Inflammatory bowel disease model derived from induced pluripotent stem cells, method for producing same, and method for evaluating drug efficacy by using same

Abstract

The present invention relates to an inflammatory bowel disease model derived from induced pluripotent stem cells and a method for producing same. The inflammatory bowel disease model simulates stable intestinal epithelial cells from induced pluripotent stem cells and remarkably exhibits the expression of inflammation-related genes according to the occurrence and improvement of inflammatory bowel disease, and thus can be effectively used for evaluating the efficacy of a drug for treating inflammatory bowel disease.

Description

Inflammatory bowel disease model derived from induced pluripotent stem cells, method for producing the same, and method for evaluating drug efficacy using the same

[0001] The present invention relates to an inflammatory bowel disease model derived from induced pluripotent stem cells and a method for producing the same.

[0002] Inflammatory bowel disease (IBD) is a chronic and relapsing disease of the digestive tract, including Crohn's disease and ulcerative colitis. Ulcerative colitis is characterized by inflammation and ulcers along the lining of the large intestine (colon) and rectum. Crohn's disease is characterized by inflammation of the digestive tract lining, often affecting the deeper layers of the digestive tract. IBD is associated with hyperactivation of immune cells, leading to the abundant secretion of certain cytokines. In particular, abnormally accelerated macrophages in CD produce inflammatory cytokines such as tumor necrosis factor-α (TNF-α), which damage intestinal epithelial cells. This, in turn, leads to invasion of intestinal bacteria and subsequent immune system stimulation, further accelerating disease progression. Recently, the treatment of active IBD has been shown to reduce intestinal inflammation and epithelial damage when combined with the TNF-α inhibitor infliximab (IFX). However, current models of mucosal damage and fibrosis in IBD have significant limitations for therapeutic drug screening. In vitro models, such as Caco-2, T-84, and CCD-18co cells, cannot reflect the complex intestinal architecture. Therefore, intestinal organoids, three-dimensional tissue structures, have recently attracted significant attention and are expected to be important tools for the study and treatment of intestinal diseases due to their structural similarity to intestinal tissue. While human-derived intestinal organoids are currently being used experimentally, their acquisition through invasive and difficult methods poses a challenge, limiting their availability for high-throughput screening.

[0003] The purpose of the present invention is to provide an inflammatory bowel disease model derived from induced pluripotent stem cells, a method for producing the same, and a method for evaluating drug efficacy using the same.

[0004] Another object of the present invention is to provide an inflammatory bowel disease drug screening system including an inflammatory bowel disease model derived from induced pluripotent stem cells.

[0005] To solve the above problem, the present invention provides an inflammatory bowel disease model derived from induced pluripotent stem cells and a method for producing the same.

[0006] In the present invention, induced pluripotent stem cells (iPSCs) are cells that have pluripotent differentiation potential, which is the ability to differentiate into all cells that make up our body, like embryonic stem cells, through artificial stimulation of multi-differentiated somatic cells other than stem cells. They are also called induced pluripotent stem cells because they are a type of pluripotent stem cell that can be directly generated from somatic cells.

[0007] The present invention comprises a step (step 1) of culturing and expanding colon organoids derived from induced pluripotent stem cells in an expansion medium for 5 to 7 days;

[0008] Step 2: Transferring the colon organoids derived from the induced pluripotent stem cells proliferated in Step 1 to differentiation media and culturing them for 3 to 4 days to differentiate them into intestinal epithelial cells; and

[0009] The present invention provides a method for producing an inflammatory bowel disease model derived from induced differentiation stem cells, characterized by including a step (step 3) of inducing inflammation by culturing colon organoids differentiated into intestinal epithelial cells in the above step 2 in a differentiation medium containing TNF-α for 1 to 3 days.

[0010] The above expansion media may include DMEM / F12, Penicillin / Streptomycin, N-Acetyl-L-cysteine, Nicotinamide, Glutamax, N-2, B-27, L-WRN culture harvest medium, A-83-1, EGF, and Primosin.

[0011] The above L-WRN culture harvest medium is a medium obtained by collecting and filtering the supernatant in which L-WRN cells are grown. The L-WRN culture harvest medium is obtained by culturing L-WRN cells in DMEM medium (10% FBS, 1% P / S) for 3 to 4 days until the culture dish is filled with L-WRN cells, removing the existing medium, adding harvest medium containing Advanced DMEM F / 12, FBS, Glutamax, and penicillin / streptomycin, and culturing for another 24 hours, collecting the medium, and filtering it using a 0.22 μm filter. It is also called conditioned media or WRN conditioned media, and may contain WNT3A, Noggin, R-spondin, etc., which are necessary for the growth of organoids.

[0012] The differentiation medium may include DMEM / F12, penicillin / streptomycin, N-acetyl-L-cysteine, Nicotinamide, Glutamax, N-2, B-27, A-83-1, EGF, and Primosin.

[0013] The colon organoid derived from induced pluripotent stem cells according to the present invention can be grown to a maximum of 400 μm after 5 to 7 days of culture in the above step 1, and can survive for up to 21 days of culture.

[0014] The present invention provides an inflammatory bowel disease model derived from induced pluripotent stem cells, manufactured using the method for manufacturing the inflammatory bowel disease model derived from induced pluripotent stem cells. The inflammatory bowel disease model derived from induced pluripotent stem cells of the present invention can be used as a differentiation model containing intestinal epithelial cells to reproduce an inflammatory bowel disease (IBD) model using TNF-α and to enhance the responsiveness of IBD drug efficacy evaluations.

[0015] In addition, the inflammatory bowel disease model derived from induced pluripotent stem cells of the present invention is characterized by increased expression of villin, E-cadherin, Mucin-2, etc. through the differentiation process.

[0016] The present invention provides a drug screening system for treating inflammatory bowel disease, comprising the inflammatory bowel disease model derived from the induced pluripotent stem cells. The drug screening system for treating inflammatory bowel disease of the present invention exhibits a significant secretion response of cytokines, such as IL-8, to drugs targeting inflammatory bowel disease and exhibits long-term survival, making it suitable for stable drug screening for treating inflammatory bowel disease.

[0017] The present invention comprises the steps of: producing an inflammatory bowel disease model derived from induced pluripotent stem cells according to the present invention; and

[0018] A step of culturing the inflammatory bowel disease model derived from the above-mentioned induced pluripotent stem cells after treating it with a drug candidate for the prevention or treatment of inflammatory bowel disease; and

[0019] A method for evaluating the efficacy of a drug candidate for the prevention or treatment of inflammatory bowel disease is provided, characterized by comprising: a step of confirming the expression level of IL-8 from the culture supernatant of a cultured induced pluripotent stem cell-derived inflammatory bowel disease model after treatment with the drug candidate;

[0020] The present invention relates to an inflammatory bowel disease model derived from induced pluripotent stem cells and a method for producing the same, which can be usefully used to evaluate the efficacy of drugs for treating inflammatory bowel disease by simulating stable intestinal epithelial cells from induced pluripotent stem cells and significantly showing the expression of inflammation-related genes according to the occurrence and improvement of inflammatory bowel disease.

[0021] Figure 1 is a microscopic photograph showing a timeline for establishing an inflammatory bowel disease model derived from induced pluripotent stem cells according to the present invention and the resulting morphological changes of organoids.

[0022] Figure 2 shows the results of immunofluorescence staining with DAPI after colon organoids cultured only in proliferation medium for 12 days and colon organoids cultured continuously in proliferation medium for 6 days and differentiation medium for 6 days were cultured for an additional 48 hours in medium containing TNF-α (+TNF-α) or without TNF-α (-TNF-α). (Nuclear: DAPI, scale bar: 100 μm)

[0023] Figure 3 shows the results of immunofluorescence staining using antibodies against Ki67, a proliferative cell marker, and Villin, a marker of intestinal epithelial cells, after colon organoids cultured only in proliferation medium for 12 days and colon organoids cultured continuously in proliferation medium for 6 days and differentiation medium for 6 days were cultured for an additional 48 hours in medium containing TNF-α (+TNF-α) or medium not containing TNF-α (-TNF-α). (Nuclear: DAPI, scale bar: 100 μm)

[0024] Figure 4 is an enlarged photograph of the immunofluorescence staining of Ki67 and Villin of the colon organoid of Figure 3. (Nuclear: DAPI, scale bar: 100 μm)

[0025] Figure 5 shows the results of immunofluorescence staining using antibodies against E-cadherin and Mucin-2 after colon organoids cultured only in proliferation medium for 12 days and continuously cultured in proliferation medium for 6 days and differentiation medium for 6 days were cultured for an additional 48 hours in medium containing TNF-α (+TNF-α) or without TNF-α (-TNF-α). (Nuclear: DAPI, scale bar: 100 μm)

[0026] Figure 6 shows the results of immunofluorescence staining using antibodies to Lgr5 and CDX2 after colon organoids cultured only in proliferation medium for 12 days and colon organoids cultured continuously in proliferation medium for 6 days and differentiation medium for 6 days were cultured for an additional 48 hours in medium containing TNF-α (+TNF-α) or without TNF-α (-TNF-α). (Nuclear: DAPI, scale bar: 100 μm)

[0027] Figure 7 shows the transwell culture timetable and microscopic photographs of colon organoids cultured in proliferation medium and differentiation medium.

[0028] Figure 8 shows microscopic and H&E stained images of colon organoids treated with TNF-α during transwell culture in proliferation medium and differentiation medium.

[0029] Figure 9 shows immunofluorescence staining of Villin and Ki67 in colon organoids treated with TNF-α during transwell culture in proliferation medium and differentiation medium. (Nuclear: DAPI, 200x)

[0030] Figure 10 is an immunofluorescence staining photograph of Mucin2 and E-cadherin in colon organoids treated with TNF-α during transwell culture in proliferation medium and differentiation medium. (Nuclear: DAPI, 200x)

[0031] Figure 11 shows immunofluorescence staining of CDX2 and LGR5 in colon organoids treated with TNF-α during transwell culture in proliferation medium and differentiation medium. (Nuclear: DAPI, 200x)

[0032] Figure 12 shows a comparison of gene expression of colon organoids according to culture method in Matrigel-coated culture dishes and Matrigel domes. (Left: Genes upregulated and genes upregulated more than 5-fold in colon organoids cultured in Matrigel-coated culture dishes after 6 days in proliferation medium and 6 days in differentiation medium, and after 6 days in proliferation medium and 4 days in differentiation medium, and after 2 days in culture with TNF-α added to the medium. Center: Genes upregulated / downregulated and genes upregulated more than 5-fold in colon organoids cultured in Matrigel domes or Matrigel-coated culture dishes after 6 days in proliferation medium and 6 days in differentiation medium, and after 2 days in culture with TNF-α added to the medium. Right: Genes upregulated / downregulated and genes upregulated more than 5-fold in colon organoids cultured in Matrigel domes or Matrigel-coated culture dishes after 6 days in proliferation medium and 4 days in differentiation medium, and after 2 days in culture with TNF-α added to the medium.)

[0033] Figure 13 shows a comparison of gene expression of colon organoids according to culture method in Matrigel domes. (Left: Genes upregulated and genes upregulated more than 5-fold in colon organoids cultured for 12 days in proliferation medium in Matrigel domes and 10 days in proliferation medium, and 2 days in culture with TNF-α added to the medium; Center: Genes upregulated / downregulated and genes upregulated more than 5-fold in colon organoids cultured for 12 days in proliferation medium in Matrigel domes and 6 days in proliferation medium and 6 days in differentiation medium; Right: Genes upregulated / downregulated and genes upregulated more than 5-fold in colon organoids cultured for 6 days in proliferation medium and 6 days in differentiation medium in Matrigel domes and 6 days in proliferation medium and 4 days in differentiation medium, and 2 days in culture with TNF-α added to the medium.)

[0034] Figure 14 is a graph showing the change in the expression of the IL-8 gene (CXCL8) according to the addition of TNF-α after 12 days of culture in proliferation medium only, 6 days in proliferation medium, and 4 days in differentiation medium.

[0035] Figure 15 is a timeline for the production of colon organoid proliferation models, differentiation models, and their TNF-α-induced inflammatory bowel disease models.

[0036] Figure 16 is a graph showing the secretion of IL-8 by each drug treatment in a TNF-α-induced inflammatory bowel disease model of a colon organoid proliferation model.

[0037] Figure 17 is a graph showing the secretion of IL-8 by each drug treatment in a TNF-α-induced inflammatory bowel disease model of a colon organoid differentiation model.

[0038] To induce inflammation in colon organoids derived from conventional induced pluripotent stem cells (iPSCs), tumor necrosis factor (TNF)-α is used to mimic inflammatory bowel disease (IBD). However, conventional TNF-α-treated colon organoids have a short survival time and low inflammation-inducing efficiency, making them unsuitable for drug screening.

[0039] In the process of studying colon organoids that mimic inflammatory bowel disease (IBD), the inventors have effectively constructed a TNF-α-based inflammatory bowel disease (IBD) model through two-step culture using proliferation media and differentiation media, and have developed an evaluation method that can clearly confirm the efficacy of drugs for inflammatory bowel disease using the same, thereby completing the present invention.

[0040] Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The contents introduced herein are provided to sufficiently convey the spirit of the present invention.

[0041]

[0042] <Example 1. Production of an inflammatory bowel disease model derived from induced pluripotent stem cells>

[0043] Preparation of expansion media

[0044] To establish an inflammatory bowel disease model derived from induced pluripotent stem cells of the present invention, a proliferation medium was prepared and used. The proliferation medium was prepared by mixing basal media and conditioned media in a 1:1 (v / v) ratio, followed by the addition of growth factors.

[0045] The basic medium was composed as shown in Table 1 below, and the growth factors were as shown in Table 2.

[0046] Reagent stockStock Conc.VolumeFinal Conc.Advanced DMEM / F121X500 ml1 ml1mMNicotinamide2.5M2ml10mMTotal volume528ml

[0047] Reagent stockStock Conc.VolumeFinal Conc.GastrinⅠ10 μM50 μl10 nMA-83-015 mM5 μl500 nMEGF50 μg / ml50 μl50 ng / mlPrimocin50 mg / ml100 μl100 μg / mlTotal volume205 μl

[0048] Conditioned medium was prepared by collecting the supernatant from L-WRN cells grown in a T75 flask, filtering it, and using it as a medium. First, L-WRN cells were cultured in DMEM medium (10% FBS, 1% P / S) for 3–4 days until the culture dish was full. The existing medium was removed, and the harvest medium (Harvest media) in Table 3 below was added and cultured for an additional 24 hours. After 24 hours, the medium was collected, filtered using a 0.22 μm filter, and used as a conditioned medium.

[0049] ReagentStock Conc.VolumeFinal Conc.Advanced DMEM F / 121

[0050]

[0051] Preparation of differentiation media

[0052] To establish an inflammatory bowel disease model derived from induced pluripotent stem cells of the present invention, the colon organoid cells were cultured in a differentiation medium to induce differentiation into epithelial cells. The differentiation medium was composed of the basic medium of Table 1, plus A83-01, EGF, and primocin, as shown in Table 4 below.

[0053] Reagent stockStock Conc.VolumeFinal Conc.Advanced DMEM / F121X50 ml1 mM100 μl1 mMNicotinamide2.5 M200 μl10 mMA-83-015 mM5 μl500 nMEGF50 μg / ml50 μl50 ng / mlPrimocin50 mg / ml100 μl100 μg / mlTotal volume52.95 ml

[0054]

[0055] The culture medium and differentiation medium for culturing colon organoids derived from induced pluripotent stem cells prepared as described above were dispensed in 40 ml portions and stored in an ultra-low temperature freezer before use during culture.

[0056]

[0057] Proliferation and culture of colon organoids derived from induced pluripotent stem cells

[0058] To establish the inflammatory bowel disease model derived from induced pluripotent stem cells of the present invention, 3dGRO™ Human iPSC Derived Colon Organoids (Millipore, #SCC300), which are autologous 3D structures capable of differentiating into intestinal epithelial cell types, were used. The colon organoids were removed from liquid nitrogen, thawed in a water bath for approximately 30 seconds to 1 minute, transferred to a pre-prepared 1.5 ml tube, and centrifuged at 8,000 rpm for 10 seconds. The separated colon organoid pellet was then placed in 1 ml of colon organoid culture medium (3dGRO™ Human Colon Organoid Expansion Medium, Millipore, #SCM304), pipetted 2-3 times, and centrifuged at 8,000 rpm for 10 seconds. After centrifugation, the culture medium was removed, the tube containing the colon organoid pellet was placed deep on ice, Matrigel (Matrigel Growth Factor reduced (GFR) Basement Membrane Matrix, Corning, #356231) was added, and then homogenized by pipetting.

[0059] The above colon organoid and GFR Matrigel mixture was dispensed into a 48-well culture dish at 20 μl per well and stabilized in a 37°C, 5% CO2 cell incubator for 10 minutes. After confirming the formation of a stabilized dome, 200 μl of the above proliferation medium was added to each well and cultured in a 37°C, 5% CO2 cell incubator.

[0060] When the Matrigel dome in each well of a 48-well culture dish was 70-90% confluent with colonic organoids, subculture was performed at a ratio of 1:3-4. First, the medium was carefully removed from each well using a pipette, and then 500 μl of gentle cell dissociation reagent (GCDR, StemcellTechnology, #100-0485) was added to each well. Then, the dome was broken by pipetting 10-15 times using a 1 ml pipette to separate the colonic organoids from the Matrigel dome. The separated colonic organoids were then transferred to a 15 ml tube and gently shaken for 15 minutes at room temperature to induce the separation reaction. The separated organoids were centrifuged at 1,100 rpm at 4°C for 5 minutes, and the supernatant was removed. After that, 1 ml TrypLE (Thermo, #12604-013) was added to a 15 ml tube and pipetted 2-3 times with a 1 ml pipette, and then the entire amount was transferred to a 1.5 ml tube, and the separation reaction was further induced for 2-3 minutes. After that, the disaggregated organoids were centrifuged at 8,000 rpm for 10 seconds to remove TrypLE and residual Matrigel in the supernatant. After that, the remaining organoid pellet was washed with 200 μl colon organoid culture medium and centrifuged at 8,000 rpm for 10 seconds to remove all residues except the organoid pellet. The tube containing only the organoid pellet was deeply fixed on ice, and the desired amount of GFR Matrigel (Corning, #356231) was added and pipetted for homogenization. At this time, 20 μl of GFR Matrigel was used per dome of a 48-well culture dish. Afterwards, the GFR Matrigel and colon organoid mixture was dispensed into a 48-well culture dish at 20 μl per well, and gelation was induced for 10 minutes in a 37°C, 5% CO2 cell incubator.After confirming the formation of a stabilized dome within the culture dish under a microscope, 200 μl of colon organoid culture medium containing 10 μM Y-27632 was added to each well and cultured overnight in a 37°C, 5% CO2 cell incubator. After subculture, the medium was replaced with 200 μl of proliferation medium containing 10 μM Y-27632 (ROCK Inhibitor, Millipore, #SCM075) for two days. From the third day of culture, the medium was replaced with 400 μl of expansion medium without Y-27632 every other day. Subculture was performed once every 6 to 7 days thereafter, and organoid morphological changes, viability, and contamination levels were evaluated under a microscope.

[0061]

[0062] Colon epithelial cell differentiation culture of colon organoids

[0063] The colon organoids grown to the desired number in the above 48-well culture dish were cultured and passaged as described above. When the Matrigel dome was 70-90% confluent with colon organoids, the dome was removed as described above, and the tube containing only the pellet was deeply fixed on ice. After adding GFR Matrigel and homogenizing by pipetting, 5-10 μl was dispensed per well into a 96-well culture dish and gelation was induced for 10 minutes in a 37°C, 5% CO2 incubator. After confirming that a stabilized dome had formed, 200 μl of colon organoid culture medium (containing 10 μM Y-27632) was added to each well and cultured overnight in a 37°C, 5% CO2 incubator. After subculture, 100 μl of the proliferation medium containing 10 μM Y-27632 was replaced daily for two days, and from the third day, 200 μl of fresh proliferation medium without Y-27632 was replaced every other day. After proliferation in this way for 5–6 days, the colon organoid culture medium was replaced with the differentiation medium described above. By replacing the medium with 200 μl of differentiation medium once every two days, the colon organoids were differentiated into intestinal epithelial cells.

[0064] Afterwards, the cells were cultured for 4 to 5 days after replacing them with intestinal epithelial cell differentiation medium, and when they were approximately 70% confluent with intestinal epithelial cells, a TNF-α-based inflammatory bowel disease model was created using the same procedure as the colon organoid proliferation model.

[0065]

[0066] TNF-α-based inflammatory bowel disease model

[0067] An inflammatory bowel disease model was created from the colon organoids of the colon organoid proliferation model or the intestinal epithelial cell differentiation model derived from the above-mentioned induced pluripotent stem cells.

[0068] First, TNF-α prepared at 1000 ng / μl was diluted with expansion medium or differentiation medium to prepare a final concentration of 200 ng / ml.

[0069] The culture medium was removed from the 96-well culture dish in which the colon organoids of the colon organoid proliferation model or the intestinal epithelial cell differentiation model obtained above were cultured to prevent the dome from breaking. Then, 200 μl of the expansion medium or differentiation medium supplemented with TNF-α diluted to 200 ng / ml was dispensed into each colon organoid of the colon organoid proliferation model or the intestinal epithelial cell differentiation model, and cultured in a 37°C, 5% CO2 cell incubator for 48 hours to induce an inflammatory response of the organoids to TNF-α. Thereafter, 120 μl of the 200 μl medium was aliquoted and used to evaluate the cytokine secretion capacity to confirm the inflammatory response or stored in an ultra-low temperature freezer.

[0070] Viability was measured by adding 20 μl of CellTiter-Glo® 3D Cell Viability Assay reagent (Promega, #G9681) to the remaining 80 μl medium after aliquoting.

[0071]

[0072] <Example 2. Characteristics of an inflammatory bowel disease model derived from induced pluripotent stem cells>

[0073] Viability and morphology confirmation

[0074] The viability and morphology of the induced pluripotent stem cell-derived inflammatory bowel disease model constructed above were observed. First, microscopic images of colon organoids cultured for 12 days in the above-mentioned expansion medium, colon organoids continuously cultured for 6 days in the expansion medium and 6 days in the differentiation medium, and colon organoids cultured for 6 days in the expansion medium and 4 days in the differentiation medium, and then continuously cultured for 48 and 72 hours in the TNF-α-supplemented medium are shown in Fig. 1. As shown in Fig. 1, colon organoids cultured only in the expansion medium for 12 days proliferated into round-shaped colon organoids, and some morphologically unstable organoids (arrows) were observed. In addition, colon organoids continuously cultured for 6 days in the expansion medium and 6 days in the differentiation medium differentiated into intestinal epithelial cells. After culturing for 6 days in proliferation medium and 4 days in differentiation medium, colon organoids cultured in TNF-α-supplemented medium progressed to inflammatory colitis, exhibiting marked morphological changes, and viability evaluation revealed almost no cell death. In particular, in the case of colon organoids cultured in TNF-α-supplemented medium for 72 hours, despite a severe inflammatory response, organoids were confirmed to maintain their morphology with minimal cell death.

[0075]

[0076] Confirmation of immunofluorescence staining

[0077] The colon organoids cultured only in the proliferation medium for 12 days and the colon organoids cultured continuously in the proliferation medium for 6 days and in the differentiation medium for 6 days were cultured for an additional 48 hours in a medium containing (+TNF-α) or not containing (-TNF-α) TNF-α, and the cell nuclei were stained with DAPI, and the results are shown in Fig. 2. Briefly, the dome-shaped organoids were fixed with formalin for 15 minutes, stored in a refrigerator in a 10% sucrose solution for 1 day, in a 20% sucrose solution for 1 day, and in a 30% sucrose solution until they sank, and then molds were made using an OCT (Optimal cutting temperature) compound, and specimens with a thickness of 8 μm were made using a cryosectioner. Afterwards, the slides were washed in PBS to remove OCT, blocked with serum, treated with primary antibodies, washed, treated with secondary antibodies, and stained for nuclei with DAPI. In Fig. 2, the upper left shows colon organoids cultured for 8 days in proliferation medium, and the middle left shows colon organoids cultured for 2 days in proliferation medium after 6 days in proliferation medium and TNF-α treatment. No morphological changes due to TNF-α were observed in the organoids. The lower left shows organoids cultured for 6 days in proliferation medium and then in differentiation medium. The shape of the nuclei was consistent and no apoptotic bodies were observed. In other words, it was confirmed that morphological changes were observed in the differentiation medium, but cell death did not occur. Meanwhile, the photo on the right shows organoids cultured for 2 days after being treated with TNF-α in differentiation medium for 6 days in proliferation medium and 4 days in differentiation medium. Although there are parts where the shape of the nucleus is consistent, morphological changes are observed to form apoptotic bodies, confirming that the organoid cells are alive even though inflammatory colitis is induced.

[0078] In the same manner as above, colon organoids cultured only in proliferation medium for 12 days and colon organoids continuously cultured in proliferation medium for 6 days and differentiation medium for 6 days were cultured for an additional 48 hours in medium containing (+TNF-α) or not containing (-TNF-α) TNF-α, and then immunofluorescent staining was performed using antibodies against the proliferation cell marker Ki67 and the intestinal epithelial cell marker Villin, and the results are shown in Fig. 3. As shown in Fig. 3, colon organoids cultured only in proliferation medium were confirmed to have a high level of Ki67, and colon organoids continuously cultured in differentiation medium showed a significant increase in the expression of the intestinal epithelial cell marker Villin. In addition, when inflammation was induced by TNF-α, the integrity of the colon organoids was found to be damaged. An enlarged view of Fig. 3 is shown in Fig. 4. When the junctions between cells become loose, the reactivity of cells to external factors may increase, which may result in an increased response to inflammatory colitis. As shown in the right picture of Fig. 4, it was confirmed that the distance between cells was constant in colon organoids cultured only in proliferation medium regardless of the presence or absence of TNF-α treatment. Therefore, it was found that TNF-α treatment in proliferation medium was difficult to induce inflammatory colitis. However, in the case of colon organoids cultured in proliferation medium and then continuously cultured in differentiation medium, the expression of the intestinal epithelial cell marker Villin significantly increased, and when inflammation was induced by TNF-α, nuclear material leaked out from inside the organoid, damaging the integrity of the colon organoid. In other words, when colon organoids were cultured in proliferation medium and then cultured in differentiation medium, the reaction of the epithelial cell differentiation marker villin became more dominant when treated with TNF-α, and the distance between nuclei increased, confirming that inflammatory colitis was effectively induced.This means that when tight junction proteins (arrows) are maintained in normal intestinal epithelial cells, the influx of external invaders such as bacteria is effectively blocked, whereas when tight junctions are dysfunctional due to inflammation, the integrity of intestinal epithelial cells is broken and external substances invade the organoid.

[0079] In addition, the expression levels of intestinal epithelial cell-specific proteins E-cadherin and Mucin-2 in colon organoids cultured in the same manner as above were stained with immunofluorescence using the same method as above, and are shown in Fig. 5. As shown in Fig. 5, the expression levels of E-cadherin and Mucin-2 were confirmed to be higher in colon organoids continuously cultured in differentiation medium than in colon organoids cultured only in proliferation medium, and when inflammation was induced by TNF-α, the integrity of colon organoids was found to be damaged.

[0080] Similarly, the expression levels of Lgr5, a marker of intestinal stem cells, and CDX2, a marker of intestinal differentiation, in colon organoids cultured in the same manner as above were stained with immunofluorescence using the same method as above, and are shown in Fig. 6. As shown in Fig. 6, the expression of Lgr5 and CDX2 was found to be high in colon organoids cultured only in proliferation medium. In addition, when inflammation was induced by TNF-α, the integrity of colon organoids was found to be damaged.

[0081]

[0082] <Example 3. Transwell culture of colon organoids>

[0083] Transwell culture is a culture method that can mimic an organ by adding a membrane insert for cell attachment and growth to a culture dish and co-culturing various types of cells centered on the membrane. The colon organoids were placed in a dissociation solution containing papain (30 unit / mL) and DNase I (125 unit / mL) in HBSS, and then shaken at 90 rpm for 30 minutes at 37°C to dissociate them into single cells. After seeding in a transwell, they were cultured in a proliferation medium for 6 days and in a differentiation medium for 4 days, and then TNF-α was added to induce inflammatory bowel disease, and the results are shown in Figure 7. As shown in Figure 7, when colon organoids were cultured in a transwell, they formed colonies and grew in both the proliferation medium and the differentiation medium, but there was a problem that the number of cells did not grow consistently, and apoptotic cells increased rapidly from the 9th day when the culture dish was full of cells. Also, as shown in Fig. 8, the morphology was not consistent due to the addition of TNF-α. In particular, after culture in differentiation medium, H&E staining showed differentiation into intestinal epithelial cells, but areas where cells were irregularly hypertrophied along the medium surface were observed, making it impossible to obtain a consistent differentiation model.

[0084] The colon organoids cultured in the above transwell were subjected to immunofluorescence staining of Villin and Ki67, Mucin2 and E-cadherin, CDX2 and LGR5 in the same manner as in the above example, and the results are shown in Figs. 9 to 11. As shown in Fig. 9, KI67 (green), a proliferative cell marker, and Villin, an intestinal epithelial cell marker, were confirmed in the colon organoids cultured in the transwell, but the expression ratio of KI67 was low compared to Villin expression, confirming that differentiation was not sufficiently achieved. There was no significant difference in the expression of mucin and cadherin and CDX and LGR5 in Figs. 10 and 11.

[0085] The above results show that 2D transwell culture was not suitable as a stable intestinal disease model because it had problems with inconsistent and insufficient differentiation and rapid cell death when the well was full.

[0086] Through the above experiment, it was confirmed that in the case of 2D culture or 2D transwell culture, it takes 10 to 12 days to reach 100% confluency, and cell death progresses rapidly around 100% confluency and the monolayer floats, whereas the inflammatory bowel disease model derived from induced pluripotent stem cells according to the present invention maintains stem cell characteristics and takes 6 to 7 days to grow to 400 μm, and can be cultured for up to a total of 21 days, so it can be usefully used as an inflammatory bowel disease model.

[0087]

[0088] <Example 4. Gene expression analysis of an inflammatory bowel disease model derived from induced pluripotent stem cells>

[0089] Comparison of induced inflammatory bowel disease models derived from induced pluripotent stem cells in Matrigel domes and Matrigel-coated culture dishes.

[0090] The culture method of the inflammatory bowel disease model derived from the induced pluripotent stem cells produced above and the changes in gene expression according to TNF-α treatment were confirmed.

[0091] First, colon organoids cultured for 6 days in proliferation medium and 6 days in differentiation medium using Matrigel-coated culture dishes, and colon organoids cultured for 6 days in proliferation medium and 4 days in differentiation medium using Matrigel-coated culture dishes, and then cultured for 2 days in TNF-α-supplemented medium were harvested, and RNA was extracted using RNeasy Mini Prep Kit (Qiagen), and the amount of RNA isolated from the organoids was measured using a NanoDrop 2000 spectrophotometer. Afterwards, cDNA was reverse-transcribed from the isolated RNA and subjected to RT. 2mRNA analysis of 81 inflammatory cytokines and receptors, excluding 5 housekeeping genes, was performed using Profiler PCR Array (#PAHS-169Z; Qiagen). Increases and decreases were identified based on a 2-fold or greater change according to the manufacturer's manual.

[0092] As shown on the left side of Figure 12, in the case of colonoids cultured for 6 days in proliferation medium, 4 days in differentiation medium, and 2 days in TNF-α-supplemented medium using culture dishes coated with Matrigel for 2D culture, the expression of 61 genes related to inflammatory factors was upregulated by more than 2-fold compared to colonoids cultured for 6 days in proliferation medium, 6 days in differentiation medium using culture dishes coated with Matrigel, and among these, the expression of 7 genes, ABCB1, ALDOB, C4BPB, IL1RN, LCN2, PCK1, and SAA1, increased by more than 5-fold.

[0093] In addition, to compare the expression of inflammation-related genes during 2D and 3D culture without TNF-α treatment, gene expression of colon organoids on Matrigel domes and Matrigel coating was compared. The gene expression of colon organoids cultured in proliferation medium for 6 days and differentiation medium for 6 days on Matrigel domes and colon organoids cultured in proliferation medium for 6 days and differentiation medium for 6 days on Matrigel-coated culture dishes was compared using the same method as above. As a result, as shown in the center of Fig. 12, compared to colon organoids cultured on Matrigel domes, the expression of inflammation-related genes in colon organoids cultured on Matrigel-coated culture dishes was upregulated by more than 2-fold in 67 genes, and 3 genes were downregulated. Among these, 17 genes, CASP1, CCL2, CH3L1, CXCL2, DEFA5, EGR3, FPR1, HLA-DQA1, IL17A, IL2RA, LTB, MMP3, MMP7, S100A9, TDO2, and UBD, were upregulated more than 5-fold, and the C3 gene was downregulated more than 5-fold. In other words, gene expression was found to be predominant under 2D culture conditions.

[0094] In addition, the gene expression of colon organoids by the addition of TNF-α during 2D and 3D culture was compared. Colonoids cultured on Matrigel domes in proliferation medium for 6 days, differentiation medium for 4 days, and then cultured in TNF-α-supplemented medium for 2 days, and colonoids cultured on Matrigel-coated culture dishes in proliferation medium for 6 days, differentiation medium for 4 days, and then cultured in TNF-α-supplemented medium for 2 days were compared using the same method as above. As shown in the right side of Fig. 12, the gene expression of colon organoids cultured on Matrigel-coated dishes was up-regulated in 2 genes and down-regulated in 6 genes compared to the gene expression of colonoids cultured on Matrigel domes in proliferation medium for 6 days, differentiation medium for 4 days, and then cultured in TNF-α-supplemented medium for 2 days. Among these, it was observed that the expression of the IL5 gene was downregulated by more than 5-fold. That is, 62 genes were relatively downregulated when inflammatory colitis was induced through TNF-α on the 2D phase.

[0095] Through the above results, it was confirmed that in the inflammatory bowel disease model derived from induced pluripotent stem cells, when the colon organoids derived from induced pluripotent stem cells were cultured on a culture dish coated with Matrigel, the inflammatory colitis-inducing genes were down-regulated when TNF-α was added, which has limitations as an inflammatory bowel disease model.

[0096]

[0097] Comparison of gene expression across culture stages of induced pluripotent stem cell-derived inflammatory bowel disease models.

[0098] Based on the results confirmed above, gene expression at each culture stage of a model of inflammatory bowel disease derived from dedifferentiated stem cells on a Matrigel dome was compared.

[0099] First, we examined the gene expression of colonoids cultured for 12 days in a growth medium with Matrigel domes and colonoids cultured for 10 days in a growth medium with Matrigel domes and then cultured for 2 days in a medium supplemented with TNF-α. As shown on the left side of Fig. 13, 53 genes were confirmed to be up-regulated more than 2-fold in colonoids cultured for 10 days in a growth medium with Matrigel domes and then cultured for 2 days in a medium supplemented with TNF-α compared to colonoids cultured only in a growth medium for 12 days. Among them, 16 genes, C3, CCL2, CCL20, CX3CL1, CXCL10, CXCL11, CXCL2, CXCL3, IL23A, CXCL8, LCN2, LTB, MMP10, TNF, and UBD, were up-regulated more than 5-fold.

[0100] In the same way, the gene expression of colonoids cultured in proliferation medium for 6 days and differentiation medium for 6 days was normalized to the gene expression of colonoids cultured in proliferation medium for 12 days, and the results are shown in the center of Fig. 13. As seen in the center of Fig. 13, in the colonoids cultured in proliferation medium for 6 days and differentiation medium for 6 days, the expression of 56 genes was upregulated more than 2-fold compared to the colon organoids cultured in proliferation medium for 12 days, and among these, the expression of 7 genes, ABCB1, ALDOB, C3, C4BPB, LCN2, PCK1, and SAA1, increased more than 5-fold.

[0101] Also, in the same way, gene expression in colon organoids cultured in proliferation medium for 6 days, differentiation medium for 4 days, and then in TNF-α-supplemented medium for 2 days was normalized to the gene expression in colon organoids cultured in proliferation medium for 6 days, and differentiation medium for 6 days, and the results are shown on the right side of Fig. 13. As shown on the right side of Fig. 13, gene expression in colon organoids cultured in proliferation medium for 6 days, differentiation medium for 4 days, and then in TNF-α-supplemented medium for 2 days was upregulated in 80 genes by more than 2-fold compared to the gene expression in colon organoids cultured in proliferation medium for 6 days, and differentiation medium for 6 days. Of these, 65 genes showed more than 10-fold increase in expression compared to the gene expression in colon organoids cultured in proliferation medium for 6 days, and differentiation medium for 6 days.

[0102] Through these results, it was confirmed that a suitable inflammatory bowel disease model could be established by culturing in a TNF-α-supplemented medium after step-by-step culture in a proliferation medium and a differentiation medium using a Matrigel dome, and then culturing in a TNF-α-supplemented medium significantly promoted proliferation, differentiation, and inflammation induction.

[0103] As a result, when colon organoids were cultured in proliferation medium and differentiation medium and inflammation was induced by adding TNF-α, the expression of the CXCL8 gene, one of the inflammation-related genes whose gene expression was significantly upregulated, was measured according to each culture condition and is shown in Fig. 14. As shown in Fig. 14, after culturing for 6 days in proliferation medium and 4 days in differentiation medium, the inflammatory bowel disease model of the present invention with the addition of TNF-α showed a significant increase in the expression of the CXCL8 gene, i.e., the expression of IL-8, indicating that it is suitable for modeling inflammatory bowel disease.

[0104] <Example 5. Confirmation of drug effects in an inflammatory bowel disease model>

[0105] The efficacy of candidate drugs for the treatment of inflammatory bowel disease was evaluated using the inflammatory bowel disease model established above.

[0106] As shown in Fig. 15, a colon organoid proliferation model, a differentiation model, and a TNF-α-induced inflammatory bowel disease model were constructed. The proliferation model was cultured only in the proliferation medium for 12 days, and the differentiation model was cultured in the proliferation medium for 6 days and in the differentiation medium for 4 days. TNF-α was added to each of these to induce inflammation, and the drugs in Table 5 below were administered together with TNF-α and cultured. After 12 days of culture, colon organoids and supernatants were collected, and the concentration of IL-8 from the supernatants was measured using ELISA analysis (R&D system) according to the manufacturer's manual, and the results are shown in Figs. 16 and 17.

[0107]

[0108] As shown in Figures 16 and 17, when each drug was treated in the colon organoid proliferation model (Expansion model), only infliximab (INF) and adalimumab (ADA) showed a significant decrease in the concentration of IL-8, but when each drug was treated in the colon organoid differentiation model (Differentiation model), the concentration of IL-8 was shown to be significantly decreased in SP01, SP02, SP03, ciprofloxacin (CIP), sulfasalazine (SSZ), dexamethasone (DEX), prednisolone (Pre), infliximab (INF), and adalimumab (ADA).

[0109] Through this, it was confirmed that the inflammatory bowel disease model of the colon organoid according to the present invention more accurately simulates high-grade inflammatory bowel disease and is useful for drug screening.

Claims

1. Step 1: Culturing and expanding colon organoids derived from induced pluripotent stem cells in expansion media for 5 to 7 days; Step 2: Transferring the colon organoids derived from the induced pluripotent stem cells proliferated in step 1 to differentiation media and culturing them for 3 to 4 days to differentiate them into intestinal epithelial cells; and A method for producing an inflammatory bowel disease model derived from induced differentiation stem cells, characterized by comprising: a step (step 3) of inducing inflammation by culturing colon organoids differentiated into intestinal epithelial cells in the above step 2 in a differentiation medium containing TNF-α for 1 to 3 days.

2. In paragraph 1, A method for producing an inflammatory bowel disease model derived from induced pluripotent stem cells, characterized in that the above expansion media contains DMEM / F12, Penicillin / streptomycin, N-Acetyl-L-cysteine, Nicotinamide, Glutamax, N-2, B-27, L-WRN culture harvest medium, A-83-1, EGF and Primosin.

3. In paragraph 1, A method for producing an inflammatory bowel disease model derived from induced pluripotent stem cells, characterized in that the differentiation medium comprises DMEM / F12, Penicillin / streptomycin, N-Acetyl-L-cysteine, Nicotinamide, Glutamax, N-2, B-27, A-83-1, EGF and Primosin.

4. In paragraph 1, A method for producing an inflammatory bowel disease model derived from induced pluripotent stem cells, characterized in that in the above step 1, colon organoids derived from induced pluripotent stem cells proliferate to a maximum of 400 μm after 5 to 7 days of culture.

5. An inflammatory bowel disease model derived from dedifferentiated stem cells manufactured by the method for manufacturing an inflammatory bowel disease model derived from dedifferentiated stem cells of clauses 1 to 4.

6. In paragraph 5, An inflammatory bowel disease model derived from induced pluripotent stem cells, characterized in that the inflammatory bowel disease model derived from induced pluripotent stem cells survives for up to 21 days.

7. A drug screening system for treating inflammatory bowel disease comprising an inflammatory bowel disease model derived from induced pluripotent stem cells of clause 5.

8. A step for producing an inflammatory bowel disease model derived from dedifferentiated stem cells of clause 5; and A step of culturing the inflammatory bowel disease model derived from the above-mentioned induced pluripotent stem cells after treating it with a drug candidate for the prevention or treatment of inflammatory bowel disease; and A method for evaluating the efficacy of a drug candidate for the prevention or treatment of inflammatory bowel disease, characterized by comprising: a step of confirming the expression level of IL-8 from the culture supernatant of a cultured induced pluripotent stem cell-derived inflammatory bowel disease model after treatment with the drug candidate;

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