gene and the chloramphenicol acetyltransferase (cat) gene.

[0087] The drug resistance or reporter gene is preferably under the control of any promoter that can function in cells. Examples include viral promoters such as the SV40-derived early promoter, cytomegalovirus (CMV) long terminal repeat (LTR), Rous sarcoma virus (RSV) LTR, murine leukemia virus (MoMuLV) LTR, and adenovirus (AdV)-derived early promoter, as well as the β-actin gene promoter, PGK gene promoter, and transferrin gene promoter. The drug resistance or other reporter gene also preferably has a poly(A) signal.

[0088] The reporter cells of the present invention can be produced by introducing the nucleic acid of the present invention into pluripotent stem cells and inducing nail matrix cells from the pluripotent stem cells carrying the nucleic acid of the present invention. Alternatively, the reporter cells of the present invention can be produced by introducing the nucleic acid of the present invention into nail matrix cells or precursor cells of the nail matrix cells and inducing differentiation as necessary. The above-mentioned nail matrix cells are preferably obtained by the production method of the present invention.

[0089] The method for introducing the nucleic acid of the present invention into cells is not particularly limited, but is typically carried out using an expression vector containing the nucleic acid of the present invention. The nucleic acid of the present invention may be integrated into the genome of the cell or may exist independently of the genome. Expression vectors that can be used in the present invention include viral vectors and plasmid vectors. Viral vectors include retroviral vectors (including lentiviral vectors and pseudotyped vectors), adenoviral vectors, adeno-associated viral vectors, herpesvirus vectors, Sendai virus, and episomal vectors. Transposon expression systems (e.g., the PiggyBac system) may also be used. Plasmid vectors include animal cell expression plasmids (e.g., pa1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo). In one embodiment, the nucleic acid of the present invention is introduced into cells using the PiggyBac system. The PiggyBac system uses a transposon vector incorporating the nucleic acid of the present invention and a transposase expression vector expressing a transposase. The genes and other components contained in the transposon vector and transposase expression vector may be contained in the above-mentioned separate vectors, or may be contained in a single vector. The transposon vector and transposase expression vector may have the following configuration, for example: To excise the nucleic acid of the present invention from the transposon vector using a transposase, the transposon vector contains terminal inverted repeats 5' upstream and 3' downstream of the nucleic acid. The transposase recognizes the terminal inverted repeats contained in the transposon vector and excises the nucleic acid of the present invention flanked by the terminal inverted repeats from the transposon vector.

[0090] Alternatively, the reporter cells of the present invention can be generated by knocking in a reporter gene downstream of the promoter of an endogenous Lgr6 gene on one or both chromosomes (alleles) using homologous recombination, such as genome editing. When using homologous recombination, donor DNA is typically used, and this donor DNA contains at least the reporter gene in the region between the 5' and 3' homology arms (hereinafter also referred to as the "intended insertion region"). The intended insertion region may be inserted downstream of the Lgr6 gene promoter on the chromosome, or it may replace a region downstream of the promoter (e.g., the Lgr6 coding region or a portion thereof). Alternatively, a fusion protein of a reporter protein and Lgr6 may be expressed. Furthermore, simultaneous expression of a reporter protein and Lgr6 can be achieved by including an internal ribosome entry site (IRES) or a 2A (e.g., T2A, P2A, E2A, F2A) coding sequence in the intended insertion region.

[0091] Examples of genome editing include a method using zinc finger nucleases (ZFNs) that link a zinc finger DNA-binding domain to a nonspecific DNA cleavage domain (Japanese Patent No. 4968498), a method using TALENs (TAL effector nucleases) that link a transcription activator-like (TAL) effector, a DNA-binding module, to a DNA endonuclease (WO 2011 / 072246), or a method using the CRISPR-Cas9 system that combines the DNA sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) with the nuclease Cas protein family, which plays an important role in conjunction with CRISPR (WO 2013 / 176772). Furthermore, the gene expression cassette may be knocked into a specific gene locus by genome editing or other methods. Examples of specific gene loci include those with an open chromatin structure, such as the human PPP1R2C locus, which are less susceptible to suppression of the inserted gene's expression.

[0092] The nucleic acid of the present invention can be introduced into cells by any of the following methods: calcium phosphate co-precipitation, electroporation, lipofection, retroviral infection, aggregation, microinjection, gene gun (particle gun), DEAE-dextran, etc.

[0093] 5. Screening Method for Preventive or Therapeutic Drugs for Fingertip Disorders A correlation can be observed between the expression levels of Lgr6 protein and the reporter protein in the reporter cells of the present invention. Increased Lgr6 protein expression reflects increased Lgr6 protein expression in nail matrix cells and / or nail matrix cell proliferation. Furthermore, stimulation of the Wnt signaling pathway is important for nail matrix cell proliferation, and Lgr6 is known to stimulate the Wnt signaling pathway. Therefore, substances that enhance Lgr6 expression could be candidate drugs for preventing or treating fingertip disorders. Therefore, the present invention also provides a screening method for preventive or therapeutic drugs for fingertip disorders, such as nail disorders, using the reporter cells of the present invention (hereinafter referred to as the screening method of the present invention).

[0094] The screening method of the present invention comprises the following steps: (1) contacting a reporter cell of the present invention with a test substance, (2) measuring the expression level of the reporter protein in the cell, and (3) selecting the test substance as a candidate drug for preventing or treating fingertip disorders when the expression level measured in step (2) is higher than the expression level in a control group not contacted with the test substance or the expression level measured in the absence of the test substance.

[0095] In the present invention, examples of test substances include cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.

[0096] In the present invention, test substances can also be obtained using any of the many approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) synthetic library methods using affinity chromatography selection. Examples of methods for the synthesis of compound libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be stored in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9), or phage (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).

[0097] In step (1), the contact between the test substance and the reporter cells of the present invention can be carried out, for example, by adding the test substance to a medium or various buffer solutions (e.g., HEPES buffer, phosphate buffer, phosphate-buffered saline, Tris-HCl buffer, borate buffer, acetate buffer, etc.) and incubating the cells for a certain period of time. The incubation period can be, for example, 10 minutes to 24 hours, but is not limited to this range.

[0098] The expression level of the reporter protein in the reporter cells of the present invention in step (2) can be measured by known methods. For example, if the reporter gene is a luciferase gene, it can be measured by luminescence intensity. If the reporter gene is a fluorescent protein gene, it can be measured by fluorescence intensity. If the reporter gene is a chromogenic enzyme gene, it can be measured by color intensity. Expression of the reporter gene can also be detected or measured using a fluorescence imaging system or a luminescence imaging system. More specifically, for example, when a luciferase gene is used as the reporter gene, the reporter cells of the present invention can be cultured with a test substance, an extract of the cells is obtained, and the extract is then reacted with luciferin and ATP to generate chemiluminescence, and the luminescence intensity is measured to measure the expression level of the luciferase gene. In this case, a commercially available luciferase reaction detection kit such as Picagene Dual Kit (registered trademark; manufactured by Toyo Ink Co., Ltd.) can be used. Furthermore, when a fluorescent protein gene is used as the reporter gene, the reporter cells of the present invention are cultured with a test substance, and then the cells are washed. The cells are irradiated with light of an excitation wavelength corresponding to the fluorescent protein used, and the intensity of the light of the corresponding fluorescence wavelength is measured, thereby measuring the expression level of the fluorescent protein gene.

[0099] In step (3), "without contact with the test substance" also includes adding a solvent (blank) in the same amount as the test substance instead of the test substance, or adding a negative control substance that does not affect the expression level of the reporter protein. Furthermore, the criteria for selecting a test substance as a candidate for a preventive or therapeutic drug for fingertip disorders can be determined appropriately by those skilled in the art. For example, in the above screening method, if the expression level of the reporter protein in the presence of the test substance increases compared to that in the absence of the test substance (e.g., if it increases by about 20% or more, preferably about 30% or more, more preferably about 50% or more), the test substance can be selected as a candidate for a preventive or therapeutic drug for fingertip disorders.

[0100] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0101] Materials and Methods 1. Human iPS cells (induced pluripotent stem cells) hiPSC lines (RIKEN-2F and 253G1) were obtained from the Pluripotent Cell Laboratory, RIKEN BioResource Research Center (Ibaragi, Japan).

[0102] 2. Maintenance of human iPS cells and differentiation into digital organoids Human iPS cells were differentiated into limb bud organoids using a previously reported method (Mori S, et al., Nat Commun. 2019;10: 3802) with some modifications. In this example, differentiation experiments for data acquisition were performed using the 253G1 cell line, but RIKEN2F cells also differentiated into similar digital organoids (data not shown). Undifferentiated human iPSCs were cultured in StemFit AK02N (Ajinomoto, Tokyo, Japan) at 0.5 g / cm. 2 Dispersed cells were maintained on plastic dishes (Corning Inc., NY, USA) coated with iMatrix511 Silk (Nippi Inc., Tokyo, Japan). Dispersed cells were cultured at 15,000 cells / cm using 10 μM Rho kinase inhibitor Y-27632 (Sellec Inc., Tokyo, Japan). 2Cells were seeded at a density of 1000 cells per well. One day later, the medium was replaced with the same medium without Y-27632. Cells were passaged every 3–4 days. Cells were detached from the culture dish by treatment with TripLE expression enzyme (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 μM Y-27632 for 20 minutes at 37°C. Five days before the initiation of differentiation (Day 0), the dispersed cells were dispersed in StemFit AK02N medium supplemented with 10 μM Y-27632 and distributed at a density of 3500 cells per well in 200 μL of medium into a non-adhesive 96-well plate (Sumitomo Bakelite, Tokyo, Japan). One day before Day 0, 100 μL of medium was aspirated from all wells and 100 μL of Essential 8 (E8) medium (Thermo Fisher Scientific) supplemented with 10 μM Y-27632 was added. On day 0, half of the medium was replaced with Essential 6 (E6) medium (Thermo Fisher Scientific) containing 20 ng / ml BMP4 and added to a 96-well plate. On days 2, 3, and 6 of differentiation, 100 μl of medium was aspirated from all wells and 100 μl of E6 medium containing 10 ng / ml BMP4 was added. On day 8 of differentiation, 100 μl of medium was aspirated from all wells and 100 μl of Dulbecco's Modified Eagle Medium (DMEM) (Fuji Film Wako Chemical Inc.) supplemented with a 1 / 100 dilution of insulin, transferrin, and selenium (ITS; Thermo Fisher Scientific) and 10 ng / ml BMP4 was added. On day 9, half of the medium was replaced with DMEM supplemented with 1 / 100 dilution of ITS, 10 ng / ml BMP4, and 2 μM all-trans retinoic acid (RA). On day 10, half of the culture medium was replaced with DMEM supplemented with 1 / 100 ITS, 10 ng / ml BMP4, and 2 μM LDN-193189. On day 11, half of the culture medium was replaced with 1 / 100 ITS, 10 ng / ml BMP4.On day 13, whole spheres were collected from the 96-well plate using a 1000 μL wide-bore tip (Axygen Corning Inc.) and cultured in organoid maturation medium (OMM) [DMEM / F12 (Fuji Film Wako Chemical Inc., Miyazaki, Japan) mixed 1:1 with Neurobasal medium (Thermo Fisher Scientific), supplemented with 0.1 mM non-essential amino acids, 1 mM glutamine, 55 μM 2-mercaptoethanol, 0.5% N-2 supplement (Thermo Fisher Scientific), 0.5% B-27 supplement (Thermo Fisher Scientific), 50 μg / ml ascorbic acid 2-phosphate (Merck KGaA, Darmstadt, Germany), 0.05% bovine serum albumin (Merck), 7 μM CHIR98014 (Sellec Inc.), 10 ng / ml FGF-basic (NacalaiTesque Inc., Kyoto, Japan), and 10 ng / ml erythritol phosphate (NacalaiTesque Inc., Kyoto, Japan)]. FGF8 (Peprotech, Thermo Fisher Scientific) and 4 μg / ml hydrocortisone (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added. The cells were then transferred to a non-adhesive 10-cm dish (Cell-Repellent Surface, Greiner Bio-One, Kremsmunster, Austria) containing 20 ml of PBS containing 100 μg / ml of PBS. Rotational culture was initiated at a stirring speed of approximately 60 rpm, with the medium replaced every 3 days until the end of the culture experiment.

[0103] 3. Development of Lgr6-GFP Reporter iPS Cells. The provisional promoter region was the 1 kb upstream sequence from the first codon of Lgr6 variant 2 (GenBank: AL356953.17; 19771 to 20838). A Kozak sequence (GCCACC) was inserted immediately above the first codon of EGFP. The PiggyBac-based reporter vector contained 5' and 3' inverted terminal repeats (ITRs) between the Lgr6 promoter, Kozak sequence, EGFP, rabbit β-globin (rBG) polyadenylation signal, and cytomegalovirus promoter (CMV), puromycin resistance gene, and bovine growth factor (BGH) polyadenylation signal. The designed vector was purchased from VectorBuilder (VectorBuilder Inc., Kanagawa, Japan). This construct was cotransfected into 253G1 and RIKEN2F cell lines with the PiggyBac transposase expression vector (System Biosciences, LLC., CA, USA). After 3–4 days, transfected cells were selected by treatment with 2 μg / ml puromycin (Thermo Fisher Scientific) for 2–3 days and then cultured for several days to allow the formation of single-cell-derived colonies. Individual colonies were manually picked and further expanded for functional testing. Small-scale digital organoid differentiation was performed, and functional clones were successfully selected by fluorescence microscopy.

[0104] 4. Quantitative Polymerase Chain Reaction (qPCR) Analysis. Total RNA was extracted from cells using ISOGEN (NIPPON GENE CO., LTD., Tokyo, Japan) according to the manufacturer's instructions. 50 ng of RNA was reverse-transcribed using SuperScript Reverse Transcriptase (Thermo Fisher Scientific) with oligo(dT)20 primers. To examine the expression of various genes, quantitative polymerase chain reaction was performed using the gene-specific primer sets shown in Table 1 with GeneAce SYBR qPCR Mix α (NIPPON GENE CO., LTD.). The expression levels of other genes were also examined. All gene expression levels were normalized to the expression level of the internal ribosomal protein S18. The sequences in Table 1 are listed in the sequence listing as SEQ ID NOS: 2 to 19, from top to bottom.

[0105]

[0106] 5. Immunofluorescence Staining. Organoids were fixed in 4% paraformaldehyde for 20 minutes at 25°C. They were then washed twice with 0.2% Tween-20 containing Tris-buffered saline (TBS-T) and incubated in 0.1% Triton-X100 containing TBS-T for 10 minutes at 25°C. They were then immersed in 30% sucrose containing TBS-T at 4°C overnight or longer. After the organoids were attached to the bottom of a cup (Cryomold No. 1, Sakura Finetek Japan Co., Ltd., Tokyo, Japan), the solution was completely aspirated using absorbent paper (Kimwipe, Nippon Paper Crecia Co., Ltd., Tokyo, Japan) through a pinhole created with the tip of a 29G syringe needle. The cup was then filled with OCT compound (Sakura Finetek Japan Co., Ltd.) and cooled to -80°C to prepare a cryoblock. Cryoblocks were cut into 8 μm-thick sections and mounted on aminosilane-treated glass slides (Matsunami Glass IND., Ltd., Osaka, Japan). After thorough drying, the slides were immersed in TBS-T to wash away residual OCT compound and rehydrate the organoids. The slides were then treated with blocking solution (Nacalai Tesque) at 25°C for 30 minutes. A primary antibody-containing blocking agent was applied, and the slides were mounted in paraffin to prevent evaporation and incubated overnight at 4°C. The cells were then washed three times with TBS-T and immersed in a secondary antibody-containing blocking agent at 25°C for 1 hour. After three washes, fluorescent signals were observed using a fluorescence microscope (Eclipse Ti2, Nikon Instruments, Tokyo, Japan) controlled by the built-in software (NIS-Elements, Nikon Instruments). The primary and secondary antibodies used are listed in Table 2. The anti-Lgr5 antibody was a rabbit antibody (GENETEX GNT-GTX130204-25) used at a dilution of 1:200. In Example 4, the anti-Lgr6 antibody was used at a dilution of 1:200.

[0107]

[0108] 6. Statistical Analysis Statistical analysis was performed using EZR software (Jichi Medical University, Japan). Significant differences compared with day 0 of differentiation were determined using one-way ANOVA, followed by post-hoc testing using Dunnett's test. Statistical significance was set at P < 0.05.

[0109] Example 1: Differentiation of Limb Bud Organoids into Finger Organoids by Rotational Expansion Culture. To differentiate fingernail tissue from human induced pluripotent stem cells (iPSCs), we modified and expanded a previous report on limb bud differentiation from human iPSCs (Non-Patent Document 1). iPSC spheres were sequentially processed in suspension culture for limb bud organoids. Rotational culture was then employed for further organoid proliferation and differentiation (Figure 1A). Sequential expression of E-cadherin, brachyury, Hand2, and Pitx1 confirmed early successful differentiation of limb bud organoids (Figure 1B). These results confirmed successful reconstitution of limb bud differentiation.

[0110] Example 2: Segmental Pattern Formation and mRNA Expression of Digit-Related Genes in Rotating Culture. Long-term culture of organoids revealed that larger organoids had necrotic cores in static suspension culture. Therefore, continuous medium flow was maintained during extended culture periods. Rotating culture was used for further proliferation and differentiation of organoids. The mRNA expression levels of various key genes were examined in relation to arm segmentation, skeletal bone formation, nail matrix differentiation, and nail formation (Figure 2). Hoxd13 is a transcription factor involved in digit formation; its gene expression level peaked on day 49 of differentiation, indicating successful differentiation of the most proximal digit segment. Pitx1 was persistently expressed in differentiated mesenchymal cells. Runx2 expression increased on day 49 of differentiation and was significantly expressed by day 77. Runx2 protein is involved in skeletal formation and supports further progression of the differentiation process to the osteogenic stage. Lgr5-expressing cells have been reported to be present in the dorsal portion of the proximal portion of hair follicles and nails (Lehoczky JA, Tabin CJ. Proc Natl Acad Sci USA. 2015;112: 13249-13254). We observed a sharp increase in Lgr5 expression on day 14 of differentiation. Lgr5 expression increased sharply on day 14 of differentiation, then rapidly decreased, and gradually increased again with increasing differentiation days. In contrast, Lgr6 expression continued to gradually increase with differentiation. Expression of the typical nail keratins KRT17 (type I; KRT17 is also known as CK-17 and PCHC1) and KRT81 (type II; also known as hHb1, MLN137, ghHkb1, and hHAKB2-1) gradually increased with differentiation.

[0111] Type I keratin KRT17 and type II keratin KRT81 are known as hard keratins because they form heterotypic complexes that form hair and nails. Lgr6-positive or Lgr5-positive cells are also known to represent hair follicle stem cell populations (Jaks V. et al., Nat Genet. 2008;40:1291-1299.; Snippert HJ. et al., Science. 2010;327:1385-1389.). Therefore, it may be difficult to determine whether the cells contained in the resulting organoids are hair follicle stem cells or nail matrix cells based solely on the above molecular markers. However, for the following reasons, the cells obtained using the production method of the present invention can be determined to be nail matrix cells. First, we tracked the developmental process leading up to nail formation and observed a time-dependent increase in finger-specific Hoxd13 (Figure 2). On the other hand, fetuses typically do not have active hair follicles on their arms or fingers (Domagala Z. et al., Adv Clin Exp Med. 2017;26:967-972). Furthermore, immunohistochemical analysis demonstrated a relatively large lack of hair follicle characteristics. These results strongly support our hypothesis that the differentiated tissue in our finger organoids is a combination of the nail matrix and nail. Second, there is a significant difference from previous studies on hair and hair follicles using organoids derived from human embryonic stem cells (Lee J. et al., Nature. 2020;582:399-404). Lee et al. reported hairy human skin produced by long-term culture of skin organoids and claimed that their skin organoids were equivalent to human facial skin. Notably, their single-cell mRNA expression analysis revealed that their organoids were composed exclusively of ectodermal cells, with no endoderm or mesodermal populations present. On the other hand, the digital organoids of the present invention clearly contained mesodermal cells. The primary spheres contained abundant brachyury-positive mesodermal mesenchymal cells (Figure 1B).In the later stages of differentiation, cells expressing Runx2 mRNA appeared (potentially osteoblasts, which form the skeleton (Karsenty G., Endocrinology. 2001;142:2731-2733.)) (Figure 2). Based on these findings, we conclude that the digital organoids of the present invention contain nail matrix cells and primitive nails.

[0112] Example 3: Adjacent protein expression of Lgr6, KRT17, and KRT81 in digital organoids Organoids were maintained in rotational culture for a period similar to the time when human embryos develop primitive digits. Serial sections of a single organoid were shown to confirm adjacent protein expression of Lgr6, KRT17, and KRT81 (Figure 3).

[0113] Example 4: Verification of the relationship between Lgr5-positive cells and Lgr6-positive cells Lgr5-positive cells and Lgr6-positive cells were detected using organoids on day 62 of differentiation. The results showed that Lgr5 protein-expressing cells were present in the vicinity of Lgr6 protein-expressing cells, and that there were also cells co-expressing both proteins (Figure 4). This suggests a strong relationship between Lgr5-positive cells and Lgr6-positive cells.

[0114] Example 5: Development of a Functional Human Lgr6-GFP Reporter Cell Line. A search of the Eukaryotic Promoter Database (EPD) identified three alternative splicing variants of Lgr6. Because variant 2 showed the clearest signal in FANTOM5 and ENCODE Cap Analysis of Gene Expression (CAGE) analyses, approximately 1 kb of upstream sequence from the first codon of variant 2 was selected for the Lgr6 reporter construct (Figure 5A). Several clones of transgenic human iPSCs were established and screened in parallel to form digital organoids. Some clones showed fluorescent signals (Figure 5B). Frozen sections of digital organoids were immunofluorescently stained for GFP and Lgr6. Colocalized expression of the reporter GFP and Lgr6 protein was confirmed (Figure 5C).

[0115] Example 6: Comprehensive Gene Expression Analysis of Finger Organoids. RNA sequencing was performed on both day 0 cells (undifferentiated iPS cells) and day 50 finger organoids (a sample not used for qPCR) to compare global gene expression. RNA (total RNA, including ribosomal RNA) was extracted and quantified from undifferentiated iPS cells (RIKEN2F line) and finger organoid samples derived from the same line at day 50 of differentiation. A 109 ng total RNA sample was processed using the NEB Next Poly(A) mRNA Magnetic Separation Module (NEB) to enrich for poly(A) mRNA and remove rRNA molecules. cDNA synthesis and subsequent transcriptome library preparation were performed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Thirteen cycles of PCR amplification were performed to increase library yield and incorporate sample barcodes into the library fragments. The resulting transcriptome libraries were loaded onto the next-generation sequencing platform NovaSeq 6000 (Illumina). Sequencing was performed according to the manufacturer's instructions using a 150-bp paired-end (PE) configuration, yielding approximately 6 GB per sample. Low-quality raw reads and adapter sequences were removed using Cutadapt v.2.9. These remaining reads were then mapped to the human genome (GRCh38 version 101) using HISAT2 v.2.2.0 (PMID: 25751142), and gene expression was quantified using the R package featureCounts v.2.6.0 (PMID: 24227677). To remove the effects of transcript length, the number of reads per 1,000 bp of transcript was calculated and normalized. Correction for the total number of reads was performed to obtain FPKM (fragments per kilobase of exon per million reads mapped).Additionally, differentially expressed genes (DEGs) were identified based on differences in expression levels between samples (log2 fold change > 1 and adjusted p value < 0.05) after removing genes with zero read counts using DESeq2 v.1.6.3.

[0116] From the DEG list, we selected genes with LogFC (fold change) >10 and LogFC <-10. Further narrowing the results by false discovery rate (FDR) ≤ 0.1 revealed significant changes in 329 genes (226 genes were upregulated and 103 genes were downregulated) (data not shown). Volcano graphs were created for genes outside both logFC10 limits (Figure 6). From the DEG list, gene categories associated with finger organoids were listed and graphs were created (Figures 7 and 8). These data revealed that both Lgr5 and Lgr6 genes, homeobox genes such as HoxD12 and HoxD13, and other genes important for arm development were all upregulated in finger organoids. Furthermore, many keratin genes were also upregulated. These data are consistent with the real-time PCR results in Example 2. Furthermore, transcription factors associated with the undifferentiated state were largely absent in finger organoids.

[0117] Example 7: Transplantation of Digital Organoids into Mice Digital organoids were prepared using the same method as in Example 5, and cells that showed green GFP fluorescence in the Lgr6 reporter (GFP-positive cells) were collected using a cell sorter (FACS AriaIIIBD) (Figure 9A). Autologous cell pellets were formed in non-adhesive 96-well round-bottom flasks (SUMILON MS-9096U, M) with 100-1000 cells each (Figure 9A). The cell pellets were transplanted subcutaneously (near the nail matrix site and in the subcutaneous tissue of the back) into NOD-SCID mice (immunodeficient mice). Anesthesia was administered by inhaling 5% vaporized forane. Digital organoids prepared using the same method as in Example 5 may also be transplanted as intact cell masses into NOD-SCID mice subcutaneously (near the nail matrix site or in the subcutaneous tissue of the back). Alternatively, the finger organ may be embedded in Matrigel (BD), AteloCell (registered trademark) (KOKEN), or the like before transplantation.

[0118] Figures 9B and 9C show sections of the Lgr6-positive cell mass formed 20 days after subcutaneous dorsal transplantation of the Lgr6-positive cell mass prepared by the above method. Figure 9B shows that the host mouse's red blood cells were surrounded by the transplanted human cells, indicating that the cells had successfully engrafted through the host's circulation. Figure 9C shows that the transplanted human cells (hNA-positive cells) contained Lgr6 reporter GFP and that the sclera keratins KRT17 and KRT81, which compose the nail, were present nearby.

[0119] Figure 10 shows tissue images taken 20 days after approximately 300 Lgr6-positive cell masses prepared by the above method were transplanted into the fingertip of an immunodeficient mouse. Figure 10 shows that cells immunostained with an antibody against the Lgr6 reporter GFP coexisted and survived in the fingertip tissue and nail matrix region.

[0120] The present invention makes it possible to provide nail matrix cells or finger organoids in vitro, enabling the development of regenerative medicine by transplanting the nail matrix cells or finger organoids into patients with fingertip disorders such as nail disorders. Nail regeneration, in particular, can be extremely important for the quality of life of patients with nail disorders. Furthermore, the present invention provides nail matrix cells carrying nucleic acids containing a reporter gene under the control of the transcriptional regulatory region of the Lgr6 gene, enabling the use of the nail matrix cells to screen for preventive or therapeutic agents for fingertip disorders. Therefore, the nail matrix cells of the present invention are extremely useful in the medical field, particularly in the field of regenerative medicine.

[0121] This application is based on patent application No. 2023-214532 filed in Japan (filing date: December 20, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing nail matrix cells, comprising a step of shaking-culture of nail stem cells under suspension culture conditions in the presence of a Wnt signaling promoter, an FGF signaling promoter and an adrenal cortical hormone.

2. The method according to claim 1, wherein the nail stem cells are in a form contained in a limb bud organoid.

3. The method according to claim 1 or 2, wherein the nail matrix cells are in a form contained in a finger organoid.

4. The method according to any one of claims 1 to 3, wherein the Wnt signaling promoter is CHIR98014.

5. The method according to any one of claims 1 to 4, wherein the FGF signaling promoter is bFGF and / or FGF8.

6. The method according to any one of claims 1 to 5, wherein the adrenal cortical hormone is hydrocortisone.

7. The method according to any one of claims 1 to 6, wherein the nail stem cells are derived from pluripotent stem cells.

8. The method according to claim 7, wherein the pluripotent stem cells are of human origin.

9. A nail matrix cell produced by the method according to any one of claims 1 to 8, or a finger organoid comprising said cell.

10. A therapeutic agent for fingertip disorders comprising the nail matrix cell or finger organoid described in claim 9.

11. The agent according to claim 10, wherein the fingertip disorder is a nail disorder.

12. A nail matrix cell having a nucleic acid containing a reporter gene under the control of the transcriptional regulatory region of the Lgr6 gene.

13. The nail matrix cell according to claim 12, produced by the method according to any one of claims 1 to 8.

14. The nail matrix cell according to claim 12 or 13, wherein the reporter gene is a gene encoding a fluorescent protein.

15. A method for screening for a preventive or therapeutic drug for fingertip disorders, comprising the following steps (1) to (3): (1) contacting the nail matrix cells according to any one of claims 12 to 14 with a test substance; (2) measuring the expression level of a reporter protein in the cells; and (3) selecting the test substance as a candidate for a preventive or therapeutic drug for fingertip disorders when the expression level measured in step (2) is higher than the expression level in a control group not contacted with the test substance or the expression level measured in the absence of the test substance.

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