Intestinal organoid and method for manufacturing the same

Abstract

To provide an intestinal organoid equipped with the same function as an intestine, and a method for manufacturing the organoid.SOLUTION: An intestinal organoid of the present invention is derived from an embryonic stem cell and / or an artificial pluripotent stem cell, has a structure including a cavity, and a length in a long axis direction is 5 mm or more. A method for manufacturing an intestinal organoid of the present invention includes: a process 1 of inoculating a cell selected from an embryonic stem cell and an artificial pluripotent stem cell onto a substrate and a cell culture substrate including a cell adhesion region and a cell non-adhesion region surrounding the cell adhesion region which are formed on the surface of the substrate; and a process 2 of culturing a cell inoculated in the process 1. The process 2 includes differentiating a part of a cell into an entodermal cell and differentiating a part of a cell into an ectodermal cell.SELECTED DRAWING: Figure 1D

Description

[Technical Field]

[0001] The present invention relates to intestinal organoids having functions similar to those of the intestine and a method for producing the same. [Background technology]

[0002] The intestine is a complex organ containing cells derived from three germ layers (endoderm, ectoderm, and mesoderm). , intestinal epithelial cells derived from the endoderm (enterocytes, goblet cells, endocrine cells, brush cells, Paneth cells) , M cells, etc.), lymphoid tissue derived from the mesoderm, smooth muscle cells, interstitial cells of Cajal, and the ectoderm The intestinal nerve plexus and other organs that it originates from are intricately combined to perform functions such as secretion, absorption, and peristalsis. .

[0003] On the other hand, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are target cells. It is possible to induce differentiation into these cells, and it is expected to be applied in the field of regenerative medicine.

[0004] In recent years, "intestinal oocytes" with intestinal-like functions have been developed from pluripotent stem cells and isolated tissue progenitor cells. A technique for producing "ganoids" has been reported (Non-Patent Document 1, etc.).

[0005] In Patent Document 1, (a) undifferentiated embryonic stem cells are collected, and the collected undifferentiated embryonic stem cells are then cultured. (b) a step of inducing embryoid bodies by hanging drop culture in a medium containing BDNF; (a) attaching the embryoid bodies induced in the step (a) to a culture dish and further culturing the embryoid bodies. A method for constructing a gut-like cell mass with an intramural nervous system is disclosed.

[0006] In Patent Document 2, embryoid bodies are formed and cultured from purified iPS cells using a three-dimensional culture system. Thereafter, the embryoid bodies are cultured using a two-dimensional adherent culture system, thereby inducing differentiation into the intestinal tract. A method for producing an artificial intestine is disclosed, comprising:

[0007] Patent Document 3 describes a method for culturing induced pluripotent stem cells into intestinal epithelial cells, which comprises the following steps (1) to (3): A method for inducing differentiation into endoderm-like cells: (1) a step of differentiating induced pluripotent stem cells into endoderm-like cells; (2) (3) differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; A step of differentiating the intestinal stem cell-like cells obtained in step (2) into intestinal epithelial cell-like cells. a MEK1 inhibitor, a DNA methylation inhibitor, and a TGFβ receptor inhibitor; A process comprising culturing in the presence of the above compound and EGF is disclosed.

[0008] Patent Document 4 describes a method for producing an intestinal structure from embryonic stem cells and / or induced pluripotent stem cells. The method comprises culturing cells on a substrate having a pattern of cell adhesion regions having a predetermined area. A method for doing so is disclosed.

[0009] Patent Document 5 discloses a method for inducing differentiation of induced pluripotent stem cells into endodermal cells, A method for culturing cells on a substrate on which a pattern of cell adhesion regions having a predetermined area is formed has been developed. is shown.

[0010] On the other hand, as a method to test the effects of drugs acting on the intestine, Caco-2 cells or specific transmembrane Bidirectional transcellular transport assay using transporter overexpressing cells (Caco-2 cell membrane permeability assay) Transient tests are known. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-239169 [Patent Document 2] WO2010 / 143747 [Patent Document 3] WO2014 / 132933 [Patent Document 4] Japanese Patent Application Laid-Open No. 2014-236716 [Patent Document 5] Japanese Patent Application Laid-Open No. 2015-15943 [Non-patent literature]

[0012] [Non-Patent Document 1] Spence JR, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 2011;470(7332):105-109 Summary of the Invention [Problem to be solved by the invention]

[0013] Caco-2 is a colon adenocarcinoma cell line that accurately reflects the absorption of small molecules by small intestinal epithelial cells. In addition, the Caco-2 cell membrane permeability test is a semi-permeable membrane (trans A monolayer is formed on the surface of the tube (swell), but there should be no leakage. The procedure was complicated because it required the measurement of the TEER (Test Electrical Resistance). -2 There was a problem that it easily damaged the cell membrane.

[0014] If we could create intestinal organoids with intestinal-like functions, we could potentially prevent intestinal-related diseases. This is expected to be useful for the development of drugs to prevent or treat intestinal diseases and for the study of the pathology of intestinal diseases. However, the intestinal organoids provided to date have not always been satisfactory.

[0015] The method described in Non-Patent Document 1 involves treating human pluripotent stem cells with activin or the like to induce mesodermal differentiation. and culturing the cells so that they differentiate into endoderm while suppressing differentiation into ectoderm to produce an intestinal epithelial structure; On the other hand, neural crest cells are produced and combined to produce intestinal organoids. This method requires complicated procedures and does not mimic the natural development of the three germ layers in vivo. It is not something that can be done.

[0016] Furthermore, the cell structures produced by the methods described in Patent Documents 1 to 5 have functions equivalent to those of the intestine. However, there was still room for improvement. [Means for solving the problem]

[0017] Therefore, the present invention provides intestinal organoids with intestinal functions equivalent to those of the intestine, a method for producing the same, and The present invention provides a medium suitable for producing intestinal organoids and a kit suitable for producing intestinal organoids. The present invention includes the following inventions.

[0018] (1) Derived from embryonic stem cells and / or induced pluripotent stem cells; It has a structure containing a cavity, Intestinal organoids characterized by a longitudinal length of 5 mm or more. (2) The intestinal organoid according to (1), comprising endodermal cells, ectodermal cells, and mesodermal cells. . (3) The intestinal organoid according to (1) or (2), comprising intestinal epithelial cells on the outer surface. (4) A substrate, a cell adhesion region formed on the surface of the substrate, and a membrane surrounding the cell adhesion region. and culturing a cell selected from embryonic stem cells and induced pluripotent stem cells on a cell culture substrate having a cell non-adhesive region. Step 1 of seeding cells to be cultured; and Step 2: Culturing the cells seeded in Step 1 Including, Step 2 is A portion of the cells seeded in step 1 differentiates into endodermal cells, and Some of the cells seeded in step 1 differentiate into ectodermal cells. A method for producing intestinal organoids comprising: (5) In step 2, the differentiation time of some of the cells seeded in step 1 into endodermal cells is The method described in (4), which is earlier than the differentiation stage of some of the cells seeded in (1) into ectodermal cells. (6) In step 2, a portion of the cells seeded in step 1 is transformed into endodermal cells within 14 days after seeding. The method according to (4) or (5), comprising differentiating the cells into cells. (7) In step 2, a portion of the cells seeded in step 1 differentiate into ectodermal cells 15 days or more after seeding. The method according to any one of (4) to (6), comprising: (8) Step 2 includes the step of preparing a fibroblast growth factor (bFGF) containing insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF). The method according to any one of (4) to (7), comprising culturing cells in a medium containing the (9) The method according to (8), wherein the medium further contains heregulin. (10) The method according to any one of (4) to (9), wherein step 2 is carried out in the absence of heterologous components. (11) A substrate, a cell adhesion region formed on the surface of the substrate, and a membrane surrounding the cell adhesion region. The present invention relates to a cell culture substrate having a non-cell-adhesive region, and a method for culturing selected cells from embryonic stem cells and induced pluripotent stem cells on the substrate. Step 1 of seeding the selected cells; Step 2: Culturing the cells seeded in Step 1 Including, Step 2 includes insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF). A method for producing intestinal organoids, comprising culturing cells in a medium. (12) The method according to (11), wherein the medium further contains heregulin. (13) The method according to (11) or (12), wherein step 2 is carried out in the absence of heterologous components. (14) Intestinal steroids, including insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF) Differentiation induction medium for producing ganoids. (15) The differentiation-inducing medium for producing intestinal organoids according to (14), further comprising heregulin. (16) The differentiation-inducing medium for producing intestinal organoids according to (14) or (15), which does not contain any xenogeneic components. (17) A differentiation-inducing medium for producing intestinal organoids according to any one of (14) to (16), and A substrate, a cell adhesion region formed on the surface of the substrate, and a cell adhesion region surrounding the cell adhesion region. A cell culture substrate having a cell non-adhesive region A kit for generating intestinal organoids. [Effects of the Invention]

[0019] The intestinal organoids of the present invention have a sufficient size, have the same functions as the intestine, and contain a cavity. It is possible to introduce substances from the outside into the sinuses, which can be used to prevent or treat intestinal-related diseases. It is useful for drug development and pathological research into intestinal-related diseases. According to the method for producing intestinal organoids of the present invention, a selection is made from embryonic stem cells and induced pluripotent stem cells. Intestinal organoids can be easily produced from the selected cells. The differentiation-inducing medium and kit of the present invention are useful for producing intestinal organoids. [Brief explanation of the drawings]

[0020] [Figure 1A] Figure 1A shows a schematic diagram of the process of generating intestinal organoids from human pluripotent stem cells on a cell culture substrate with a patterned cell adhesive region. [Figure 1B]The left image in Figure 1B shows a cell culture substrate coated with a hydrophilic polymer and patterned with numerous circular cell adhesion areas, each 1.5 mm in diameter (scale bar: 15 mm). The right image in Figure 1B shows that human ES cells adhere and proliferate exclusively on each circular cell adhesion area (scale bar: 500 μm). [Figure 1C] Figure 1C shows the time course of organoid culture. In Figure 1C, D3, D7, D14, D33, D43, and D63 represent days 3, 7, 14, 33, 43, and 63 of culture, respectively. The scale bars in the upper and lower right panels of Figure 1C indicate 200 μm, while the scale bar in the lower left panel of Figure 1C indicates 50 μm. [Figure 1D] Figure 1D shows photographs of six intestinal organoids, showing intestinal-like motility maintained at day 129 of culture. The scale bar in Figure 1D indicates 5 mm. [Figure 2A] Figure 2A shows the results of analyzing the expression levels of biomarker genes at various time points in human embryonic stem cells (hESCs). Expression levels are expressed relative to GAPDH as mean ± SEM. On the horizontal axis, ES represents undifferentiated ES cells, D7 represents organoids at day 7 of culture, D14 represents organoids at day 14 of culture, and D21 represents organoids at day 21 of culture. The significance of the difference in expression levels in organoids at each time point relative to undifferentiated ES cells was tested by Student's t-test (**P < 0.01) (n = 3-6). [Figure 2B] Figure 2B shows the results of H&E staining of intestinal organoids with peristaltic activity at day 100 of culture. The scale bar on the left in Figure 2B is 2 mm, and the scale bar on the right in Figure 2B is 200 μm. [Figure 2C] Figure 2C shows the results of Alcian Blue staining of differentiated organoids on day 60 of culture. The scale bar in Figure 2C indicates 100 μm. Goblet cells were confirmed at the positions indicated by the white arrowheads. [Figure 2D]Figure 2D shows the relative expression levels of cell marker genes in differentiated intestinal organoids at day 50 of culture and in the adult human small intestine. The significance of the difference in expression levels in organoids compared to the adult human small intestine was tested by the Mann-Whitney rank-sum test (* P < 0.05, ** P < 0.01). In Figure 2D, each expression level is shown as the mean ± SEM of values ​​obtained from three independent experiments (n = 3-4). [Figure 3A] Figure 3A shows immunostaining results for intestinal differentiation markers in organoids derived from human embryonic stem cells (hESCs) at 50–60 days of culture. The intestinal differentiation markers used were villin, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), CDX2, E-cadherin (ECAD), chromogranin A (CGA), mucin-2 (MUC2), Paneth cell-specific defensin alpha-6 (DEFA6), α-smooth muscle actin (SMA), and protein gene product 9.5 (PGP9.5). Nuclei were counterstained with DAPI. In the lower right panel of Figure 3A, arrowheads indicate PGP9.5-positive enteric neurons in the α-SMA-positive smooth muscle layer. The scale bars in Figure 3A indicate 50 μm for the upper left, upper middle, and lower right panels, and 100 μm for the upper right, lower left, and lower middle panels. [Figure 3B] Figure 3B shows electron microscopic images of enterocytes with characteristic brush border microvilli (left), Paneth cells with secretory granules (black arrowheads), and goblet cells with mucin granules (yellow arrowheads). The scale bar indicates 10 μm in the left image and 5 μm in the right image. [Figure 3C]Figure 3C shows that organoids formed from human embryonic stem cells (hESCs) transfected with pPB-hLgr5p-EGFP-neo expressed EGFP under the control of the LGR5 promoter. In this experimental system, EGFP-expressing cells are LGR5-positive. A general image of organoids on day 34 of culture is shown in the upper left of Figure 3C. A magnified image of the intestinal-like structure boxed in the upper left of Figure 3C is shown in the upper right of Figure 3C. A fluorescence microscopy image of the boxed area in the upper right of Figure 3C is shown in the lower left of Figure 3C. The image in the lower left of Figure 3C reveals that EGFP-positive cells are relatively few in the intestinal organoids on day 34 of culture. An increase in EGFP-positive cells was confirmed in the intestinal organoids on day 41 of culture (lower right of Figure 3C). The scale bar in the upper left of Figure 3C indicates 300 μm, while the scale bars in the upper right, lower left, and lower right of Figure 3C indicate 100 μm. [Figure 4A] Figure 4A shows an image of intestinal organoids on day 60 of culture, immunohistochemically stained for α-smooth muscle actin (SMA). The scale bar indicates 200 μm. [Figure 4B] Figure 4B shows the results of immunostaining of Cajal cells. The scale bar indicates 50 μm. Arrowheads indicate cells double-positive for CKIT and S-100. Double-positive cells for CKIT and S-100 were identified in the nerve plexus between the myenteric layer and in the submucosal layer. [Figure 4C] Figure 4C shows the distribution of the neurotransmitter serotonin, a marker for enteroendocrine cells, in human embryonic stem cell (hESC) intestinal organoids cultured on day 60. The scale bar indicates 100 μm. The serotonin-positive cells indicated by the arrowheads in Figure 4C were found in the tegmental epithelium and had a triangular shape. [Figure 4D] Figure 4D shows the contractility of peristaltic intestinal organoids. The aspect ratio (vertical axis) was calculated for each frame of the video based on the ratio of the longest to shortest diameter of the intestinal organoids. The video was recorded at 30 frames per second. Contraction waves were recorded before drug treatment (frames 1-160). Figure 4D shows that histamine treatment increased the frequency of contractions, while atropine treatment decreased the frequency of contractions. [Figure 4E] Figure 4E shows that organoids without peristaltic activity did not contract even after treatment with histamine. [Figure 4F] Figure 4F shows the results of histamine H1 receptor immunostaining of human intestinal tissue, organoids with peristaltic activity, and organoids without peristaltic activity. Cell nuclei were counterstained with DAPI. Histamine H1 receptor-positive cells were observed in the epithelial and mesenchymal regions of both organoids with and without peristaltic activity. The scale bar in Figure 4F indicates 20 μm. [Figure 5A] Figure 5A shows the results of quantitative RT-PCR analysis of the expression levels of the intestinal oligopeptide transporter (PEPT1) and the major ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 in intestinal organoids derived from human embryonic stem cells (hESCs) at day 50 of culture, compared with those in healthy adult small intestines. Statistical analysis was performed using t-tests or Mann-Whitney rank-sum tests (**P < 0.01). Values ​​are shown as mean (%) ± SEM (n = 3). [Figure 5B] The upper left and lower left panels of Figure 5B show the results of an experiment in which intestinal organoids were treated with the fluorescently labeled dipeptide β-Ala-Lys-AMCA in the presence or absence of the angiotensin-converting enzyme inhibitor captopril. In each of the upper left and lower left panels of Figure 5B, the intestinal organoids on the left were treated with the dipeptide in the presence of captopril, while the intestinal organoids on the right were treated with the dipeptide in the absence of captopril. The upper left panel of Figure 5B shows a brightfield image, the upper right panel shows a fluorescent image, and the lower left panel shows a superimposition of the two. Each group was analyzed using a n=3 sample. The scale bar indicates 200 μm. It was confirmed that intestinal organoids could internalize the dipeptide, and that this uptake was inhibited by captopril. The lower right panel of Figure 5B shows a schematic diagram of the dipeptide uptake assay procedure. [Figure 5C]To quantify the uptake of β-Ala-Lys-AMCA, intestinal organoids were cultured with or without 10 μM, 100 μM, or 1 mM captopril. The AMCA fluorescence signal was observed using a fluorescence microscope (BZ-X710; Keyence) equipped with a top-stage incubator (5% CO2, 37°C). The fluorescence signal intensity was quantified using a Hybrid Cell Count / BZ-H3C (Keyence). No captopril concentration dependency was observed. [Figure 6A] Figure 6A shows images of intestinal organoids derived from human embryonic stem cells (hESCs) at day 115 of culture, immunostained with a marker for the cystic fibrosis transmembrane conductance regulator (CFTR), and observed under a fluorescence microscope. The left image of Figure 6A shows the intestinal organoids, and the right image shows the human intestine. CFTR is shown in green, actin in red, and DAPI in blue. [Figure 6B] Figure 6B shows that forskolin induces expansion of intestinal organoids. Human embryonic stem cell (hESC)-derived intestinal organoids were monitored by time-lapse fluorescence laser confocal microscopy (Keyence). The surface area of ​​intestinal organoids was measured using a Hybrid Cell Count / BZ-H3C (Keyence). The normalized total surface area of ​​organoids was calculated as the average of measurements from three separate wells for each experimental condition. Forskolin-treated organoids (19 min) were overlaid with untreated organoids (0 min, 569 μm). [Figure 7A] Figure 7A shows the results of analyzing the expression levels of cellular biomarkers in differentiated intestinal organoids without peristaltic activity (culture day 52). The expression levels of each marker are shown as values ​​normalized by GAPDH expression levels. Biomarker gene expression levels in intestinal organoids without peristaltic activity were comparable to those in the adult small intestine. Statistical analysis was performed using t-tests or Mann-Whitney rank-sum tests (* P < 0.05, ** P < 0.01). Values ​​are shown as mean (%) ± SEM (n = 3). [Figure 7B]Figure 7B shows the expression levels of albumin and insulin in intestinal organoids without peristaltic activity. Expression of these genes was not detectable in intestinal organoids. Data are from three independent experiments (n = 3-6). [Figure 7C] Figure 7C shows the results of immunostaining non-peristaltic intestinal organoids for CDX2, E-cadherin (ECAD), and α-smooth muscle actin (SMA). Cell nuclei were counterstained with DAPI. The scale bar represents 100 μm. In non-peristaltic intestinal organoids, a CDX2- and ECAD-positive epithelial layer was confirmed, but SMA expression levels were low in the mesenchymal region. [Figure 7D] The left panel of Figure 7D shows the results of immunostaining for neurofilament and CDX2 in intestinal organoids without peristaltic activity. Cell nuclei were counterstained with DAPI. The right panel of Figure 7D shows the results of similar immunostaining in intestinal organoids with peristaltic activity. The scale bar indicates 50 μm. In intestinal organoids with peristaltic activity, the development of neuronal networks with neurofilaments was observed in the mesenchymal region, whereas in intestinal organoids without peristaltic activity, neurofilaments were barely detectable. [Figure 8A] Figure 8A shows that a single intestinal organoid from day 35 of culture was transplanted under the kidney capsule of an adult immunodeficient mouse. The scale bar indicates 200 μm. [Figure 8B] Figure 8B shows that 6 weeks after transplantation, organoids constitutively expressing EGFP were present under the mouse kidney capsule. The outline of the mouse kidney is indicated by a dotted line in the right image of Figure 8B. The scale bar indicates 1 mm. [Figure 8C] Figure 8C shows hematoxylin-eosin (H&E) stained images of the transplanted intestinal organoids. The scale bar in the left image indicates 500 μm, and the scale bar in the right image indicates 100 μm. The formation of luminal and laminated structures was confirmed. [Figure 8D]Figure 8D shows that the transplanted intestinal organoids formed a layered intestinal muscle structure (α-SMA staining). MUC2 and CDX2 were expressed in the epithelial layer. E-cadherin (ECAD) and Na+ / K+-ATPase were found to be distributed in the same region as chromogranin A (CGA)-positive highly differentiated enteroendocrine cells and DEFA6-positive Paneth cells. Expression of the neural marker PGP9.5 was also observed in the mesenchymal region of smooth muscle. The scale bar in the upper panel of Figure 8D indicates 100 μm, and the scale bar in the lower panel indicates 50 μm. [Figure 9A] Figure 9A shows that human iPS cells adhered and proliferated only to the cell adhesion area on culture day 10. The scale bar indicates 500 µm. [Figure 9B] Figure 9B shows that human iPS cell-derived intestinal organoids formed self-organized cystic spheroids on day 42 of culture. Similar to the human ES cell-derived intestinal organoids, the human iPS cell-derived intestinal organoids were also classified as either simple cystic spheroids surrounded by a thin cell wall or bicomponent spheroids with solid portions and partially cystic protrusions. The scale bar indicates 200 μm. DETAILED DESCRIPTION OF THE INVENTION

[0021] 1. Intestinal Organoids In the present invention, "intestinal organoid" refers to the intestine of the organism of origin of cells, particularly mammals such as humans. intestines, especially those with functions similar to those of the human intestine (specifically, peristaltic movement, mucus secretion, and substances) It refers to a tissue structure that has functions such as absorption.

[0022] The intestinal organoids of the present invention are derived from embryonic stem cells (ES cells) and / or induced pluripotent stem cells (iPS cells). ) intestinal organoids.

[0023] The embryonic stem cells (ES cells) used in the present invention are preferably mammalian-derived ES cells. For example, ES cells derived from rodents such as mice or primates such as humans are used. It is particularly preferable to use ES cells derived from mouse or human. ES cells are A stem cell line derived from the inner cell mass of a blastocyst-stage embryo, an early stage in animal development. It refers to a type of stem cell that can theoretically differentiate into any tissue outside of a body while retaining its pluripotency and can grow almost infinitely. As for ES cells, for example, it is possible to easily confirm the degree of differentiation. To achieve this, cells with a reporter gene introduced near the Pdx1 gene can be used. For example, a 129 / Sv-derived ES cell line incorporating the LacZ gene into the Pdx1 locus or a Pdx1 promoter-controlled ES cell line SK7 carrying a GFP reporter transgene can be used. , mRFP1 reporter transgene and Pdx under the control of Hnf3β endoderm-specific enhancer fragment The PH3 ES cell line, which carries a GFP reporter transgene under the control of the 1 promoter, was used. It is also possible to establish a new system at the Reproductive and Cellular Medicine Research Department of the National Center for Child Health and Development, and to H, et al. Regen Ther. 2015;1:18-29, which are ES cell lines SEES1, SEES2, and SEES 3. SEES4, SEES5, SEES6, or SEES7, or cells in which additional genes have been introduced into these ES cell lines Stocks can also be used.

[0024] The induced pluripotent stem cells (iPS cells) used in the present invention are capable of initializing somatic cells. The creation of induced pluripotent stem cells was initiated by Yamaguchi University's Professor Shinya Naka's group and Rudolf Janisch of the Massachusetts Institute of Technology Enisch et al. group, and James Thomson et al. at the University of Wisconsin group, Konrad Hochedlinger of Harvard University Several groups have been successful in this area, including the group of WO2007 / 06966. Publication No. 6 contains the genes of Oct family genes, Klf family genes, and Myc family genes. Somatic cell nuclear reprogramming factors, including gene products, as well as Oct family genes and Klf family genes Nuclear reprogramming of somatic cells involves the gene products of the Sox family genes and the Myc family genes The present invention describes a method for the treatment of somatic cells comprising contacting the nuclear reprogramming factor with a somatic cell. A method for producing induced pluripotent stem cells by nuclear reprogramming of cells is described.

[0025] There are no particular limitations on the type of somatic cells used to generate iPS cells, and any somatic cells can be used. That is, the somatic cells referred to in the present invention are all cells that constitute a living body except for germ cells. The term "somatic cells" refers to cells that are differentiated or undifferentiated stem cells. The mammals may be any of mammals, birds, fish, reptiles, and amphibians, but are not particularly limited thereto. Preferably, Mammals (e.g., rodents such as mice, or primates such as humans) are particularly preferred. In addition, when human somatic cells are used, fetal, newborn or adult somatic cells are used. Any somatic cells may be used. Specific examples of somatic cells include fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, liver epithelial cells, alveolar epithelial cells), endothelial Cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreas cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiac muscle cells), Hepatocytes, non-hepatocytes, adipocytes, osteoblasts, and cells that make up periodontal tissue (e.g., teeth) Ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), cells that make up the kidneys, eyes, and ears Examples include:

[0026] iPS cells can be cultured for a long period of time under specific culture conditions (e.g., conditions for culturing ES cells). They have the ability to self-renew and differentiate into ectoderm, mesoderm and endoderm under specific differentiation induction conditions. In the present invention, iPS cells refer to stem cells that have the ability to differentiate into various types of cells, such as mice. The stem cells may be capable of forming teratomas when transplanted into the test animal.

[0027] To produce iPS cells from somatic cells, we first need to transduce at least one type of reprogramming gene. The reprogramming gene is introduced into somatic cells. It has the effect of reprogramming somatic cells to become iPS cells. These genes encode reprogramming factors. Specific examples of combinations of reprogramming genes include: The following combinations may be mentioned, but are not limited to: (i) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 largeT gene (iv) Oct gene, Klf gene, Sox gene

[0028] In one embodiment, the intestinal organoids have a structure containing a cavity. Preferably, the cavity is closed so that the cavity can hold a liquid. The id can take in substances present outside into the cavity, making it possible to evaluate drug metabolism. It is useful for applications such as:

[0029] In one embodiment, the intestinal organoid has a longitudinal length of 5 mm or more, preferably 8 mm or more. More preferably, the thickness is 10 mm or more, more preferably, 12 mm or more, and even more preferably Including the reports mentioned in the Background Art section, the length in the long axis direction has conventionally been There have been no examples of such large-sized intestinal organoids being produced. Higher accuracy in drug effect testing compared to smaller intestinal organoids For example, it is preferable to use a large-sized intestinal organelle. The cavity contained within the id can hold a large amount of fluid, making it suitable for the large intestinal organelles. The test drug was added to the buffer solution while the intestinal tract was suspended in the buffer solution. After a certain period of time, Conventional intestinal organoid analysis involves extracting the organoids and extracting the fluid in the cavity for analysis. It is also possible to carry out analyses that were previously impossible with a conventional lid. Therefore, simple evaluation methods such as fluorescence observation can be used without using mass spectrometry. .

[0030] Here, the "longitudinal length" is measured by visually or optically observing the intestinal organoids in an appropriate buffer solution. In the observation image when observed using It refers to the longest distance between two points that can be connected by a straight line passing through the intestine. The outline of the organoid may be deformed by peristaltic movement, but the maximum measured value was taken as the length in the longitudinal direction. Just do that.

[0031] The overall shape of the intestinal organoids is not particularly limited, but they are usually granular. The term "spherical" also includes spherical. The intestinal organoids preferably comprise endodermal cells, ectodermal cells and mesodermal cells.

[0032] The endoderm gives rise to the tissues of organs such as the digestive tract, lungs, thyroid gland, pancreas, and liver, as well as the tissues that open into the digestive tract. Cells of the glands, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, urinary tract (bladder, most of the urethra, ureters) The differentiation of ES cells or iPS cells into endodermal cells is a process that is specific to the endoderm. This can be confirmed by measuring the expression levels of endoderm-specific genes. In addition to the genes described below, other genes include AFP, SERPINA1, SST, ISL1, IPF1, and IAPP. , EOMES, HGF, ALBUMIN, PAX4, TAT, etc.

[0033] Endodermal cells that can be contained in intestinal organoids include intestinal epithelial cells. The intestinal epithelial cells of the ganoids consist of enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. It is preferable that the intestinal epithelial cells include one or more selected from the group consisting of enterocytes, goblet cells, and intestinal epithelial cells. It is particularly preferred that the intestinal organoids contain both secretory cells and Paneth cells. The presence of cysts can be determined based on the positive expression of endodermal cell markers. CDX2 as an intestinal cell marker, MUC2 as a goblet cell marker, and enteroendocrine cell marker CGA is a marker for vasoconstriction, and DEFA6 is a marker for Paneth cells. K+-ATPase and villin are markers for intestinal epithelial cells. In addition, definitive endoderm markers FOXA2 and SO X17 or CXCR4 can also be used as a marker for distinguishing endodermal cells. The expression of GATA4, GATA6, or T (Brachyury), which are markers of germ layer and mesoderm, also distinguishes endodermal cells. It can be used as a marker for

[0034] The ectoderm forms the epidermis of the skin, the epithelium of the distal part of the male urethra, hair, nails, and skin glands (including mammary glands and sweat glands). ), sensory organs (including the epithelium of the oral cavity, pharynx, nose, and distal rectum, salivary glands), and lenses. During development, part of the ectoderm invaginates into a groove to form the neural tube, which then gives rise to the central nervous system, such as the brain and spinal cord. They also form the peripheral nervous system. The differentiation of iPS cells into ectodermal cells can be confirmed by measuring the expression levels of genes specific to the ectoderm. In addition to the genes described below, the genes specific to the ectoderm can also be identified by For example, β-tublin, nestin, galanin, GCM1, GFAP, neurod1, olig2, synaptophysin , DESMIN, TH, etc.

[0035] Ectoderm cells that can be contained in intestinal organoids include cells that make up the enteric nerve plexus. The presence of ectodermal cells in intestinal organoids is confirmed by the expression of ectodermal cell markers. This can be determined based on the positive expression of the marker. The myenteric plexus marker PGP9.5 and the neural progenitor cell marker SOX1 can be used.

[0036] The mesoderm forms the body cavity and its lining, the mesothelium, muscles, skeleton, skin dermis, connective tissue, heart, and blood. ducts (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, gonads ( The differentiation of ES cells or iPS cells into mesodermal cells is This can be confirmed by measuring the expression levels of mesoderm-specific genes. Specific genes include those described below, as well as FLK-1, COL2A1, FLT1, HBZ, Examples include MYF5, MYOD1, RUNX2, and PECAM1.

[0037] Mesodermal cells that can be contained in intestinal organoids include smooth muscle cells and interstitial cells of Cajal. The presence of mesodermal cells in intestinal organoids is confirmed by the expression of mesodermal cell markers. The mesodermal cell markers include smooth muscle cells. The cell marker α-smooth muscle actin (SMA), the interstitial cell markers CD34 and CKIT ( In addition, GATA4 and GAT, which are markers of early endoderm and mesoderm, can be used. A6 or T (Brachyury) can also be used as a marker for distinguishing mesodermal cells.

[0038] The intestinal organoids further preferably comprise intestinal stem cells. The presence of intestinal stem cells may be This can be determined by using the marker LGR5 as a positive indicator.

[0039] Preferably, the intestinal organoids further comprise serotonin-positive enteroendocrine cells. Intestinal organoids also contain transporter-positive cells, which allow transporter-mediated It is preferable that the transporter is capable of uptake of the substance. ATP-binding cassette (ABC) transporters ABCB1 and ABCB2 Examples include CG2.

[0040] Intestinal organoids also contain cystic fibrosis transmembrane conductance regulator (CFTR)-positive intestinal epithelial cells CFTR-positive intestinal epithelial cells are involved in mucus secretion. Intestinal organoids containing CFTR-positive intestinal epithelial cells have mucus secretion ability similar to that of the intestine. Preferably, the intestinal organoids further comprise histamine H1 receptor-positive cells.

[0041] The intestinal organoids of the present invention preferably comprise intestinal epithelial cells on at least a portion of their outer surface. According to this embodiment, substances outside the intestinal organoids are transported through the intestinal epithelial cells on the outer surface. In this embodiment, the outer surface When the intestinal epithelial cells are also transporter-positive, the transport of substances via the transporter It is more preferable that the transporter-positive intestinal epithelial cells can take up the serotonin. Intestinal organoids containing the above have the ability to absorb substances similar to the intestine. Intestinal epithelial cells are oriented toward the inside of the hollow intestinal tract, and the intestinal organoids according to this embodiment is different.

[0042] The intestinal organoid of the present invention is preferably one in which the development of microvilli and crypts is observed on the outer surface. can be.

[0043] The intestinal organoids of the present invention preferably have the ability to perform contractile movements similar to peristalsis. These functions are achieved through the development of neural networks and smooth muscles. In the explanation below, the ability to perform contraction movements similar to peristalsis may be referred to as "peristaltic activity." Intestinal organoids having peristaltic activity are particularly preferably those in which the frequency of contractions is reduced by histamine treatment. The frequency of contractions was increased by atropine treatment, and the frequency of contractions was decreased by atropine treatment. Intestinal organoids, which have peristaltic activity and show drug responsiveness similar to that of the intestine, are able to inhibit the peristaltic movement of the intestine. The present invention can be suitably used for evaluating the influence of the temperature on the movement of the object.

[0044] As described above, the intestinal organoids of the present invention have a sufficient size and have functions equivalent to those of the intestine. The cavity inside the capsule can absorb substances from the outside, preventing or treating intestinal diseases. The intestinal organoids of the present invention are useful for the development of drugs to treat intestinal diseases and for the pathological research of intestinal diseases. By using this method, it is possible to perform drug testing on a semi-permeable membrane, as in the conventional drug testing method using Caco-2. The formation of a fragile monolayer and measurement of transepithelial electrical resistance (TEER) as an indication of membrane integrity were performed. There is no need to perform complicated steps such as determining the

[0045] Furthermore, if the intestinal organoids of the present invention are produced from disease-specific iPS cells, it will be possible to identify genetic factors and This will facilitate research into enteric diseases and drug evaluation involving the gut.

[0046] 2. How to prepare intestinal organoids The intestinal organoids of the present invention can be prepared by the following steps: A substrate, a cell adhesion region formed on the surface of the substrate, and a cell adhesion region surrounding the cell adhesion region. and a cell culture substrate having a non-adhesive region and a cell-culture method thereon. Step 1 of seeding cells to be cultured; and Step 2: Culturing the cells seeded in Step 1 The method can be prepared by a method comprising the steps of:

[0047] The cells selected from embryonic stem cells and induced pluripotent stem cells used in this method are as described above. be.

[0048] Preferred embodiments of the cell culture substrate used in the present invention are as follows. In the cell culture substrate used in the present invention, preferably, a cell adhesive region is present in the cell non-adhesive region. There are several islands of these.

[0049] The cell culture substrate used in the present invention preferably has a cell non-adhesive region made of polyethylene glycol. The cell adhesion region is immobilized on the substrate. Formed by oxidation and / or decomposition of at least a portion of polyethylene glycol is.

[0050] In the present invention, "cell adhesiveness" refers to the strength of cell adhesion, i.e., the ease with which cells adhere. The cell adhesive region means a region with good cell adhesiveness, and the cell non-adhesive region means a region with good cell adhesiveness. The area with poor cell adhesion is therefore a pattern of cell adhesion areas and non-cell adhesion areas. When cells are seeded on the surface of the substrate, the cells adhere to the cell-adhesive areas, but the non-cell-adhesive areas Since cells do not adhere to these regions, cells are arranged in a pattern on the surface of the cell culture substrate. becomes.

[0051] The cell adhesion spreading rate during actual cell culture is used as an index to judge cell adhesion. The cell adhesive surface is preferably a surface with a cell adhesion spreading rate of 60% or more. It is more preferable that the surface has a cell adhesion spreading rate of 80% or more. In the present invention, the cell adhesion spreading rate is determined by the seeding density. 4000 cells / cm 2 More than 30000 cells / cm 2 The cells to be cultured within the range of After seeding on the surface and storing in an incubator at 37°C and 5% CO2, the cells were cultured for 14.5 hours. The percentage of cells that adhered and spread ({(number of adhered cells) / (number of seeded cells)} × 100(%)) Define.

[0052] In the above measurement, cells were suspended in DMEM medium containing 10% FBS and seeded on the object to be measured. After that, shake the object to be measured so that the cells are distributed as uniformly as possible. Furthermore, the cell adhesion spreading rate is measured by Immediately before the measurement, the medium is changed to remove unadhered cells. So, where the density of cells tends to be specific (for example, where the density tends to be high) The measurement points are areas excluding the center of the specified area and the periphery of the specified area where the density tends to be low. do.

[0053] On the other hand, cell non-adhesiveness refers to the property that makes it difficult for cells to adhere. The chemical and physical properties of the surface determine whether or not cell adhesion and spreading are difficult to occur. The non-cell-adhesive surface is preferably a surface having a cell adhesion spreading rate of less than 60% as defined above. Preferably, the surface is less than 40%, more preferably less than 5%. It is preferable that the surface area is 2% or less.

[0054] The cell culture substrate used in the present invention preferably has a cell non-adhesive region made of polyethylene glycol. The cell adhesion region is immobilized on the substrate. Formed by oxidation and / or decomposition of at least a portion of polyethylene glycol Such a cell culture substrate may be prepared by, for example, coating the entire surface of the substrate with polyethylene glycol. (PEG) thin film is formed, and then oxidized and / or treated in the area where cell adhesion is desired. The cell adhesive property can be imparted to the surface by decomposition treatment. indicates a non-cell adhesive region where PEG is immobilized.

[0055] Polyethylene glycol (PEG) consists of one or more ethylene glycol units ((CH2)2-O). The ethylene glycol chain (EG chain) may be either linear or branched. The ethylene glycol chain may, for example, be of the following formula: -((CH2)2-O)m- (m is an integer indicating the degree of polymerization) m is preferably an integer of 1 to 13, more preferably an integer of 1 to 10. It's a number.

[0056] PEG also includes ethylene glycol oligomers. The functional group includes, for example, an epoxy group, a carboxyl group, an N-hydroxyl group, and the like. Hydroxysuccinimide group, carbodiimide group, amino group, glutaraldehyde group, (meth The functional group may be optionally linked via a linker, preferably The functional group is introduced into the terminal of the PEG. For example, PEG(meth)acrylate is acrylate, and PEG di(meth)acrylate.

[0057] The substrate used for cell culture is a substrate on which a thin PEG film can be formed. There are no particular limitations on the material as long as it is made of such a material. Specifically, metal, glass, etc. Inorganic materials such as ceramics and silicon, elastomers, plastics (e.g., polystyrene) Resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, Iron, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methyl Pentene resin, phenolic resin, melamine resin, epoxy resin, vinyl chloride resin) The shape of the organic material is not limited, and examples thereof include a flat plate, a flat film, a film, and the like. Flat shapes such as films and porous membranes, cylinders, stamps, multi-well plates, and microplates are also available. When using a film, there is no particular limit to its thickness. However, it is usually 0.1 to 1000 μm, preferably 1 to 500 μm, and more preferably 10 to 200 μm. be.

[0058] The average thickness of the PEG thin film formed on the substrate is preferably 0.8 nm to 500 μm, and more preferably 0.8 nm to 100 μm. The thickness is more preferably 1 μm, further preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, the PE on the substrate surface is effective for protein adsorption and cell adhesion. It is preferable because it is less affected by the area not covered by the thin film. If the thickness of the PEG thin film is less than a certain value, coating is relatively easy. This reduces the cell non-adhesiveness, allowing cells to adhere and spread to areas other than the cell adhesive area. Furthermore, by keeping the thickness of the PEG thin film below a certain level, the cell viability in the culture medium can be reduced. This allows factors necessary for cell growth to reach cells close to the substrate within the cell adhesion region.

[0059] There are two methods for forming a thin PEG film on the surface of a substrate: direct adsorption of PEG onto the substrate, and a method of directly coating PEG onto a substrate, a method of coating PEG onto a substrate and then performing a crosslinking treatment, To improve adhesion to the substrate, a primer layer is formed on the substrate, and then PEG is coated. and a method in which polymerization initiation points are formed on the surface of a substrate and then PEG is polymerized. A preferred method is to form an underlayer on the substrate and then coat it with PEG.

[0060] The underlayer can be formed by, for example, the method described in JP-A-2012-175983, and is preferably or reacts with the hydroxyl group at the end of PEG or with an introduced functional group to form a covalent bond. or a silane cup having a functional group that can be converted to such a functional group. Such a functional group can be formed using a ring-linking agent. Examples of such a functional group include (meth)acryloyl. group, (1H-imidazol-1-yl)carbonyl group, succinimidyloxycarbonyl group, Lysidyl group, epoxy group, aldehyde group, amino group, thiol group, carboxyl group, azide group hydroxyl group, cyano group, active ester group (1H-benzotriazol-1-yloxycarbonyl group, Pentafluorophenyloxycarbonyl group, paranitrophenyloxycarbonyl group, etc. ), a halogenated carbonyl group, an isocyanate group, a maleimide group, etc., among which ( A methacryloyl group, a glycidyl group or an epoxy group is preferred, and a glycidyl group or an epoxy group is preferred. An oxy group is most preferred.

[0061] For example, a silane coupling agent having a methacryloyl group at the end (methacryloylsilane) For example, the water contact angle of the substrate surface to which methacryloylsilane is added is typically 45° or more, preferably 47° or more, more preferably 48° or more, and even more preferably 50° or more This allows for the formation of sufficient cell-nonadhesive regions by subsequently immobilizing PEG. It is possible.

[0062] The density of PEG immobilized on the substrate and the cell non-adhesiveness are determined by the contact angle of water on the surface. For example, the water contact angle of the surface after PEG immobilization is typically If the angle is 48° or less, preferably 40° or less, and more preferably 30° or less, the PEG will have a sufficient density. In the present invention, the water contact angle is the angle at 23°C. This refers to the water contact angle measured at the surface.

[0063] In the present invention, "oxidation" has a narrow meaning, and refers to the reaction of an organic compound, i.e., PEG, with oxygen. In the present invention, "decomposition" refers to a reaction in which the oxygen content increases as a result of the reaction. This refers to the reaction in which the bond of the organic compound, i.e., PEG, is broken. Examples of decomposition include, but are not limited to, decomposition by oxidation and decomposition by ultraviolet irradiation. If "decomposition" is decomposition accompanied by oxidation (i.e., oxidative decomposition), what is the difference between "decomposition" and "oxidation"? Refers to the same process.

[0064] Decomposition by UV irradiation refers to the process in which PEG absorbs UV light, goes into an excited state, and decomposes. In addition, when PEG is exposed to ultraviolet light in a system where it is present together with oxygen-containing molecular species (oxygen, water, etc.), When irradiated with UV light, the UV rays are absorbed by PEG, causing decomposition. In addition, the molecular species is activated and PEG The latter reaction can be classified as "oxidation." The reaction in which PEG decomposes due to oxidation by UV irradiation is not "decomposition due to UV irradiation" but "decomposition due to oxidation." As mentioned above, "oxidation" and "decomposition" can overlap as operations. Therefore, in this specification, the term "oxidation and / or decomposition" is used. "The term "

[0065] The oxidation and / or decomposition methods include ultraviolet irradiation of the PEG thin film, photocatalytic treatment, etc. and methods such as partially oxidizing and / or treating with an oxidizing agent. When disassembling, use a mask such as a photomask or stencil mask, or use a stamp. In addition, it is recommended to use a direct writing method such as a method using a laser such as an ultraviolet laser to prevent oxidation and and / or may be disassembled.

[0066] For UV irradiation treatment, mercury lamps that emit UV rays with wavelengths of 185 nm or 254 nm or 172 nm are used. The light source is a lamp that emits ultraviolet light in the VUV to UV-C range, such as an excimer lamp. When photocatalyst treatment is performed, it is preferable to use light that emits ultraviolet light with a wavelength of 365 nm or less. It is preferable to use a light source that emits ultraviolet light with a wavelength of 254 nm or less. Examples of photocatalysts include titanium oxide photocatalysts, and acids activated by metal ions or metal colloids. It is preferable to use a titanium dioxide photocatalyst. As the oxidizing agent, organic acids and inorganic acids can be used without any particular limitation. However, since highly concentrated acids are difficult to handle, they should be diluted to a concentration of 10% or less. The optimum UV treatment time, photocatalyst treatment time, and oxidizing agent treatment time depend on the light source used. The amount of oxidation is determined appropriately according to various conditions such as the ultraviolet intensity, the activity of the photocatalyst, and the oxidizing power and concentration of the oxidizing agent. It is possible.

[0067] The carbon content of the cell adhesion region (including the underlayer if present) is It is preferable that the carbon content is lower than that of the base layer (including the base layer if present). Specifically, the carbon content of the cell adhesion region is 20 to 99% of the carbon content of the non-cell adhesion region. In addition, it is preferable that the cell adhesion region (including the base layer if present) The percentage of carbon bound to oxygen in the carbon is the percentage of carbon in the non-cell-adhesive region (where no underlying layer is present). The percentage of carbon bonded to oxygen in the carbon layer (including the underlayer if present) Specifically, it is preferable that the carbon sac in the cell adhesion region is smaller than the value. The percentage of carbon bound to oxygen in the non-cell-adhesive region is the percentage of carbon bound to oxygen in the non-cell-adhesive region. The ratio (%) of carbon bonded to atoms is preferably 35 to 99%. As the amount of UV exposure increases during incubation, cell adhesion increases, but adhesion remains high during cell recovery. This is because the cells become difficult to detach and are difficult to recover.

[0068] In the present invention, the "amount of carbon" is determined by the "resolution of the C1s peak obtained using an X-ray photoelectron spectrometer." The "amount of carbon obtained from the analysis value" is defined as "the proportion of carbon bonded to oxygen" and the "proportion of carbon bonded to oxygen" is defined as "the amount of carbon obtained from the analysis value" and "the proportion ... The oxygen-bound carbon determined from the analysis value of the C1s peak obtained using a photoelectron spectrometer It is defined as the "percentage of carbon."

[0069] In the cell culture substrate used in the present invention, the area of ​​each cell adhesion region is not particularly limited. A specific example of the area of ​​each cell adhesion region is 0.1 mm 2 The above are examples, and preferably 0.5 mm 2 End , preferably 0.785 mm 2 More preferably, 1.0 mm 2 More preferably, 1.2 mm 2 End , more preferably 1.5 mm 2 More than 1.7 mm, most preferably 2 More than 25 mm, preferably 2 Less than or equal to 15 mm, preferably 2 Less than 10 mm, more preferably 2 Less than or equal to 5 mm, most preferably 2 The pattern is formed so that the area of ​​the cell adhesion region is in the following range. It is easy to culture large intestinal organoids with a longitudinal length exceeding 5 mm.

[0070] The shape of each cell adhesion region is not particularly limited, but may be polygonal, including square, circular, or elliptical. The shape can be circular, and the diameter of the circular shape is preferably The diameter can be within the above area range, and specifically, the diameter of the circle is 0.35 mm or more, for example. It can be shown that the thickness is preferably 0.8 mm or more, preferably 1.0 mm or more, preferably 1.2 mm or more, more preferably Preferably, it is 1.5 mm or more, preferably 6 mm or less, more preferably 4 mm or less, and even more preferably The diameter is preferably 3 mm or less, and more preferably 2 mm or less. It is preferable that the existing cell adhesion regions all have the same area, and the same area and shape. It is more preferable that the areas and shapes are different from each other, but different areas and shapes may be mixed.

[0071] In addition, in the cell culture substrate, each cell adhesive region is surrounded by a cell non-adhesive region. That is, they are separated from each other, preferably by at least 0.75 mm, more preferably by at least 1.5 mm. In other words, the shortest distance between cell adhesion regions (in the case of circles, the distance between two circles) The distance between the centers is preferably 0.75 mm or more, the sum of the radii being the sum of the radii. More preferably, the distance between the cell adhesion regions is 1.5 mm or more. By doing so, cells in each cell adhesion region form intercellular junctions with cells in other cell adhesion regions. They are cultured uniformly at regular intervals without any disruption, allowing for the construction of highly reproducible experimental systems.

[0072] The ratio of the cell adhesion area in the cell culture substrate is usually 5 to 80%, preferably 20 to 70%, more preferably The preferred ratio is 40 to 60%. This ratio is also applicable when the substrate is placed in a dish or the like. The cell adhesion area is the ratio of the cell adhesion area to the entire substrate, not including the bottom of the dish. By keeping the number of cells above a certain level, cell death can be prevented, and by keeping it below a certain level, cell survival can be prevented. This prevents the depletion of factors necessary for cell proliferation and the resulting damage to cells.

[0073] In addition, each cell adhesion region is arranged at regular intervals, for example, in a grid pattern with the same pitch vertically and horizontally. It is preferable that the cells in each cell adhesion region are arranged in a paracrine fashion. By keeping the ion effect constant, the effect on differentiation can be made constant.

[0074] For example, a pattern having a plurality of circular cell adhesion regions may be formed by forming a frame having a plurality of circular openings. Using a photomask, the glass substrate on which the PEG thin film was formed was placed opposite the photomask. The PEG thin film was placed on the substrate and irradiated with ultraviolet light from the photomask side. This can be formed by subjecting the corresponding area to an oxidation treatment.

[0075] The cell culture substrate used in the present invention is a substrate for culturing embryonic stem cells (ES cells) and / or induced pluripotent stem cells ( It is known that the surface is pre-coated to promote adhesion of iPS cells to the cell adhesion area. Pre-coating treatment is preferred. proteoglycan, laminin, vitronectin), gelatin, lysine, peptides, This can be done by coating the cell culture substrate with a gel matrix containing serum, etc. Pre-coating treatment reduces the cell adhesion area of ​​ES cells and iPS cells, which have low adhesiveness. This can promote adhesion to the target area, allowing for effective cell adhesion culture and differentiation induction.

[0076] Similarly, in order to promote the adhesion of ES cells and iPS cells to the cell adhesion region, Before seeding the ES cells, feeder cells were seeded and cultured for about 24 hours, and then ES cells and iP cells were grown on the feeder cells. It is preferable to culture S cells. Any cell that can be used can be used, and there is no particular limitation, but examples thereof include fibroblasts. The number of feeder cells was 1.26 × 10 per cell culture substrate. 5 cells / cm 2 Density less than 6.3x 10 4 cells / cm 2 At a density of less than 3.15 x 10 4 cells / cm 2 Sow at a density of at least 1000.

[0077] In the present invention, both pre-coating treatment and seeding of feeder cells may be performed. Only pre-coating treatment may be performed, or the feeder cells may be seeded without pre-coating treatment. It is also acceptable to carry out the test.

[0078] Next, steps 1 and 2 of the method for producing intestinal organoids of the present invention will be described. In step 1, embryonic stem cells and / or induced pluripotent stem cells before seeding on a cell culture substrate The cells are maintained in an undifferentiated state using a non-differentiation-inducing medium. The medium is switched to a differentiation-inducing medium before and after, and the cells are seeded onto the surface of the substrate.

[0079] Non-differentiation-inducing medium is a medium that does not induce differentiation of embryonic stem cells and / or induced pluripotent stem cells. Although not particularly limited, for example, undeveloped mouse embryonic stem cells and mouse induced pluripotent stem cells It contains leukemia inhibitory factor, which is known to have the property of maintaining differentiation. Examples of media include:

[0080] In step 1, the seeding density of embryonic stem cells and / or induced pluripotent stem cells on the cell culture substrate In one embodiment of the present invention, embryonic stem cells are and / or induced pluripotent stem cells to a cell culture substrate at a density of 3 × 10 4 cells / cm 2 Sow at a density of more than Seeds are preferably 3 x 10 4 ~5×10 5 cells / cm 2 It is more preferable to sow at a density of , 3×10 4 ~2.5×10 5 cells / cm 2 It is more preferable to sow the seeds at a density of 1000 to 15000.

[0081] Step 2 is a step of culturing the cells seeded in step 1. The incubation temperature in step 2 is usually 37°C. Using a CO2 cell culture device, etc., the incubation temperature can be adjusted to about 5%. It is preferable to culture the cells in an atmosphere with a CO2 concentration of 1000 or more.

[0082] Step 2 is carried out in a differentiation-inducing medium. The differentiation-inducing medium is a medium containing embryonic stem cells and / or artificial multicellular The medium is not particularly limited as long as it can induce differentiation of the potential stem cells. For example, Examples include serum-containing media and serum-free media containing known components with properties that can replace serum. Depending on the type of cells used, MEM medium, BME medium, DMEM medium, DMEM-F12 medium, αMEM medium , IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, and RPMI1640 medium, etc. The medium may contain various growth factors, antibiotics, amino acids, and the like. For example, 0.05 mM to 1.0 mM non-essential amino acids, 1 mM to 5 mM GlutaMAX-I, 0.01 mM to 0.1 mM of β-mercaptoethanol, 0.1 mM to 2 mM pyruvate, 10 U / ml to 200 U / ml penicillin, 10μg / ml~200μg / ml streptomycin, 10μg / ml~200μg / ml L-ascorbic acid 2-lysine Examples of the anti-inflammatory agent include phosphate, and 1 μM to 20 μM of a ROCK inhibitor (for example, Y-27632).

[0083] In step 2, when cells are cultured in differentiation-inducing medium, the cell adhesion area increases within about 3 days after seeding. The cells become confluent within the plate and form a cell pattern. Then, the culture is continued. When the cell pattern is formed, it becomes a hemispherical dome-shaped cell mass on the cell adhesion area, and the cell Differentiation progresses within the cell clusters. Approximately 30 days after seeding, the cell clusters detach from the cell adhesion area and float in the medium. If necessary, further culture in a floating state can be continued to obtain the intestinal organoids of the present invention. The culture period is not particularly limited, and the culture may be terminated when the cells are detached from the cell adhesion region. Typically, the culture is carried out for 30 to 130 days after seeding. During this period, the medium is changed as appropriate. Unlike the method described in Non-Patent Document 1, the method of the present invention allows autonomous cell growth within the cell mass. The procedure is simple and the resulting intestinal organoids are Loganoids are preferred because they are believed to function more similarly to the natural intestine.

[0084] In one preferred embodiment of step 2, A portion of the cells seeded in step 1 differentiates into endodermal cells, and Some of the cells seeded in step 1 differentiate into ectodermal cells. Includes.

[0085] In this embodiment, more preferably, in step 2, a portion of the cells seeded in step 1 is The differentiation into endodermal cells occurs when some of the cells seeded in step 1 differentiate into ectodermal cells. In natural embryonic development, the intestinal tract is formed first, followed by the development of the saccharin. The enteric nervous system develops as a result of the invasion of the endodermal cells. Differentiation into cells progresses, and epithelial structures such as the intestinal epithelium derived from the endoderm are formed, and then The progression of differentiation into ectodermal cells and the development of the nervous system derived from the ectoderm is a developmental mechanism Therefore, the method for producing intestinal organoids according to the present embodiment can be said to mimic the intestinal It is expected that this technology can be used to elucidate the mechanisms of nervous system development in the brain. Furthermore, the intestinal organoids obtained by this method underwent a process similar to that of natural development. Because they are intestinal organoids, their responses to drugs are thought to be closer to those of the living body, making them suitable for drug discovery. It is expected to be a suitable model for research.

[0086] In step 2, the differentiation time of some of the cells seeded in step 1 into endodermal cells is determined to be the same as that of step 1. It is known that the differentiation of some of the cells seeded in the culture medium into ectodermal cells is earlier than that of other cells. The period when the increase in the expression level of the endoderm markers per unit time is greatest is the period when the increase in the expression level of the ectoderm markers This is before the time when the increase in the expression level of CAR per unit time is maximum. In this case, the endoderm marker is a gene specific to the endoderm. Specific examples include fetal endoderm markers FOXA2, SOX17, and CXCR4. The ectoderm marker may be any gene specific to the ectoderm. The expression level of each marker in a cell culture is determined by its mRNA expression level. It can be expressed as the relative amount of GAPDH mRNA expression level per unit time. The increase in the amount of rice produced per week can be used as the increase in the amount of rice produced per week. By measuring the expression level of each marker every week, it is possible to know the extent of increase in expression level every week. can.

[0087] In this embodiment, more preferably, step 2 is carried out by dissolving a portion of the cells seeded in step 1 in a solution containing 100% ethanol. The differentiation of the cells into endodermal cells by 14 days after seeding is specifically performed in cell culture. The period from seeding to seeding when the increase in the expression level of endoderm markers per unit time is greatest is During this period, differentiation into endodermal cells occurs, forming a gut-like structure. It is easy to obtain functional intestinal organoids. Even in this case, Although some cells may subsequently differentiate into endodermal cells, the majority of differentiation into endodermal cells It is preferable that this occurs by 14 days after sowing.

[0088] In this embodiment, more preferably, step 2 is carried out by dissolving a portion of the cells seeded in step 1 in a solution containing 100% ethanol. The differentiation into ectodermal cells is carried out after 15 days of culture, specifically, differentiation into ectodermal cells in cell culture. The maximum increase in marker expression per unit time occurs 15 days after sowing. During this period, differentiation into ectodermal cells occurs, resulting in the formation of intestinal organelles with intestinal-like functions. Even in this case, the cells were differentiated into ectodermal cells by 14 days after seeding. Although there may be some cells that differentiate, most of the differentiation into ectodermal cells occurs after 15 days from seeding. It is preferable to do so.

[0089] In another preferred embodiment of step 2, the differentiation-inducing medium is different from that of a mammalian cell to be cultured. It does not contain components derived from mammals of any species (xenogeneic components). Examples of such a medium include serum-free media containing known components that have properties that can replace serum. Intestinal organoids differentiated in a medium free of ATP are highly safe for transplantation into living organisms and are suitable for regenerative medicine. Suitable for medical use.

[0090] In another preferred embodiment of step 2, the differentiation-inducing medium contains insulin-like growth factor (IGF). and basic fibroblast growth factor (bFGF). Embryonic stem cells and / or induced pluripotent stem cells are cultured in the differentiation-inducing medium containing these two components. When cultured, it has a structure containing a cavity and is characterized by a length of 5 mm or more in the longitudinal direction. Intestinal organoids can be produced that have the above-mentioned advantageous characteristics and preferably further have the above-mentioned advantageous characteristics. It was found that IGF-1 is particularly preferable as the IGF. The concentration of bFGF in the differentiation-inducing medium is preferably 20 ng / ml to 2 μg / ml. Preferably, the IGF and bFGF are derived from the same species as the cells to be cultured. In addition, IGF and bFGF are not limited to those consisting of natural amino acid sequences, and It may be a functionally equivalent variant or fragment, for example, IGF-1, LONG R 3 -IGF-1 (Sigma- The differentiation-inducing medium may contain, in addition to IGF and bFGF, It is particularly preferred that the heregulin contained heregulin-1β is preferred. The Glin-1β may contain at least the EGF domain. The concentration of heregulin is preferably 1 ng / ml to 100 ng / ml. If heregulin is included, the heregulin is preferably derived from the same species as the cells to be cultured. It is not limited to those consisting of natural amino acid sequences, but also includes functionally equivalent variants and It may be a fragment.

[0091] In step 2, differentiation into intestinal organoids is carried out using a differentiation induction medium containing IGF and bFGF. In this embodiment, the differentiation-inducing medium does not need to contain IGF and bFGF at the time of seeding in step 1. For example, in step 1, cells are seeded on a cell culture substrate, and the cells grow confluently in the cell adhesion region. The cells were cultured in a differentiation-inducing medium that did not contain IGF or bFGF until they became fluent (for example, about 3 days after seeding). After reaching confluence, the medium was changed to a differentiation-inducing medium containing IGF and bFGF. The culture can be continued after the culture medium is replaced.

[0092] 3. Differentiation induction medium for producing intestinal organoids, kit for producing intestinal organoids The present invention also provides a differentiation-inducing medium for producing intestinal organoids, comprising IGF and bFGF. Details of this differentiation-inducing medium for producing intestinal organoids have been described above.

[0093] Embryonic stem cells and / or induced pluripotent stem cells are grown in this differentiation-inducing medium for producing intestinal organoids. When cultured, it has a structure containing a cavity and is characterized by a longitudinal length of 5 mm or more. Intestinal organoids can be produced that preferably further comprise the above-mentioned preferred characteristics. .

[0094] The present invention also relates to the above-mentioned differentiation-inducing medium for producing intestinal organoids, a substrate, and a method for producing intestinal organoids using the substrate. The cell adhesion region is formed on the surface, and the cell non-adhesion region surrounds the cell adhesion region. The present invention relates to a kit for producing intestinal organoids, which includes a cell culture substrate. The cell culture substrate in this kit is as described above for the method for producing intestinal organoids. do.

[0095] The present invention will be described below with reference to specific experimental results, but the scope of the present invention is not limited to the scope of the experimental results. It is not limited to the area. [Example]

[0096] 1. Preparation of cell culture substrate (First stage reaction) Toluene 58.5g, epoxy silane TSL8350 (Momentive Performance Materials, Inc.) 20.25 g of the product was mixed, and a catalytic amount of triethylamine was added to the mixture while stirring. The mixture was stirred at room temperature for several minutes. A 5-inch square glass substrate that had been UV-cleaned in advance was placed in the above epoxy resin solution. The glass substrate was immersed in a silane solution and left at room temperature for 20 hours. The substrate was washed with ethanol, then washed with water, and dried. The average value of the water contact angle of the substrate surface after drying was The angle was 56°. Thus, an epoxysilane-treated substrate was obtained.

[0097] (Second-stage reaction) While stirring 45 g of polyethylene glycol (molecular weight 400), slowly add a catalytic amount of concentrated sulfuric acid. The epoxy silane-treated substrate was added to the above-mentioned The substrate was immersed in polyethylene glycol and reacted at 120°C for 30 minutes. After the reaction, the substrate was thoroughly washed with water. This resulted in the creation of a glass substrate with a hydrophilic thin film. .

[0098] (Patterning process) The photomask is a 5-inch mask with a pattern of multiple circular openings of the same size. The photomask had circular openings of 1.5 mm in diameter, and the spacing between the openings was The spacing between the openings, i.e., the shortest distance between the openings, was 0.35 mm. Ta.

[0099] The mask was gently placed on the film-forming surface of the glass substrate on which the hydrophilic thin film was formed, and the back side of the mask from a xenon excimer lamp (172 nm, 10 mW / cm 2 ) for 1 minute. As a result, the areas on the surface of the hydrophilic thin film that corresponded to the openings of the photomask were oxidized. The substrate was cut into 5 cm squares and used for cell culture.

[0100] The shape of the cell adhesion area on the obtained cell culture substrate was circular, with a diameter of 1.5 mm. The space between the cell adhesion regions, i.e., the shortest distance between the cell adhesion regions, was 0.35 mm in all cases. The percentage of cell adhesion area on the cell culture substrate was 51.8%.

[0101] 2. Culturing intestinal organoids 2.1. Cell lines Human embryonic stem cell (hESC) lines SEES1, SEES2, and SEES3 were obtained from the National Center for Child Health and Development's Reproductive Medicine Center. The cells established by the Department of Cell Therapy Research were used (Akutsu H, et al. Regen. Ther. 2015;1:18-2 9) These ES cells were cultured in a commercially available serum-free DMEM (trade name: KnockOut TMD-MEM (Thermo Fisher Scientific ientific), 20% KnockOut TM Serum Replacement(KnockOut Serum Replacement)(Life Technol ogies), 0.1 mM non-essential amino acids (NEAA), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.055 mM β-mercaptoethanol, 50 U / ml penicillin / 50 μg / ml streptomycin (Pen-St rep), and 8 ng / ml recombinant human bFGF (all purchased from Life Technologies). The cells were maintained on a gamma-irradiated mouse embryonic fibroblast (MEF) feeder layer in the same medium. The medium was changed every two days. Approximately once a week, the cells were treated with an enzymatic method (dispase; Wako Pure C Cells were passaged using either a mechanical method (EZPassage; Life Technologies) or a microcentrifuge (Microelectronic Industries). Substituted.

[0102] Human iPS cells (hiPSCs) are generated at the Department of Reproductive and Cellular Medicine, National Center for Child Health and Development. In this study, four Yamanaka factors (Oct3 / 4, Sox2, Klf4, and c-Myc) were transfected into the MRC5 cell line, a human fetal lung fibroblast. The gene was established by expression using a retroviral vector (Makino H, et al. Exp Ce ll Res. 2009;315(16):2727-2740; Nishino K, et al. PLoS ONE. 2010;5(9):e13017; To Yoda M, et al. Genes Cells. 2011;16(1):1-11). These human iPS cells were cultured in the presence of 10 ng / ml bFGF. In iPSellon medium (Cardio Incorporated) supplemented with γ-irradiated MEF feeder layers, was maintained.

[0103] 2.2. Formation of human intestinal organoids In previous research, the inventors have developed xeno-free culture media for establishing and growing human ES cells. Xeno-free (XF) conditions have been established (Akutsu H, et al. Regen. Ther. 2015;1:18-29) 85% knockout of human ES cells TM D-MEM, 15% KnockOut TM Serum replacement (Knockout Serum R replacement) XF CTS (XF-KSR; Life Technologies), 1 mM pyruvate, 2 mM GlutaMAX-I , 0.1 mM NEAA, Pen-Strep, 50 μg / ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), 10 ng / ml Heregulin-1β (recombinant human NRG-β1 / HRG-β1 EGF domain; R&D System s), 200 ng / ml recombinant human IGF-1 (LONG R 3 -IGF-1; Sigma-Aldrich), and 20 n The cells were stably maintained in XF hESC medium containing 100 μg / ml human bFGF (Life Technologies).

[0104] The following three types of differentiation-inducing media were prepared. Regarding the medium composition, "%" refers to volume % unless otherwise specified. Differentiation-inducing medium 1 (sometimes referred to herein as "XF-KSR(-) medium"): 80% KnockOut TM D -MEM, 20% KnockOut TMKnockout Serum ReplacementXF CTS (XF-KSR; Life Technologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 0.055 m Medium containing 10 μM β-mercaptoethanol and 10 μM Y-27632 Differentiation-inducing medium 2 (sometimes referred to herein as "XF hESC medium"): 85% KnockOut TM DM EM, 15% KnockOut TM Knockout Serum ReplacementXF CTS (XF-KSR; Life T technologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 50 μg / ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), 10 ng / ml heregulin-1β (recombinant Human NRG-β1 / HRG-β1 EGF domain (R&D Systems), 200 ng / ml recombinant human IGF -1 (LONG R3-IGF-1; Sigma-Aldrich) and 20 ng / ml human bFGF (Life Technologies). medium containing Differentiation-inducing medium 3 (sometimes referred to herein as "XF-KSR medium"): 80% KnockOut TM D-ME M, 20% KnockOut TM Knockout Serum ReplacementXF CTS (XF-KSR; Life Te technologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 0.055 mM Medium containing β-mercaptoethanol

[0105] To generate intestinal organoids, undifferentiated hESCs or hiPSCs are treated with dispase. The cell culture substrate prepared by the above procedure was placed in a 90 mm culture dish. , coated with 0.1% human recombinant type I collagen peptide (RCP) (Fujifilm) Other materials such as vitronectin (Life Technologies) were used in this study. The matrix-coated culture dish may be preheated at 37°C. After 1 hour of incubation, the coating solution was removed and the substrates were washed three times with PBS. x10 6 The cells were seeded on the substrate and left for 10 minutes. Then, the medium was removed by aspiration. Then, 10 ml of fresh differentiation-inducing medium 1 was gently added. Differentiation-inducing medium 1 was used to induce the differentiation of Rho-associated proteins as described above. It contains the protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) and inhibits growth factors. hESCs or hiPSCs were cultured in differentiation-inducing medium 1 containing Y-27632 but not containing growth factors for 1 day. Then, the cells were cultured in differentiation induction medium 3 containing no growth factors or Y-27632. After 3 days, the medium 3 was replaced with differentiation-inducing medium 2 containing growth factors. The cells were cultured while changing the medium as needed. Differentiation-inducing medium 2 was gently changed every 3 to 4 days. After 3 days, multiple organoids exhibiting peristaltic movements similar to those of the intestine were collected and cultured in 60-mm Ultralow A The cells were cultured together in differentiation-inducing medium 2 on a differentiation plate (NOF Corporation). In another experiment, undifferentiated hESCs or hiPSCs were seeded on the substrate in the same manner and allowed to grow for 4 days. The cells were cultured in differentiation-inducing medium 1 on day 1, and the same procedure was performed except that the medium was changed to differentiation-inducing medium 2 on day 4. Ta.

[0106] Unless otherwise specified, the cells were cultured in a stationary incubator at 37°C and 5% CO2. went.

[0107] 2.3. Video recording of human intestinal organoids The floating organoids were transferred to a culture plate containing the differentiation-inducing medium 2, and ZILOS-t Video recording was performed using an inverted microscope equipped with a camera (Cohu 3600) from the K system (Hamilton Thorne). To count the number of contracting intestinal organoids, the number of cells generated on one plate was recorded and analyzed. All the human embryonic stem cell (hESC) floating organoids were transferred to a new dish and incubated for 10 minutes. Organoids exhibiting peristaltic contraction were evaluated as positive.

[0108] 2.4. Quantitative RT-PCR analysis RNA was isolated from organoids using the RNeasy Mini Kit (Qiagen) and contaminating DNA was removed. The cDNA was purified using SuperScript III reverse transcriptase and DNase (Life Technologies). and oligo-dT primers (Life Technologies) were synthesized according to the manufacturer's instructions. QuantStudio 12K Flex Real-Time PCR System was used for quantitative RT-PCR. The PCR was performed using stem (Life Technologies) (n=3). The primer sequences are shown in the table below.

[0109] [Table 1]

[0110] After amplification, a dissociation curve was obtained to confirm that each PCR product was amplified. GAPDH was used as the housekeeping gene. QuantStudio 12K Flex software v1.0 The mRNA expression level of the target gene was normalized to the mRNA expression level of GAPDH using the standard The relative expression levels of mRNA of target genes were quantified. in Institute) and adult pancreatic and liver cDNAs (HA-188 and HA-149, respectively, Alpha Diagnostics A fluoroscopy specimen (Osmotic International) was used as a positive control.

[0111] 2.5. Immunocytochemical analysis Organoids were cultured in 4% paraformaldehyde-containing PBS (Wako Pure Chemical Industries). The cells were fixed with 0.2% Triton X-100 for 5 minutes at 4°C, permeabilized with 0.2% Triton X-100 for 2 minutes at room temperature, and then If necessary, each antibody was subjected to blocking treatment with PBS containing 5% normal serum. The organoids were incubated overnight at 4°C with primary antibodies against the following antigens: The antigens were as follows: villin (sc-7672, 1:50, Santa Cruz Biotechnology); E-cain (SC-7672, 1:50, Santa Cruz Biotechnology); Doherin (610181, 1:50, BD Pharmingen); CGA (ab16007, 1:100), CDX2 (ab76541, 1:10 0), and PGP9.5 (ab8189, 1:10) (from Abcam); DEFA6 (HPA019462, 1:500) and SMA (A25 47, 1:400) (from Sigma-Aldrich); MUC2 (sc-7314, 1:50, Santa Cruz Biotechnology); LGR5 (LMC-1235, 1:100, Medical & Biological Laboratories); CKIT (NB100-77477AF48 8, 1:10, Immuno-Biological Laboratories); Na+ / K+-ATPase (NB300-146, 1:100, Novus Biologicals); S-100 (422091, 1:100, Nichirei Biosciences); neurofilament (M076229, 1:50, Dako); β-actin (A5316, 1:1,000, Sigma-Aldrich); histamine H1 receptor (aa471-484, 1:200, LSBio); and CFTR (ab131553, 1:100, Abcam). Alexa 4 88- or Alexa 546-labeled anti-mouse, anti-rabbit, or anti-goat secondary antibodies (BD Biosciences) Cell nuclei were counterstained with DAPI. Fluorescence was analyzed using a fluoroscopic microscope (Carl Zeiss Microscopy).

[0112] 2.6. Histochemical analysis Organoids were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced ​​at 4 μm thickness. Every other section was mounted on a slide, stained with H&E, and analyzed. For blue staining, the cells were incubated in 1% Alcian Blue in 3% acetic acid solution at pH 2.5 for 20 minutes. bated, then incubated in 0.1% Nuclear Fast Red for 2 minutes, and The sections were dehydrated and cleared with xylene.

[0113] Histochemical analysis of organoids was performed using antibodies against SMA and serotonin (ab16007, 1:20, A The study was performed using sections stained with a polyclonal rabbit anti-rabbit antibody. Usual immunoglobulin-HRP (1:100; Dako) was used.

[0114] 2.7. Electron Microscopy Electron microscopy of intestinal organoid samples was performed according to standard protocols (Tokai University). Electron Microscopy). Organoids were cultured in 2% paraformaldehyde in PBS and The tissue was fixed with 2% glutaraldehyde, dehydrated, and embedded in epoxy resin. The sample was then cut into 70 nm sections, mounted on copper grids, and imaged with a Veleta CCD camera (Olympus). The observation was performed using a JEM-1200EX TEM (Jeol Ltd.) equipped with a TEM.

[0115] 2.8. Cell transfection To investigate differentiation during intestinal organogenesis, we specifically targeted intestinal cell lines. A reporter construct (pPB-hLgr5p-EGFP-neo) was constructed. kb LGR5 promoter and phosphoglycerol kinase (PGK) promoter, respectively. The expression of EGFP and neomycin resistance gene (neo) was promoted in SEES1 cells. After 24 hours of culture, they were subjected to electroporation, washed with PBS, and then soaked in Accuutase solution (L The cells were collected using iPlane Technologies and resuspended in iPSellon medium. The cells were separated into single cell suspensions by 1-2 × 10 6 The cells were pelleted and resuspended in Opti-MEM ( pPB-hLgr5p-EGFP-neo reporter vector and hyperactive PiggyBac Transposase expression vector (pCMV-hyPBase) (A. Bradley, Wellcome Trust Sanger Institute) (provided by the Institute, Hinxton, Cambridge, United Kingdom). The resulting cell suspension was transferred to a cuvette and electroporated using a NEPA21 Super Electroporator (Nepa Gene). The transfected cells were obtained by electroporation using the Cells were selected with G418 (Sigma-Aldrich).

[0116] To visualize the in vivo function of transplanted intestinal organoids, we used human embryonic stem cells (hESCs). The vector (pmGENIE-EGFP) expressing EGFP constitutively was used (S. Moisyadi, University of Hawaii). aii, Honolulu, Hawaii, USA) and screened. Through gene expression analysis, we established stable GFP-positive human embryonic stem cell (hESC) lines.

[0117] 2.9. Assessment of β-Ala-Lys-AMCA uptake by human intestinal organoids in vitro Intestinal organoids grown in differentiation-inducing medium 2 were washed with PBS and then treated with 25 μM fluorescently labeled dipeptide. The cells were cultured in DMEM containing the peptide β-Ala-Lys-AMCA (Biotrend Chemicals) for 4 hours at 37°C. For inhibition experiments, 1 mM of the angiotensin-converting enzyme inhibitor captopril (Sigma-Aldrich) was used. The cells were cultured for 1 hour with or without the addition of PBS, rinsed with PBS, and then placed in the same dish. The cells were placed in a tube and observed under a fluorescence microscope (Olympus). To quantify captopril levels, intestinal organoids were incubated with 10 μM, 100 μM, or 1 mM captopril. The cells were cultured under conditions with or without additives, and AMCA-related signals were detected in the top stage incubator. The fluorescence signals were observed using a fluorescent microscope (BZ-X710; Keyence) equipped with a 5% CO2, 37°C (5% CO2, 37°C) The intensity was quantified using Hybrid Cell Count / BZ-H3C (Keyence). Three independent assays were performed for each concentration condition.

[0118] 2.10. In vitro contractility of human intestinal organoids One of the human embryonic stem cell (hESC)-derived intestinal organoids (culture days 80-90) exhibited peristaltic-like movement. , histamine (0.2 μM), which stimulates peristalsis, and atropine sulfate (0.2 μM), an anticholinergic agent. Contractile responses were recorded using an inverted microscope. Image analysis software CL-Qua The movement of intestinal organoids was visualized using nt version 3.10 (Nikon Corporation). First, in each frame of the time-lapse image, organoids were detected using the software. Second, the software fits an ellipse to the region. The aspect ratio was calculated by dividing the maximum diameter by the minimum diameter. The changes were plotted on a chart and videotaped at 30 frames per second.

[0119] 2.11. CFTR transport activity in intestinal organoids Place one ol in one well of an ART Culture Dish 12 (NIPRO) containing 100 μl of medium. The ganoids were placed in EGFP-human embryonic stem cells, which constitutively express EGFP under the control of the CMV promoter. Volume changes were visualized using organoids derived from stem cells (hESCs). Phosphorus was added, and organoid morphology was observed using a time-lapse fluorescence laser confocal microscope (Keyence). To inhibit CFTR, organoids were incubated with 50 μM of the CFTR inhibitor CFT Pre-incubated with Rinh172 and 50 μM GlyH-101 (TOCRIS) for 3 hours. Images were acquired every minute for 20 minutes in a stage incubator (5% CO2, 37°C). Each experimental condition was evaluated in triplicate. The DMSO concentration in all experimental conditions was 0.2 The surface area of ​​the organoids was determined by the Hybrid Cell Collection (HCC). The total surface area of ​​the normalized organoids was calculated using nt / BZ-H3C (Keyence). Measurements from three separate wells per experimental condition were averaged.

[0120] 2.12. Transplantation of human intestinal organoids To confirm the in vivo growth of intestinal organoids, immunodeficient colonic organoids were purchased from CLEA Japan. The organoids were transplanted into nude mice (BALB / cAJcl-nu / nu). One intestinal organoid was transplanted into a nude mouse (BALB / cAJcl-nu / nu) on day 35 of culture. The transplanted mice were euthanized by cervical dislocation after 6 weeks. The kidneys were observed under an MVX10 fluorescence microscope (Olympus). , and further analyzed by H&E staining and immunocytochemistry.

[0121] 2.13. Statistics Quantitative data are presented as mean ± SEM values ​​from at least three independent experiments. The statistical analysis was performed using an unpaired, two-tailed t-test or the Mann-Whitney rank sum test. P < A difference of 0.05 was considered statistically significant.

[0122] 3. Results Representative results are shown in Figures 1A to 9B. 1A to 1D show the results of culturing human pluripotent stem cells on a cell culture substrate with a patterned cell adhesion region. FIG. 1 illustrates the formation of peristaltic intestinal organoids from cells. 2A to 2D are diagrams illustrating the differentiation characteristics of intestinal organoids. Figures 3A to 3C show the characteristics of intestinal organoids and the role of LGR5-E in the formation of intestinal organoids. FIG. 1 shows the results of detecting GFP-positive cells. 4A to 4F are diagrams illustrating the peristaltic activity of intestinal organoids. 5A to 5C are diagrams illustrating the absorptive function of intestinal organoids. Figures 6A and 6B illustrate CFTR transport activity in intestinal organoids. Figures 7A to 7D show the characteristics of intestinal organoids that do not have peristaltic activity. Figures 8A to 8D show that when intestinal organoids (culture day 35) were transplanted, a highly structured intestinal tract was formed. Indicates that it will be. Figures 9A and 9B show changes during the culture process of intestinal organoids derived from human iPS cells.

[0123] The experimental results will be explained below with reference to the drawings as appropriate. This is described in the "Brief description of the surface" section.

[0124] 3.1. Intestinal organoids grow autonomously on cell culture substrates with patterned cell adhesion regions formed in Generally, the self-organization of tissues consists of three main steps: autonomous assembly, self-patterning, and self-assembly. morphogenesis (Sasai Y. Nature. 2013;493(7432):318-326). Using the technique, the intestinal morphology is developed through the stages of cell pattern swelling and automorphogenesis. The first step is to induce the formation of erythrocytes in XF medium (Akutsu H, et al. Regen. Th Human embryonic stem cells (hESCs) or human iPS cells (hiPSCs) cultured in a medium containing 100% soluble soluble erythrocytes (Molecular Biology, 2015;1:18-29). However, in differentiation induction medium 2, cell culture substrate (Okochi N, Okazaki T, Hattori H. Langmuir. 20 09;25(12):6947-6953) assembled into circular patterns of cell adhesion areas on the glass surface. The cells that had gathered in the cell adhesion area formed hemispherical dome-shaped structures after the 7th day of culture (Figure 1C Subsequently, large structures with cavities were formed. These structures were composed of self-assembled cells. The epithelium was folded by the cyst clusters, forming cyst-like protrusions, and by the 20th day of culture, the cells were differentiated into cuboidal epithelial cells. By day 30 of culture, self-formed cystic spheroids had formed over the cell culture medium. The spheroids were separated from the substrate. They were roughly divided into two types. The other was a simple cystic spheroid with a solid part and a thin wall. It was a bicomponent spheroid with cystic protrusions. It was these two-component spheroids that had the ability to move. Only 4% of the isolated spheroids (34 of 791, n=3) were maintained over the long term. This indicates that cell types derived from different germ layers can form functional cell networks. This suggests that organoids formed by forming a network were aggregated.

[0125] Next, we examined the expression of germ layer markers at different time points during differentiation. During differentiation, the proliferating patterning cells express the definitive endoderm markers FOXA2, SOX17, and CXCR4. , as well as early endoderm and mesoderm markers GATA4, GATA6, and T (Brachyury) (Figure 2A). The expression of these markers increased until day 14 of culture, when hemispherical dome-shaped structures were formed. CXCR4 expression decreased on the 21st day of culture, when differentiation had progressed further (Fig. 1A and Fig. 2A). is the downregulation of gene expression during hindgut formation in mouse embryonic development. This was consistent with the report by McGrath KE, Koniski AD, Maltby KM, McGann JK, Pali s J. Dev Biol. 1999;213(2):442-456). The expression level of the hindgut marker CDX2 was significantly higher in the early proliferation stage. The expression of neural progenitor cell marker SOX1 increased during differentiation and remained relatively high even as differentiation progressed (Fig. 2A). Expression increased relatively late (day 21) compared with other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 was significantly reduced during the early differentiation stage. Thus, our procedure resulted in the development of definitive endoderm and endothelial cells from human ES cells by day 21 of culture. The differentiation of the cells into the hindgut and posterior intestine was promoted, forming a hemispherical dome-shaped structure. The aggregates formed hollow structures and were detached from the cell culture substrate (Figure 1C). The epithelium had a folded structure. The organoids exhibited peristaltic movement and were embedded in paraffin. H&E staining confirmed the intestinal structure consisting of the mucosa and submucosa (Fig. 2B). The intestinal epithelium is made up of two types of cells: absorptive enterocytes and secretory cells. These cells include mucus-producing goblet cells, enteroendocrine cells, and Paneth cells. Including (Gracz AD, Magness ST. Am J Physiol Gastrointest Liver Physiol. 2014;307(3): G260-G273.) Alcian Blue staining revealed that goblet cells containing mucopolysaccharides stained purple. It was shown that the cells were present in the epithelial layer of the organoids (Figure 2C). Mature organoids show a significant difference in the relative expression levels of several late differentiation markers. The expression of CDX2 (a transcription factor in the intestine) and ECs was comparable to that of the adult intestine (Fig. 2D). AD (epithelial cell-specific E-cadherin) and brush border-specific villin are expressed in intestinal organoids. The expression level in the human adult intestine was similar to that in the human adult intestine. It was confirmed that phospholipids are localized on the apical side of the epithelium, and CDX2 is present in the epithelial layer. TEM (transmission electron microscope) images of the organoids showed that the apical side of the microvilli The formation of the brush border was confirmed (Fig. 3B). Paneth cell-specific defensin α Paneth cells were identified by staining with DEFA6 (Figure 3A), and the Paneth cells were identified by TEM. The presence of secretory granules was confirmed (Fig. 3B). H&E staining and Alcian Blue staining revealed that the epithelial layer The presence of goblet cells was identified in the spleen. The presence of goblet cells was confirmed by mucin-2 (MUC2) staining ( These cells were identified by the presence of mucin granules in TEM images (Fig. 3A) and mucin granules in TEM images (Fig. 3B). The expression level of the marker chromogranin A (CGA) was higher in intestinal organoids than in human adult intestine. Although the levels were low (Fig. 2D), immunostaining for ECAD and CGA showed that both of these proteins were expressed in the epithelium. In intestinal organoids, the intestinal stem cell marker LGR5 was expressed in CDX2 (Fig. 3A). The cells were co-expressed with the EGFP expression vector under the control of the LGR5 promoter (Fig. 3A). LGR5 expression was also confirmed during intestinal organoid formation from human embryonic stem cells (hESCs) (Figure 3C). Therefore, the self-formed intestinal organoids of the present invention can be used to treat various types of highly These results suggest that the intestinal lining of the spleen is composed of differentiated intestinal cell types.

[0126] 3.2. Intestinal organoids have functions specific to the mature intestine Intestinal organoids possess intestinal-specific functions such as peristalsis, peptide absorption, and mucus secretion The intestinal organoids exhibited contractile movements, suggesting the presence of a functionally mature mesenchymal layer. Intestinal motility is regulated by pacemakers such as the enteric nervous system and interstitial cells of Cajal (ICC). - Cellular regulation (Sanders KM, Koh SD, Ward SM. Annu Rev Physiol. 2006;68:3 07-343; uizinga JD, Lammers WJ. Am J Physiol Gastrointest Liver Physiol. 2009;29 6(1):G1-G8). To identify which cell types are involved in intestinal contractile movements, immunohistochemistry was performed. Mesoderm-derived smooth muscle cells were stained for α-smooth muscle actin (SMA). These results suggest that mesoblasts are essential for organoid formation. Subepithelial myofibroblasts were shown to be involved in the in vivo and in vivo function of the human intestinal epithelium. Supports in vitro growth (Lahar N, et al. PLoS One. 2011;6(11):e26898). Quantitative R Using T-PCR, we detected the protein gene product 9.5 (PGP9.5), a marker of the enteric nervous system (Fig. 3A), We also identified cells expressing the ICC markers CD34 and CKIT (Figure 2D). Using staining, double positive cells for glial cell markers CKIT and S-100 were detected in the submucosal We identified that serotonin is localized in the gastrointestinal tract (GI) region (Fig. 4B). It is the main neurotransmitter that controls the immune system and is synthesized by enteroendocrine cells in the intestinal mucosa (Mawe GM, Hoffman JM. Nat Rev Gastroenterol Hepatol. 2013;10(8):473-486). Immunohistochemistry The analysis revealed the presence of serotonin-positive cells in the epithelial layer of the intestinal organoids. The contraction rate of intestinal organoids was increased by histamine treatment and decreased by atropine treatment (Fig. 4C). Thus, contractility was reduced after treatment with histamine or atropine (Fig. 4D). These changes indicate that intestinal organoids possess motility similar to that of the mature intestine. (Mittal RK, Padda B, Bhalla V, Bhargava V, Liu J. Am J Physiol Gastrointes Intestinal organoids with peristaltic activity are capable of such a function. Intestinal organoids lack peristaltic activity and respond to drugs, whereas in ... which stimulate contractions. On the other hand, histamine H1 receptors were not responsive to peristaltic steroids (n = 6, Figure 4E). It was expressed in the epithelial and mesenchymal regions of the human intestine and in the oocytes lacking peristaltic activity (Fig. 4F). The ganoids also expressed histamine H1 receptors, and quantitative RT-PCR and intestinal tissue-specific Immunostaining analysis of genes confirmed that the tissue resembled the intestine (Fig. 7A). Albumin and insulin, markers of endoderm-derived cells, were not expressed in intestinal organoids. The organoids lacking peristaltic activity were genetically similar to the human adult intestine. Although the expression level of the IL-1 receptor was high, immunohistochemistry showed that the mesenchymal layer of the organoids lacked peristaltic activity. Smooth muscle marker SMA staining in organoids lacking peristaltic activity was not observed. , but at a lower level compared to organoids with peristaltic activity (Figure 7C). In organoids with peristaltic activity, the ATP was distributed throughout the mesenchymal region, whereas in peristaltic organoids, the ATP was distributed throughout the mesenchymal region. In the non-motile organoids, the number was sparse (Figure 7D). Insufficient development of the rectum may be the cause of the lack of peristaltic function.

[0127] To assess peptide uptake by intestinal organoids, we performed intestinal oligopeptide transfection. transporter (PEPT1) and the major ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 The expression levels of these genes were similar between intestinal organoids and adult intestine. The uptake of the active peptide was evaluated by an in vitro assay (Gronebe rg DA, Doring F, Eynott PR, Fischer A, Daniel H. Am J Physiol Gastrointest Liver Physiol. 2001;281(3):G697-G704). Fluorescently labeled dipeptide β-Ala-Lys-N(ε)-7-amino-4- Intestinal organoids cultured with methylcoumarin-3-acetic acid exhibited peptide absorption and The peptide absorption was decreased by treatment with Captopril (Fig. 5B). However, the inhibitory effect of captopril on peptide absorption was not confirmed to be concentration-dependent. (Figure 5C).

[0128] To investigate the secretory activity of the organoids, we transfected them with cystic fibrosis transmembrane conductance regulator (CFTR). The expression level of TR was confirmed and forskolin-induced swelling (FIS) was measured. CFTR is important for mucus secretion in intestinal epithelial cells. In organoids, we confirmed the presence of CFTR in the epithelial layer, similar to that in the human intestine (Figure 6A). The FIS assay (Dekkers JF, et al. Nat Med) is used in the art to confirm the function of TR. 2013;19(7):939-945), and found that forskolin caused the intestinal organoids to expand. Furthermore, a CFTR blocker completely inhibited this swelling (Fig. 6B). Based on these results, the present inventors concluded that intestinal organoids have the same absorptive and secretory capabilities as the mature intestine. And so he concluded.

[0129] The experimental results showed that stem cell-derived intestinal organoids generated under xeno-free conditions were able to function in the intestine. It was confirmed that the major cell types involved have an organized structure and mature intestinal functions. Previous studies have shown that when intestinal organoids are transplanted into the kidney capsule of mice, they undergo maturation and differentiation. It has been shown that this occurs (Watson CL, et al. Nat Med. 2014;20(11):1310-1314). To confirm that intestinal organoids formed from stem cells in vitro are mature We developed a human embryonic stem cell (hESC)-derived gene that constitutively expresses EGFP under the control of the CMV promoter. One intestinal organoid from day 35 of culture was transplanted under the kidney capsule of an immunodeficient nude mouse. All intestinal organoids were successfully transplanted (n=4), and exhibited the intestinal characteristics of a luminal and laminated structure (Fig. 8A). The cells were highly structured (Fig. 8B and 8C). CDX2 (enterocytes), MUC2 (goblet cells), CGA (enteroendocrine cells), DEFA6 (Paneth cells) lls), as well as ECAD and Na+ / K+-ATPase (epithelial), and PGP9.5, an enteric nerve marker. It was confirmed that the protein was expressed in the multilayered SMA-positive mesenchyme (Fig. 8D). All intestinal cell types were present in the transplanted intestinal organoids. The ids did not grow significantly after transplantation, indicating that they were highly differentiated in vitro. It was suggested that this is the case.

[0130] 4. Discussion The above experimental results demonstrate that human stem cell-derived intestinal organoids were generated under xeno-free conditions. The intestinal saccharin has a highly organized structure of the major cell types associated with the intestine, and functions equivalent to those of the mature intestine. The intestinal organoids obtained in this experiment have at least three advantages: do.

[0131] First, intestinal organoids can be stably maintained for long periods in xeno-free conditions and are capable of regenerating. Suitable for medical applications.

[0132] Second, intestinal organoids have a complex tissue structure and function equivalent to that of the mature intestine. Unlike epithelial-derived organoids, intestinal organoids contain both the intestinal epithelial and mesenchymal layers. Intestinal organoids have epithelial functions such as peptide absorption and mucus secretion, and they also have the ability to mature. Similar to the mature human intestine, it exhibits peristaltic movements in response to histamine and atropine. Intestinal organoids can be used as "mini-guts" and are useful in the study of gut-related diseases. It is highly useful.

[0133] Third, this experiment demonstrated that stem cells can be derived in vitro into three germ layers: endoderm, mesoderm, and ectoderm. This is the first example of creating a functional organoid that combines the cells that grow within it. [Industrial Applicability]

[0134] The intestinal organoids provided by the present invention can be used to study the mechanisms of intestinal diseases and to develop therapeutic drugs. It is useful for

[0135] The method for producing intestinal organoids provided by the present invention can be used for disease research and drug development. Therefore, intestinal organoids can be easily generated in vitro.

Claims

[Claim 1] 1. A method for retaining a substance, comprising: Intestinal organoids containing intestinal epithelial cells on at least a portion of the outer surface and a liquid containing the substance, Placing the intestinal epithelial cells in contact with the liquid; and retaining the substance within the intestinal organoids; The method comprising:

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