Cell capable of regulating production of inflammation regulatory factor and / or niche factor for improving tissue environments

Abstract

[Problem] The present invention addresses the problem of providing a niche factor production regulation cell which can supply an inflammation regulation factor capable of suppressing inflammations and an epithelial cell growth factor in an ulcer site-specific manner. [Solution] A cell capable of recognizing an inflammatory site and an ulcer site and also capable of releasing an inflammation regulatory factor and / or a niche factor was successfully produced using a synNotch, and on this basis, the present invention has been accomplished.

Description

Inflammatory control factor and / or niche factor producing regulatory cells that improve tissue environment

[0001] The present invention relates to inflammatory control factor and / or niche factor-producing regulatory cells that improve the tissue environment. More specifically, the present invention relates to inflammatory control factor and / or niche factor-producing regulatory cells, a transplantable intestinal organoid composition, a composition for promoting intestinal epithelial tissue regeneration, and a composition for suppressing intestinal inflammation. This application claims priority to Japanese Patent Application No. 2023-188993, which is incorporated herein by reference.

[0002] (Current Status of Organoid Transplantation) Recent advances in stem cell research have led to the development of organoid cultures that replicate parts of organs in vitro, raising hopes for their application in regenerative medicine. Indeed, transplantation therapy, which aims to regenerate tissue by transplanting organoids into diseased tissues damaged by inflammatory or degenerative diseases, is being tested. However, regenerating tissue in diseased areas where the niche where tissue formation occurs has collapsed is challenging. Furthermore, administering potent anti-inflammatory factors or growth factors that promote stem cell proliferation to promote tissue regeneration is not practical due to the risk of side effects on healthy tissue.

[0003] (synNotch) When cells sense their surrounding environment through receptors, they have the information processing ability to output new gene expression and morphological changes through signal transduction by intracellular molecules. The well-known technology of synthetic Notch receptor (synNotch) is an artificial receptor that is a modified version of the Notch receptor that transmits intercellular signals, and both the "molecule that the cell recognizes" and the "gene that the cell induces" after molecular recognition can be freely designed (Non-Patent Document 1).

[0004] Patent Document 1 discloses a chimeric Notch polypeptide comprising, "covalently linked from the N-terminus to the C-terminus: a) an extracellular domain comprising an antigen-binding domain of an antibody; b) a Notch regulatory region comprising a transmembrane domain comprising Lin-12 Notch repeats, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site; and c) an intracellular domain heterologous to the Notch regulatory region and comprising a transcriptional activator or transcriptional repressor, wherein binding of the antigen-binding domain to an antigen induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain." Patent Literature 2 discloses "a plasmid vector pair comprising a first vector and a second vector, wherein the first vector comprises a Notch core and further comprises nucleic acid molecules encoding other extracellular and intracellular segments, the intracellular segment preferably comprising a transcriptional activator, and the second vector comprises a nucleic acid molecule that specifically recognizes and binds to the intracellular segment in the first vector and a nucleic acid molecule encoding IL12." Patent Literature 3 discloses "a method for treating a disease in a subject, comprising the step of administering engineered cells to the subject, wherein the cells express on their surface an exogenous construct comprising an extracellular antigen recognition domain that recognizes an antigen on a target cell, the cells activate an immune response when the extracellular antigen recognition domain binds to the target cell, and the cells are plasmacytoid dendritic cells." Non-Patent Literature 2 discloses "an artificial morphogen system that enables the design of cell differentiation patterns."

[0005] The above-mentioned prior art and non-patent literature does not disclose or suggest "cells or organoids that recognize inflamed or ulcerated sites and further release inflammation control factors."

[0006] Patent 6784687, Patent 2021-511784, Patent 2023-538463

[0007] Cell 164,780-791 (2016) Science. 2018 Jul 13;361(6398):156-162

[0008] Inflammatory bowel disease (IBD) is known to be an intractable disease that is difficult to cure once it develops. While recent advances in the use of anti-inflammatory drugs and antibody preparations have enabled the suppression of inflammation to some extent, some patients still experience intractable ulcers, preventing epithelial regeneration and leaving them with no other treatment option than total colectomy. Therefore, the objective of this study is to provide niche factor-producing regulatory cells that can supply inflammation-suppressing inflammatory factors and epithelial cell growth factors specifically to the ulcer site.

[0009] In order to solve the above problems, the present inventors have completed the present invention by using synNotch to generate cells that recognize sites of inflammation or ulcers and release inflammation-regulating factors and / or niche factors.

[0010] The present invention is as follows: 1. A cell capable of recognizing a surface antigen of an immune cell and releasing an inflammatory regulator and / or a niche factor, wherein the cell expresses a chimeric peptide comprising: (a) an extracellular domain including a domain that recognizes the surface antigen of an immune cell; (b) a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease; and (c) an intracellular domain comprising a transcription activator, and the cell has a nucleic acid encoding the inflammatory regulator and / or niche factor and a transcription control element operably linked to the nucleic acid, wherein binding of the domain that recognizes the surface antigen of the immune cell to an antigen causes cleavage at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain comprising the transcription activator, the released transcription activator binds to the transcription control element, the nucleic acid encoding the inflammatory regulator and / or niche factor is translated, and the inflammatory regulator and / or niche factor is released extracellularly. 2. 2. The cell according to the preceding item 1, wherein the ability is to control the release of an inflammation control factor and / or a niche factor. 3. The cell according to the preceding item 1 or 2, wherein the inflammation control factor is IL-10. 4. The cell according to the preceding item 1 or 2, wherein the cell is a colon stromal cell. 5. The cell according to the preceding item 1 or 2, wherein the immune cell is an immune cell involved in inflammation. 6. The cell according to the preceding item 1 or 2, wherein the inflammation control factor is IL-10, the cell is a colon stromal cell, and the immune cell is an immune cell involved in inflammation. 7. A composition for suppressing enteritis or promoting regeneration of intestinal epithelial tissue, comprising the cell according to the preceding item 6.8. A vector composition comprising (1) or (2) below: (1) a vector composition comprising Vector 1 and Vector 2 below, wherein Vector 1 comprises a nucleic acid encoding an extracellular domain comprising a domain that recognizes a surface antigen of an immune cell, a nucleic acid encoding a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease, and a nucleic acid encoding an intracellular domain comprising a transcription activator, and Vector 2 comprises a nucleic acid encoding an inflammation regulator and / or a niche factor, and a nucleic acid encoding a transcription control element operably linked to the nucleic acid, or (2) a vector composition comprising Gene Expression Cassette 1 and Gene Expression Cassette 2 below, wherein Gene Expression Cassette 1 comprises a nucleic acid encoding an extracellular domain comprising a domain that recognizes a surface antigen of an immune cell, a nucleic acid encoding a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease, and a nucleic acid encoding an intracellular domain comprising a transcription activator, and Gene Expression Cassette 2 comprises a nucleic acid encoding an inflammation regulator and / or a niche factor, and a nucleic acid encoding a transcription control element operably linked to the nucleic acid. 9. An intestinal organoid composition for transplantation, comprising intestinal organoids and cells that regulate niche factor production, wherein the cells release one or more of the following niche factors: (1) Rspondin1 (2) Noggin (3) EGF (4) Wnt3a 10. A composition for promoting intestinal epithelial tissue regeneration, comprising intestinal organoids and cells that regulate niche factor production, wherein the cells release one or more of the following niche factors: (1) Rspondin1 (2) Noggin (3) EGF (4) Wnt3a 11. An intestinal organoid composition for transplantation, comprising intestinal organoids that regulate niche factor production or colon stromal cells that regulate niche factor production, wherein the cells or intestinal organoids release one or more of the following niche factors.(1) Rspondin1 (2) Noggin (3) EGF (4) Wnt3a 12. A composition for promoting intestinal epithelial tissue regeneration, comprising niche factor production-controlled intestinal organoids or niche factor production-controlled colon stromal cells, wherein the cells release one or more of the following niche factors: (1) Rspondin1 (2) Noggin (3) EGF (4) Wnt3a.

[0011] The present invention can provide cells that recognize an inflammation site or an ulcer site and further release an inflammation-regulating factor and / or a niche factor.

[0012] Establishment of communication between intestinal organoids and niche factor production control cells via secreted substances {The top three figures show "wild-type organoids," and the bottom three figures show "GFP-secreting organoids"} Creation of intestinal organoids using niche factor production control cells Creation of intestinal organoids by controlling niche factor production Niche factor selection Confirmation of spatial manipulation of niche factor production control cells Control of niche factor production Confirmation of niche factor production by colonic stromal cells Confirmation of colonic stromal cell engraftment at the ulcer site Confirmation of the localization within the connective tissue of colonic stromal cells engrafted at the ulcer site Confirmation of colonic stromal cell engraftment in the colon 5 days after transplantation {The left figure shows "bright field + luminescence," and the right figure shows "luminescence signal"} Creation of synNotch "inflammatory Notch" that recognizes immune cells associated with inflammation and induces gene expression Confirmation of activation of inflammatory Notch-expressing cells at the ulcer site Confirmation of activation of inflammatory Notch-expressing colonic stromal cells at the ulcer site (colitis mice) (1) Transplantation of colonic stromal cells into the submucosa of the colon. (2) Confirmation of the induction of mild inflammation by cell transplantation.) Confirmation of the induction of inflammation by transplantation of stromal cells into the submucosa of the colon and activation of inflammatory Notch-expressing cells. {(1) Transplantation of colonic stromal cells into the submucosa, (2) Confirmation of the induction of mild inflammation by cell transplantation.} Confirmation of the induction of inflammation by transplantation of stromal cells into the submucosa of the colon and activation of inflammatory Notch-expressing cells. {(1) Confirmation of luminescence signals in colon slices two days after transplantation, (2) Confirmation of the activation of inflammatory Notch due to inflammation associated with cell transplantation.} Confirmation of the activation of inflammatory Notch-expressing cells by increased expression of LFA-1 in activated immune cells. {(1) Schematic diagram of RAW cells activated by LPS stimulation and inflammatory Notch-expressing cells, (2) Confirmation that RAW cells activated by LPS stimulation strongly activated inflammatory Notch.}

[0013] (Subject of the present invention) The present invention relates to an inflammation control factor and / or niche factor production control cell, a transplantable intestinal organoid composition, a composition for promoting intestinal epithelial tissue regeneration, and a composition for suppressing enteritis. These are described in detail below. The compositions of the present invention also include various agents (therapeutic, alleviating, preventative, and recurrence prevention agents), foods (supplements, beverages), and the like.

[0014] (synNotch System) The synNotch system allows for the design of both the molecules that cells recognize and the genes that they induce after molecular recognition. Using this system, it is possible to design and generate cells that recognize cells or components specifically present at target sites (e.g., ulcers or inflammation) and produce anti-inflammatory factors or niche factors (growth factors). Therefore, synNotch makes it possible to develop "designer cells" that recognize diseases within the body and perform therapeutic activities against those diseases. Using designer cells, therapeutic factors that are not suitable for systemic administration (e.g., anti-inflammatory cytokines and niche factors) can be delivered to the necessary location and time. The principles and construction methods of the synNotch system are disclosed in Non-Patent Documents 1-2 and Patent Documents 1-3, etc. References to these documents are made throughout this specification as necessary.

[0015] (Inflammation Regulatory Factor and / or Niche Factor Production Control Cells) The inflammation regulator and / or niche factor production control cells of the present invention (hereinafter sometimes simply referred to as "control cells of the present invention") do not simply release inflammation regulator and / or niche factor into the environment, but rather recognize a specific environment (site of inflammation, site of ulcer) and release a target protein (inflammatory regulator and / or niche factor) in that specific environment by using the synNotch system described above. Therefore, the cells can control (stop, interrupt, reduce, increase) the release of the target protein in response to changes in the environment (such as inflammation suppression or activation).

[0016] The regulatory cells of the present invention are not limited to any particular cellular components, as long as they are capable of recognizing a surface antigen on an immune cell and releasing an inflammatory regulator and / or a niche factor using the synNotch system. The regulatory cells of the present invention express a chimeric peptide comprising: (a) an extracellular domain comprising a domain that recognizes the surface antigen on an immune cell; (b) a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease; and (c) an intracellular domain comprising a transcriptional activator. Additionally, the regulatory cells of the present invention have a nucleic acid encoding the inflammatory regulator and / or niche factor and a transcriptional control element operably linked to the nucleic acid. When the domain that recognizes the surface antigen on an immune cell binds to the antigen, cleavage occurs at the one or more proteolytic cleavage sites, releasing the intracellular domain comprising the transcriptional activator. The released transcriptional activator binds to the transcriptional control element, leading to transcription and translation of the nucleic acid encoding the inflammatory regulator and / or niche factor, and the inflammatory regulator and / or niche factor being released extracellularly. The "domain" of the present invention may be heterogeneous, i.e., may be composed of sequences derived from different protein chains. Furthermore, when the "domain" of the present invention includes a nucleic acid encoding a heterologous protein, it is not necessary to encode the full-length amino acid sequence of the protein, but it is sufficient to have only the site that expresses the necessary function of the protein (e.g., antigen recognition function).

[0017] (Extracellular domain including a domain that recognizes a surface antigen on an immune cell) The domain that recognizes a surface antigen on an immune cell is a site that recognizes an antigen on the immune cell, and can be designed using known techniques according to the desired therapeutic application or target cell / tissue. Immune cells are not particularly limited, but include T cells, B cells, NK cells, NKT cells, DNAT cells, etc., and particularly include immune cells contained in splenic tissue, such as T cells, B cells, and dendritic cells. Furthermore, the present invention preferably targets immune cells involved in inflammation. Immune cell surface antigens are not particularly limited, but include the well-known LFA-1 (lymphocyte function-associated antigen-1). The domain that recognizes a surface antigen on an immune cell is not particularly limited as long as it can recognize (bind to) the antigens described above. For example, the following combinations of antigen and domain that recognizes the antigen are possible: LFA-1-Icam-1

[0018] (Proteolytically cleavable Notch receptor containing one or more proteolytic cleavage sites) In the present invention, the "proteolytically cleavable Notch receptor containing one or more proteolytic cleavage sites" preferably uses a known Notch used in the synNotch system or a variant thereof. Examples include Notch1, Notch2, Notch3, and Notch4, with mouse-derived Notch1 being preferred. More specifically, the Notch receptor of the present invention preferably retains the important core regulatory region of a native Notch receptor. This region includes one or more of the Lin12-Notch repeat (LNR), heterodimerization domain (HD), and transmembrane domain (TMD), etc.

[0019] (Transcription activator) In the present invention, the "transcription activator" preferably uses a known transcription activator used in the synNotch system or a modified version of the transcription activator. The transcription activator preferably includes a DNA-binding factor and a transcription activation domain. The DNA-binding factor specifically recognizes and binds to a transcription control element. The transcription activation domain promotes RNA polymerase activity near the transcription start site, potently inducing gene expression. Examples of DNA-binding factor-transcription activation domains include TetR-VP64, Gal4-VP64, Gal4-VP16, and LacI-VP64, with TetR-VP64 being preferred.

[0020] (Transcriptional control element) In the present invention, the "transcriptional control element" has the function of binding to a transcriptional activator and inducing the transcription of a nucleic acid encoding an inflammatory control factor and / or a niche factor, which is a target protein. For example, the transcriptional activator-transcriptional control element combination is as follows: TetR-Vp64 - TRE Gal4-VP64 - UAS

[0021] (Cells) The "cells" of the present invention are not particularly limited as long as they can realize the synNotch system, and examples thereof include well-known cells such as colon stromal cells, NIH3T3 cells, and intestinal organoids.

[0022] (Niche Factors) Niches are tissue microenvironments that are created when cells surrounding stem cells supply signaling molecules, scaffolding, nutrients, etc., and are involved in the control of stem cell proliferation and differentiation, making them important in tissue formation and regeneration. In the present invention, "niche factors" refer to factors necessary for promoting niche formation and repair. Examples include Respondin1, Noggin, EGF, and Wnt3a. Niche factors have effects such as inhibiting enteritis and promoting the regeneration of intestinal epithelial tissue. In addition, niche factors also include inflammation-regulating factors. In the present invention, "inflammatory regulatory factors" refer to factors that have the effect of suppressing inflammation at inflamed or ulcerated sites in tissues (small intestine (duodenum, jejunum, ileum) and large intestine (cecum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum)). Specific examples include IL-10, IL-12, IL-17, IL-22, IL-37, IL-38, TGF-β, GM-CSF, etc.

[0023] (Vector Composition) The "vector composition" of the present invention comprises either (1) or (2) below. (1) A vector composition comprising the following vectors: vector 1 and vector 2. vector 1 comprises a nucleic acid encoding an extracellular domain comprising a domain that recognizes a surface antigen of an immune cell, a nucleic acid encoding a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease, and a nucleic acid encoding an intracellular domain comprising a transcription activator. vector 2 comprises a nucleic acid encoding an inflammation regulator and / or a niche factor, and a nucleic acid encoding a transcription control element operably linked to the nucleic acid. (2) A vector composition comprising the following gene expression cassette 1 and gene expression cassette 2. gene expression cassette 1 comprises a nucleic acid encoding an extracellular domain comprising a domain that recognizes a surface antigen of an immune cell, a nucleic acid encoding a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease, and a nucleic acid encoding an intracellular domain comprising a transcription activator. gene expression cassette 2 comprises a nucleic acid encoding an inflammation regulator and / or a niche factor, and a nucleic acid encoding a transcription control element operably linked to the nucleic acid. The regulatory cells of the present invention can be prepared by transfecting the "vector composition" of the present invention into cells capable of expressing a known synNotch system.

[0024] (Composition for inhibiting enteritis) The "composition for inhibiting enteritis" of the present invention can inhibit ulcers and inflammation at the ulcer site by administering and / or transplanting the composition to the site of intestinal inflammation or ulcer. This composition contains the regulatory cells of the present invention.

[0025] (Intestinal Organoid Composition for Transplantation) Regarding the "intestinal organoid composition for transplantation" of the present invention, by administering and / or transplanting the composition to diseased tissues or the like where the niche environment has been disrupted, the niche environment is improved and intestinal organoids can be engrafted. This composition contains intestinal organoids and cells that regulate the production of niche factors (Rspondin1, Noggin, EGF, and Wnt3a). Furthermore, this composition contains intestinal organoids that regulate the production of niche factors (Rspondin1, Noggin, EGF, and Wnt3a) or colonic stromal cells that regulate the production of niche factors (Rspondin1, Noggin, EGF, and Wnt3a).

[0026] (Composition for Promoting Intestinal Epithelial Tissue Regeneration) With regard to the "composition for promoting intestinal epithelial tissue regeneration" of the present invention, by administering and / or transplanting the composition to diseased tissue, etc., where intestinal epithelial tissue has been damaged or destroyed, niche factors can promote the regeneration and repair of intestinal epithelial tissue. The composition comprises intestinal organoids and cells that regulate the production of niche factors (Rspondin1, Noggin, EGF, and Wnt3a). The composition also comprises intestinal organoids that regulate the production of niche factors (Rspondin1, Noggin, EGF, and Wnt3a) or colonic stromal cells that regulate the production of niche factors (Rspondin1, Noggin, EGF, and Wnt3a).

[0027] (Methods for Administration and Transplantation of Regulatory Cells and Compositions of the Present Invention) The regulatory cells and compositions of the present invention can be administered parenterally. Parenteral administration includes injection into the site of inflammation and rectal administration using suppositories, etc. For example, rectal formulations such as suppositories contain common excipients such as polyalkylene glycol, petrolatum, and cocoa oil. Vaginal formulations may contain absorption enhancers such as bile salts, ethylenediamine salts, and citrate salts. Inhalation formulations may be solid and may contain excipients such as lactose, and intranasal drops may be aqueous or oil solutions. Nasal administration, which is a local administration, is thought to have fewer side effects than systemic administration. The exact dosage and administration schedule of the regulatory cells and compositions of the present invention can be adjusted depending on the individual treatment subject's requirements, treatment method, disease, or severity of need. The dosage can be determined specifically depending on the age, body weight, general health condition, sex, diet, administration time, administration method, excretion rate, drug combination, and patient's condition, and may also be determined taking other factors into consideration.

[0028] (Modified Products) The modified products of the present invention may be those having mutations in naturally occurring proteins or those obtained by introducing mutations into naturally occurring genes. Methods for introducing mutations are known per se, and include, for example, site-directed mutagenesis, homologous recombination, primer extension, and polymerase chain reaction (hereinafter abbreviated as PCR), which can be used alone or in appropriate combinations. For example, methods described in textbooks (Sambrook et al., eds., "Molecular Cloning, A Laboratory Manual, 2nd Edition," 1989, Cold Spring Harbor Laboratory; Muramatsu Masami, ed., "Laboratory Manual of Genetic Engineering," 1988, Maruzen Co., Ltd.) can be used, or modified versions of these methods can be used. Ulmer's technique (Ulmer, KM, Science, 1983, Vol. 219, pp. 666-671) can also be used. In the case of peptides, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations, for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) are easily conceivable. More specifically, examples of the modified product of the present invention include the following: (1) A polypeptide consisting of an amino acid sequence in which 1 to 20 (1 to 15, 1 to 10, 1 to 5, 1 to 3, or 2) amino acids in a naturally occurring protein have been substituted, deleted, inserted, and / or added, and which has substantially the same function as the protein; (2) A polypeptide consisting of an amino acid sequence which has 90% or more, 95% or more, 98% or more, or 99% or more identity with a naturally occurring protein and which has substantially the same function as the protein.

[0029] (Factors required for membrane protein production) Factors required for membrane protein production (e.g., CD8 signal sequence, immunoglobulin light chain kappa-derived signal sequence, etc.) are used to ensure that the SynNotch receptor is correctly transported to the cell membrane and can recognize antigens there. Specifically, factors required for membrane protein production play a role in accurately positioning the receptor's extracellular domain and transmembrane domain on the cell surface, enabling it to detect external signals.

[0030] (Mechanism by which the regulatory cells of the present invention release inflammatory regulators and / or niche factors) Examples of the mechanism by which the regulatory cells of the present invention recognize antigens and further release inflammatory regulators and / or niche factors are exemplified below, but are not limited to these. When the Notch receptor contained in the regulatory cells of the present invention recognizes an antigen via a domain that recognizes a surface antigen on an immune cell, tension from the surface antigen causes a conformational change in the receptor. This change exposes a cleavage site on the receptor where a protease acts. Next, a protease present in the cell membrane (e.g., γ-secretase, ADAM17) cleaves the exposed proteolytic cleavage site in the transmembrane domain (TMD). Next, the intracellular domain containing the DNA-binding factor and the transcriptional activation factor containing the transcriptional activation domain cleaved by the protease translocates through the cytoplasm to the cell nucleus. The DNA-binding factor specifically binds to a transcriptional regulatory element, and the transcriptional activation domain potently promotes transcriptional activity, resulting in the transcription and translation of nucleic acids encoding the inflammatory regulators and / or niche factors. The translated inflammatory regulatory factors and / or niche factors are released extracellularly. As confirmed in the following examples, the regulatory cells of the present invention can increase the release of inflammatory regulatory factors and / or niche factors when immune cells are activated. Furthermore, the release of inflammatory regulatory factors and / or niche factors can be reduced or stopped when immune cells are inactivated or eliminated.

[0031] (Examples of regulatory cells of the present invention) Combinations of regulatory cells of the present invention include, but are not limited to, the following: "Niche factor (inflammatory regulatory factor)"-"regulatory cell"-"immune cell" "IL-10"-"colon stromal cell"-"immune cell involved in inflammation" "Wnt3a"-"colon stromal cell"-"immune cell involved in inflammation" "Wnt3a and IL-10"-"colon stromal cell"-"immune cell involved in inflammation"

[0032] The present invention will be specifically described below with reference to examples and comparative examples to deepen understanding of the present invention, but these are not intended to limit the scope of the present invention. All animal experiments were conducted with the approval of the Kanazawa University Animal Experiment Committee (approval number: AP-214277).

[0033] (Materials and Methods) The following Examples 2 to 8 were carried out using the following materials and methods.

[0034] (Plasmid Construct Design of Inflammatory Notch System) The design of the Inflammatory Notch system is based on the known synthetic Notch receptor (see Cell 164, 780-791 (2016)). The Inflammatory Notch system consists of the recognition domain of inflammation-associated antigens in the extracellular domain, the regulatory region of the transmembrane region of mouse Notch1 (NM_008714) (Ile1427 to Arg1752 (ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAA This system was constructed by fusing three proteins: the CD8α signal sequence (MALPVTALLLPLALLLHAARP; SEQ ID NO: 2) and the intracellular domain of TetR-VP64. The CD8α signal sequence (MALPVTALLLPLALLLHAARP; SEQ ID NO: 2) and Myc tag (EQKLISEEDL; SEQ ID NO: 3) were inserted at the N-terminus of the system. The inflammatory Notch system was cloned under the PGK promoter and linked to the puromycin resistance gene via a P2A ribosomal skipping peptide. For inducible expression of target genes using the inflammation-Notch system, the target genes were cloned under a tetracycline response element (TRE; TCCCTATCAGTGATAGAGA: SEQ ID NO: 4) fused to a minimal CMV promoter. Both constructs were loaded into a modified pHR'SIN:CSW vector for lentiviral transduction. The extracellular domain containing Icam-1 was used as the recognition domain for inflammation-associated antigens. To create an inflammation-Notch system that recognizes soluble inflammation-associated antigens, a combination of two recognition domains was used (see Science. 370, 327-331 (2020)).Target genes are designed to contain fluorescent reporter proteins and therapeutic factors (anti-inflammatory factors such as IL10, growth factors / niche factors such as Wnt, and any genetically encoded factors such as engineered proteins, biologics, and enzymes).

[0035] (Isolation and Culture of Colonic Stromal Cells) Mouse colons were opened longitudinally and washed in PBS to remove feces. The opened colons were placed in 5 mL of HBSS / 5 mM EDTA in a 15 mL tube and incubated at 37°C for 15 minutes with shaking (110 rpm on an orbital shaker). The colons were then transferred to a 6 cm dish and vigorously shaken with tweezers in 5 mL of PBS to remove epithelial cells. The colonic tissue was transferred to a new 6 cm dish containing 5 mL of PBS and shaken several times to remove almost all of the epithelium. The colonic tissue was then cut into small pieces in 5 mL of RPMI-1640 containing 4% FBS in the 6 cm dish and placed in a 15 mL tube containing 0.5 mg / mL collagenase D, 0.5 mg / mL dispase, and 0.1 mg / mL DNase I. The pieces were then incubated at 37°C for 1 hour with shaking (110 rpm on an orbital shaker). The digested tissue was centrifuged, resuspended in 10 mL of HBSS / 5 mM EDTA, and then passed through a 40 μm cell strainer to remove debris. Isolated cells were washed in RPMI-1640 / 4% FBS, suspended in 12 mL of RPMI-1640 / 10% FBS / Penstrep, and plated onto 10 cm dishes. Colon stromal cells were cultured in RPMI-1640 / 10% FBS / Penstrep medium at 37°C in a humidified atmosphere with 5% CO2.

[0036] (Generation of niche factor-producing regulatory cells) Colon stromal cells were stably transduced with the inflammatory Notch system and an inducible gene cassette using lentivirus. To produce lentivirus, HEK293T cells were transfected with the lentiviral packaging plasmids pCMVdR8.91 and pMD2.G, which encode both the inflammatory Notch system and the inducible target gene, using PEI MAX (Polysciences, #24765). Colon stromal cells were then co-cultured with various amounts of lentiviral supernatant in 6 cm dishes. Three days after infection, colon stromal cells were selected with 1 μg / mL puromycin. Stepwise cell sorting was performed to establish a pure cell population that recognized immune cells and had high target gene induction. Specifically, activated cells were selected after stimulation with splenocytes, and cells lacking target gene expression in the absence of stimulation were selected. In addition, to detect transplanted colonic stromal cells in the mouse colon, Venus-Akaluc was also expressed in colonic stromal cells.

[0037] (In vitro activation of niche factor-producing regulatory cells) We confirmed whether niche factor-producing regulatory cells, which were generated by introducing the inflammatory Notch system into colon stromal cells, recognize and activate immune cells in vitro and induce target gene expression. 5 Niche factor-producing control cells were seeded into two wells of a 12-well plate. The next day, 4.0x10 cells were seeded into one well. 6 Splenocytes were added to the wells. After overnight culture, the reporter fluorescent protein was analyzed by flow cytometry for background expression levels without stimulation and for induced expression levels upon splenocyte stimulation.

[0038] (Transplantation of niche factor-producing regulatory cells into the colonic lumen) This was performed using a modified method for transplanting intestinal organoids into the colon described in Nature Protocols 17, 649-671 (2022). To induce colitis, dextran sodium sulfate (DSS) was administered in drinking water for 5 consecutive days (days -7 to -2). The DSS concentration was optimized for each lot. Two days after DSS treatment (day 0), niche factor-producing regulatory cells were injected into the colonic lumen using the following method: 5x10 6 1x10 7 Niche factor-producing regulatory cells were resuspended in 350 μL of PBS containing 5% Matrigel. A winged-needle silicone tube was used as a flexible catheter for transplantation. The needle was removed from the winged needle to create a flexible catheter approximately 4 cm long. The inside of the catheter was washed with PBS containing 0.1% BSA. Wild-type mice were anesthetized with isoflurane (5% isoflurane until immobility was detected, then maintained at 2–3%). An empty catheter was inserted 2 cm into the anus before injection and removed after confirming smooth insertion. Next, 350 μL of cell suspension was aspirated into the catheter, and while holding the anus with tweezers, the catheter was inserted 2 cm into the anus and injected into the colonic lumen. Immediately after catheter removal, the anus was closed with the embolizing agent Histoacryl. The Histoacryl naturally detached from the anus in most mice by the next morning, but in mice where it had not, it was removed the following morning.

[0039] (Analysis of Transplanted Niche Factor-Producing Regulatory Cells) For flow cytometry analysis, the colon (approximately 2-3 cm from the anus) was opened longitudinally and washed with saline. The colon was immersed in 2 mL of TrypLE in a 3.5 cm dish and treated at 37°C for 30 minutes. The colon was shaken to remove detached cells, which were then resuspended in PBS / 5% FBS and analyzed by flow cytometry. For bioluminescence imaging, the colon (approximately 2-3 cm from the anus) was opened longitudinally and washed with saline. The colon tissue was placed in a culture dish with the lumen facing up, and saline containing 200 μM acarmine was added. Bioluminescence images were captured using the Chemiluminescence Imaging System Fusion. For fluorescence imaging, the opened colon was placed in a culture dish with the lumen facing down and observed using a Keyence fluorescence microscope. For RT-PCR analysis, the opened colon was cut into small pieces in a 1.5 mL tube, 1 mL of ISOGEN was added, and the colon tissue was sheared with an 18-gauge needle, lysed, and RNA was purified.

[0040] (Method for generating intestinal organoids) Intestinal organoids were generated from crypts isolated from mouse small intestines. Crypt isolation and culture were performed according to the protocol for IntestiCult Organoid Growth Medium (Mouse) (STEMCELL Technologies). To generate intestinal organoids secreting GFP, a lentiviral vector was constructed that contained a secreted GFP gene under the EF1a promoter (see Science. 370, 327-331 (2020)) and a puromycin resistance gene linked via a P2A ribosomal skipping peptide. Lentivirus was prepared using this vector, and intestinal organoids were infected and selected with 5 μg / mL of puromycin.

[0041] (Culture method for intestinal organoids using niche-producing regulatory cells) We introduced the synNotch system (see Science. 370, 327-331 (2020)), which recognizes GFP secreted into NIH3T3 cells, to generate niche factor-producing regulatory cells that induce fluorescent reporter proteins and niche factors (Rspondin1, Noggin, EGF, Wnt3a) as target genes. To examine the proliferation of intestinal organoids using NIH3T3 cells generated as niche factor-producing regulatory cells, 2x10 cells were cultured. 3 The cells were seeded in a 96-well plate. The next day, the supernatant was removed from each well, and 50 μL of Advanced DMEM / F-12 solution containing 50% Matrigel, containing either wild-type or GFP-secreting intestinal organoids, was added. The cells were incubated at 37°C for 10 minutes to allow the Matrigel to solidify. Then, 150 μL of Advanced DMEM / F-12 containing 20% ​​FBS, 10 μM Y-27632, and 2 mM valproic acid was added, and the cells were cultured for 4 days.

[0042] (Generation of niche factor-producing regulatory cells that induce intestinal organoid formation) We investigated whether niche factor-producing regulatory cells can induce intestinal epithelial tissue regeneration in an in vitro culture system using intestinal organoids and a mouse fibroblast cell line. First, we generated intestinal organoids that secrete GFP, a green fluorescent molecule used as an inflammation-related factor, as a model of intestinal tissue under inflammatory conditions. We also used the synNotch system to generate a mouse fibroblast cell line, NIH3T3, that induces a fluorescent reporter upon GFP recognition. When these cells were co-cultured, we observed that GFP diffusion activated fibroblasts surrounding the intestinal organoids (Figure 1). Next, we generated niche factor-producing regulatory cells engineered to produce niche factors (Rspondin1, Noggin, EGF, and Wnt3a) required for intestinal organoid growth upon GFP recognition. These cells were then co-cultured with wild-type and GFP-secreting intestinal organoids. As a result, in a medium lacking niche factors, most wild-type intestinal organoids died after four days of culture. However, in the case of GFP-secreting intestinal organoids, niche factor-producing regulatory cells recognized and activated GFP, producing niche factors before the intestinal organoids died, successfully culturing the intestinal organoids (Figure 2). The niche factor-producing regulatory cells that recognize the intestinal organoids enable intestinal organoid culture in an environment lacking niche factors. In other words, the intestinal organoids and the niche factor-producing regulatory cells that recognize the intestinal organoids can recognize the intratissue environment and promote tissue regeneration (intestinal epithelial tissue regeneration).

[0043] We hypothesized that the current intestinal organoid transplantation therapy could be further improved by modifying intestinal organoids themselves to generate niche factor-regulated intestinal organoids that also function as niche factor-regulated cells. Therefore, we introduced the synNotch system into intestinal organoids to generate niche factor-regulated intestinal organoids. Specifically, when the synNotch system, which induces a fluorescent reporter upon GFP recognition, was introduced into intestinal organoids, the synNotch system functioned normally, and the intestinal organoids responded to GFP and induced the fluorescent reporter (Figure 3A). Therefore, we generated niche factor-regulated intestinal organoids that produce niche factors (Rspondin1, Noggin, EGF, and Wnt3a) upon GFP recognition. When cultured in niche-free medium, the intestinal organoids grew significantly upon the addition of GFP (Figure 3B). This suggests that niche factor-regulated intestinal organoids can recognize the tissue environment and promote tissue regeneration (intestinal epithelial tissue regeneration).

[0044] (Selection of Niche Factors) In this example, we identified niche factors essential for organoid culture. NIH3T3 cells were generated that express various combinations of four niche factors (Rspondin1, Noggin, EGF, and Wnt3a) required for mouse intestinal organoid culture. We confirmed that culture with two, three, and four factors was possible (Figure 4). Furthermore, intestinal organoids proliferated most vigorously when co-cultured with NIH3T3 cells that simultaneously expressed all four factors (Figure 4). Therefore, in the following examples, cells or intestinal organoids that regulate the production of the four factors were used.

[0045] (Confirmation of spatial manipulation of niche factor-producing cells) Intestinal organoid formation using niche factor-producing cells can significantly reduce culture costs because the cells themselves supply niche factors, eliminating the need to purchase purified proteins. Furthermore, signaling distances can be manipulated by changing whether the niche factor-producing cells recognize cell surface ligands (membrane-bound GFP) or secreted factors (secreted GFP). Furthermore, the spatial distribution of activated niche factor-producing cells could be manipulated by plating the cells on the surface of a dish or within Matrigel (Figure 5). When producing niche factors that interact with the cell surface or extracellular matrix, manipulating the positioning of niche factor-producing cells alters the spatial distribution of niche factors, enabling spatial control of organoid morphology and cellular composition.

[0046] (Consideration of cell types for niche factor production-regulating cells for transplantation) We investigated whether stromal cells resident in the mouse small intestine and large intestine could be used as a source of niche factor production-regulating cells for transplantation. Both small intestinal and colonic stromal cells could be transduced with lentiviruses. However, small intestinal stromal cells ceased proliferation after approximately one month of passage, whereas colonic stromal cells could be cultured long-term, enabling the necessary cell numbers for transplantation experiments to be obtained. Next, to convert colonic stromal cells into niche factor production-regulating cells, we confirmed the function of the synNotch receptor in colonic stromal cells. When colonic stromal cells were transfected with the synNotch system, which induces a fluorescent reporter upon recognition of GFP, and co-cultured with K562 cells expressing GFP on the cell surface, the colonic stromal cells were activated and induced the fluorescent reporter (Figure 6). These results demonstrate the feasibility of generating niche factor production-regulating colonic stromal cells based on the synNotch system.

[0047] (Confirmation of engraftment of niche factor-producing regulatory cells when transplanted into the intestinal tract) In a mouse model of enteritis in which colonic ulcers were formed by administration of dextran sodium sulfate (DSS), colonic stromal cells, the source of niche factor-producing regulatory cells, were transplanted into the colon, and engraftment of the colonic stromal cells into the colon was confirmed. Colonic stromal cells were labeled by expressing the fluorescent molecule Venus and the luminescent molecule Akaluc, and 5x10 6 We transplanted colonic stromal cells into the colons of mice with enteritis and confirmed their survival. First, the colons were harvested the day after transplantation, and their surfaces were observed under a fluorescence microscope. While no colonic stromal cells survived on normal epithelium with villi present on the colonic surface, colonic stromal cells were observed to survive on ulcerated areas where villi had been lost (Figure 7A). Furthermore, the colon was sectioned into 5-mm sections, and the luminescence of each tissue section was detected using a plate reader. We confirmed that the transplanted cells survived on ulcerated areas within 2 cm of the anus (Figure 7B). Next, we prepared sections of the colon transplanted with colonic stromal cells and visualized the transplanted cells by immunostaining with a GFP antibody that recognizes Venus. We found that colonic stromal cells not only adhered to the ulcerated surface but also localized within the connective tissue (Figure 8; the arrowheads indicate transplanted cells visualized by immunostaining). Furthermore, 1 x 10 7 After transplantation of colonic stromal cells, approximately 2 cm of the colon was removed from the anus five days later, and bioluminescence imaging analysis of the entire colonic lumen surface was performed. Surviving colonic stromal cells were detected even five days after transplantation (Figure 9). These results demonstrate that colonic stromal cells that survive in the ulcer site for more than five days are suitable as a source of niche factor-producing regulatory cells for the treatment of enteritis.

[0048] (Construction of synNotch Recognizing Immune Cells Associated with Inflammation and Inducing Gene Expression, and Evaluation of Cells Expressing the Notch) To generate niche factor-producing regulatory cells that recognize and activate ulcer sites (producing niche factors), we designed an artificial receptor that recognizes and activates immune cells infiltrating the ulcer site in large numbers. Specifically, we engineered an artificial receptor by inserting the extracellular domain of Icam-1, a cell adhesion molecule that binds to the immune cell surface antigen LFA-1, into the extracellular domain of the synNotch receptor (Figure 10). NIH3T3 cells expressing this artificial receptor were activated and induced fluorescent reporters when co-cultured with immune cells collected from mouse spleens (Figure 10). Furthermore, to confirm whether the synNotch receptor bearing extracellular Icam-1 recognizes and activates immune cells in vivo, we transplanted NIH3T3 cells expressing this artificial receptor into the intestines of mice with enteritis. The NIH3T3 cells efficiently engrafted into the ulcer site, similar to colon stromal cells, and simultaneously activated the artificial receptor, inducing the fluorescent reporter (Figure 11). Based on these results, the synNotch receptor, which has an extracellular molecule (Icam1) that recognizes surface antigens on immune cells, is an artificial receptor system that can recognize immune cells and induce target genes both in vitro and in vivo. We named this system "inflammatory Notch."

[0049] Next, we introduced inflammatory Notch into colonic stromal cells and confirmed the effects as described above. In vitro, colonic stromal cells expressing inflammatory Notch were activated by co-culture with mouse spleen-derived immune cells, resulting in the induction of fluorescent reporter expression. Furthermore, when transplanted into mice with enteritis, the cells engrafted at the ulcer site, became activated, and induced the expression of fluorescent reporters (Figure 12). These results suggest that niche factor-producing regulatory cells expressing inflammatory Notch can recognize ulcer sites (inflammation sites) and respond by producing niche factors.

[0050] (Evaluation of Niche Factor-Producing Regulatory Cells That Recognize Immune Cells and Produce Anti-Inflammatory Factors) In this example, we generated niche factor-producing regulatory cells that recognize immune cells and produce anti-inflammatory factors (IL-10), and confirmed the anti-inflammatory effects of these cells. Colon stromal cells expressing the inflammatory Notch gene shown in Figure 13 were generated. Specifically, artificial receptors were created by inserting the extracellular domain of the cell adhesion molecule Icam-1, which binds to the surface antigen LFA-1 on immune cells recovered from the spleen (Figure 13). The culture supernatant obtained by co-culturing these cells with immune cells recovered from the spleen was added to a co-culture system of a macrophage cell line (RAW264.7 cells) and lipopolysaccharide (LPS), and the expression level of the inflammatory cytokine IL6 was analyzed (Figure 13). Macrophages recognized LPS, activated the innate immune response, and produced the inflammatory cytokine IL6. Niche factor-producing regulatory cells, which recognize immune cells and produce anti-inflammatory factor (IL-10), were activated when spleen-derived immune cells were added, producing IL-10 and suppressing IL6 induction in macrophages. Thus, when niche factor-producing regulatory cells recognize immune cells, they produce IL-10, suppressing the expression of inflammatory cytokines induced by macrophage activation, resulting in a therapeutic effect against enteritis.

[0051] (Induction of inflammation and activation of inflammatory Notch-expressing cells by colonic stromal cell transfer into the colonic submucosa) 2x10 colonic stromal cells were transferred into the colonic submucosa of wild-type mice. 6 cells or 5x10 5When cells were directly transplanted, luminescence signals from the transplanted cells were detected in the colon tissue two days after transplantation, confirming the engraftment of colon stromal cells into the colon tissue of wild-type mice. We observed increased expression of the inflammatory cytokine IL6 in the colon transplanted cells, and found that transplantation of colon stromal cells into the submucosal layer induced milder inflammation than DSS administration (see Figures 14(1)(2)). Therefore, we harvested the colon transplanted with inflammatory Notch-expressing colon stromal cells into the submucosa, isolated the transplanted cells by enzymatic treatment, and analyzed the results. We found that the inflammatory Notch-expressing colon stromal cells were activated (see Figures 15(1)(2)). These results confirmed that inflammatory Notch-expressing colon stromal cells are activated by inflammation induced in vivo.

[0052] (Confirmation of Activation of Inflammatory Notch-Expressing Cells by Increased LFA-1 Expression in Activated Immune Cells) The expression level of LFA-1 expressed by monocytes and leukocytes is known to increase in response to inflammatory stimuli and contribute to adhesion to vascular endothelial cells. The macrophage-like cell line RAW also expresses LFA-1, and its expression has been reported to increase upon activation by LPS stimulation. In this example, RAW cells were cocultured with inflammatory Notch-expressing colonic stromal cells to determine whether the activation level of inflammatory Notch changed with the presence or absence of inflammatory stimulation by LPS (see Figure 16(1)). As a result, inflammatory Notch-expressing cells recognized and weakly activated steady-state RAW cells, whereas LPS-activated RAW cells strongly activated inflammatory Notch-expressing cells. Therefore, inflammatory Notch strongly responded to activated immune cells present at the site of inflammation (see Figure 16(2)). These findings suggest that niche factor-producing regulatory cells can regulate (adjust) the niche factor-producing ability of immune cells depending on their activation state.

[0053] (General Discussion) Niches, tissue microenvironments, are generated by the supply of signaling molecules, scaffolding, and nutrients by surrounding cells. They are involved in the regulation of stem cell proliferation and differentiation and are important in tissue formation and regeneration. In this example, we developed "niche factor-producing regulatory cells," cells that produce niche factors in response to changes in the surrounding environment, in order to generate cells that specifically generate niches in diseased tissues where the niche has collapsed. We demonstrated that niche-producing regulatory cells, which recognize intestinal organoids and produce niche factors, induce intestinal organoid formation in a niche factor-free environment. Furthermore, niche cells in living tissues not only supply niche factors but also secrete niche factors to form concentration gradients, providing positional information to surrounding cells and spatially regulating cell differentiation. Therefore, we demonstrated the advantage of using niche-producing regulatory cells by manipulating the spatial pattern of activated niche-producing regulatory cells, allowing them to be positioned in various patterns relative to organoids. Furthermore, niche factor production regulatory cells that express "inflammatory Notch" can recognize and activate ulcer sites (inflammation sites) and produce designated niche factors. They can also produce anti-inflammatory factors as niche factors to suppress inflammation at ulcer sites and promote tissue regeneration. In addition, niche factor production regulatory cells can control (adjust) the ability to produce niche factors depending on the activation state of immune cells.

[0054] The present invention can provide cells that recognize an inflammation site or an ulcer site and further release an inflammation-regulating factor and / or a niche factor.

Claims

1. A cell having the ability to recognize a surface antigen of an immune cell and release an inflammatory regulator and / or a niche factor, wherein the cell expresses a chimeric peptide comprising: (a) an extracellular domain comprising a domain that recognizes the surface antigen of an immune cell; (b) a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a proteolytic enzyme; and (c) an intracellular domain comprising a transcription activator, wherein the cell has a nucleic acid encoding the inflammatory regulator and / or niche factor and a transcription control element operably linked to the nucleic acid, wherein upon binding of the domain that recognizes the surface antigen of the immune cell to an antigen, cleavage occurs at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain comprising the transcription activator, the released transcription activator binds to the transcription control element, the nucleic acid encoding the inflammatory regulator and / or niche factor is translated, and the inflammatory regulator and / or niche factor is released extracellularly.

2. The cell of claim 1, wherein the ability is capable of controlling the release of inflammatory regulators and / or niche factors.

3. The cell described in claim 1 or 2, wherein the inflammation control factor is IL-10.

4. The cell according to claim 1 or 2, wherein the cell is a colon stromal cell.

5. The cell according to claim 1 or 2, wherein the immune cell is an immune cell involved in inflammation.

6. The cell according to claim 1 or 2, wherein the inflammation control factor is IL-10, the cell is a colon stromal cell, and the immune cell is an immune cell involved in inflammation.

7. A composition for suppressing enteritis or promoting regeneration of intestinal epithelial tissue, comprising the cells described in claim 6.

8. A vector composition comprising the following (1) or (2): (1) a vector composition comprising the following vector 1 and vector 2, wherein the vector 1 comprises a nucleic acid encoding an extracellular domain comprising a domain that recognizes a surface antigen of an immune cell, a nucleic acid encoding a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease, and a nucleic acid encoding an intracellular domain comprising a transcription activator, and the vector 2 comprises a nucleic acid encoding an inflammation regulator and / or a niche factor, and a nucleic acid encoding a transcription control element operably linked to the nucleic acid; or (2) a vector composition comprising the following gene expression cassette 1 and gene expression cassette 2, wherein the gene expression cassette 1 comprises a nucleic acid encoding an extracellular domain comprising a domain that recognizes a surface antigen of an immune cell, a nucleic acid encoding a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and cleavable by a protease, and a nucleic acid encoding an intracellular domain comprising a transcription activator, and the gene expression cassette 2 comprises a nucleic acid encoding an inflammation regulator and / or a niche factor, and a nucleic acid encoding a transcription control element operably linked to the nucleic acid.

9. A transplantable intestinal organoid composition comprising an intestinal organoid and a niche factor production control cell, wherein the cell releases any one or more of the following niche factors: (1) Respondin1 (2) Noggin (3) EGF (4) Wnt3a 10. A composition for promoting intestinal epithelial tissue regeneration, comprising intestinal organoids and niche factor production regulatory cells, the cells releasing one or more of the following niche factors: (1) Respondin1 (2) Noggin (3) EGF (4) Wnt3a 11. A transplantable intestinal organoid composition comprising niche factor production-regulated intestinal organoids or niche factor production-regulated colon stromal cells, wherein the cells or the intestinal organoids release any one or more of the following niche factors: (1) Respondin1 (2) Noggin (3) EGF (4) Wnt3a 12. A composition for promoting intestinal epithelial tissue regeneration, comprising niche factor production-controlled intestinal organoids or niche factor production-controlled colon stromal cells, the cells releasing one or more of the following niche factors: (1) Respondin1 (2) Noggin (3) EGF (4) Wnt3a

Images