Method for establishing and culturing organoid

Abstract

Provided is a method for establishing and culturing an organoid, a composition used for establishing and culturing an organoid, and a culture medium. The composition comprises a histone deacetylase (HDAC) inhibitor, vitamin C and a derivative thereof, and a platelet-derived growth factor receptor (PDGFR) inhibitor.

Description

Methods for establishing and culturing organoids Technical Field

[0001] This application relates to the field of biomedicine, specifically to a composition and method for organoid culture. Background Technology

[0002] Adult stem cell (ASC)-derived organoids are generated by mimicking the complex processes of tissue development, homeostasis, and regeneration in vitro. Their exceptional ability to reproduce tissue structure, cellular composition, and function makes them a highly attractive platform for studying in vitro development and disease. Despite significant efforts, previous attempts to culture ASC-derived organoids to replicate the complex and dynamic processes occurring in vivo have encountered considerable challenges. Traditional organoid culture systems for many tissues are optimized for maintaining stem cell self-renewal and expansion, resulting in low functional cellular diversity due to cells remaining in an undifferentiated state. Conversely, attempts to promote differentiation and maturation often lead to increased cellular diversity but cause organoids to cease proliferation or experience decreased proliferative capacity. This problem exists for organoids from organs of the liver, pancreas, lung, and other tissues, for example. Therefore, typical organoid culture systems require separate expansion and differentiation steps, hindering their scalability and practicality in large-scale, high-throughput screening.

[0003] Stem cells are capable of self-renewal and differentiation. Some tissues and organs in the body can maintain proliferation while simultaneously producing various differentiated functional cell types. Maintaining a balance between the self-renewal and differentiation of tissue stem cells is essential in this process. However, in homogeneous culture systems for adult stem cell-derived organoids, achieving this balance is difficult due to the lack of a signal gradient similar to the in vivo microenvironment. Summary of the Invention

[0004] This application develops an optimized culture condition that captures the delicate balance between cell self-renewal and differentiation, thereby achieving greater cell diversity, higher cell proliferation rate, higher organoid formation efficiency, and greater scalability in a single culture system.

[0005] By enhancing the stemness of stem cells or precursor cells in organoids, their differentiation potential can be increased, thereby increasing cell diversity in organoids without the need to establish artificial spatiotemporal signal gradients. Furthermore, by modulating microenvironmental and intracellular signals in organoids, the dynamic regulation of cell fate observed in vivo can be reproduced in organoid systems, which will help achieve a balance between stem cell self-renewal and multi-lineage differentiation in organoids. A combination of small molecule pathway regulators can achieve a balance between stem cell self-renewal and multi-lineage differentiation in organoids, and further promote a controllable shift of this balance towards enhanced self-renewal or enhanced directed differentiation. The compositions or culture media of this application, when used to culture cells, can yield novel organoids with both strong proliferative capacity and cell diversity, achieving a balance between self-renewal and differentiation. In addition, this application also provides a novel organoid that simultaneously possesses strong proliferative capacity and cell diversity.

[0006] On one hand, this application provides a composition comprising a histone deacetylase (HDAC) inhibitor, vitamin C and its derivatives, and a platelet-derived growth factor receptor (PDGFR) inhibitor.

[0007] In some implementations, the HDAC inhibitors include pan-HDAC inhibitors and selective HDAC inhibitors.

[0008] In some implementations, the concentration of the HDAC inhibitor used is about 1 nM to about 50 mM.

[0009] In some embodiments, the HDAC inhibitor includes trichostatin A, valproic acid (VPA), CAY10683, tubastatin A, and any combination thereof.

[0010] In some embodiments, the composition contains: an HDAC inhibitor of nystatin A at a concentration of about 1 to about 2000 nM; an HDAC inhibitor of valproic acid at a concentration of about 0.01 to about 50 mM; an HDAC inhibitor of CAY10683 at a concentration of about 0.05 to about 50 μM; an HDAC inhibitor of LMK235 at a concentration of about 5 to about 5000 nM; or an HDAC inhibitor of Tubastatin A at a concentration of about 0.05 to about 50 μM.

[0011] On the other hand, this application provides a composition comprising a nucleosome remodeling and deacetylase (NuRD) complex inhibitor, vitamin C and its derivatives, and a platelet-derived growth factor receptor (PDGFR) inhibitor.

[0012] In some embodiments, the NuRD complex inhibitor includes histone deacetylase 1 / 2 (HDAC1 / 2) inhibitors and / or methylated CpG binding protein 2 / 3 (MBD2 / 3) inhibitors.

[0013] In some embodiments, the HDAC1 / 2 inhibitor includes trichostatin A. In some embodiments, the concentration of trichostatin A is about 1 to about 2000 nM.

[0014] In some embodiments, the MBD2 / 3 inhibitor includes KCC-07. In some embodiments, the concentration of KCC-07 is from about 0.1 to about 100 μM.

[0015] In some embodiments, the concentration of the PDGFR inhibitor used is from about 0.05 μM to about 50 μM. In some embodiments, the PDGFR inhibitor includes CP673451 and / or crenolanib.

[0016] In some embodiments, the composition contains: CP673451 as a PDGFR inhibitor at a concentration of about 0.05 to about 50 μM; or crenolanib as a PDGFR inhibitor at a concentration of about 0.05 to about 50 μM.

[0017] In some embodiments, the concentration of vitamin C and its derivatives used is from about 10 μg / mL to about 1000 μg / mL.

[0018] In some embodiments, vitamin C and its derivatives include L-ascorbic acid-2-phosphate. In some embodiments, vitamin C and its derivatives are L-ascorbic acid-2-phosphate at a concentration of about 10 to about 1000 μg / mL.

[0019] In some embodiments, the composition comprises nystatin A, L-ascorbic acid-2-phosphate and CP673451.

[0020] In some embodiments, the concentration of nystatin A is about 1 to about 2000 nM, the concentration of L-ascorbic acid-2-phosphate is 10 to 1000 μg / mL, and the concentration of CP673451 is about 0.05 to about 50 μM.

[0021] In some embodiments, the composition further comprises a BET inhibitor. In some embodiments, the BET inhibitor comprises iBET-151.

[0022] In some embodiments, the BET inhibitor is iBET-151, at a concentration of about 0.01 to about 50 μM.

[0023] In some embodiments, the composition further comprises one or more components from the group consisting of: Wnt signaling activator, BMP signaling inhibitor, EGF activator, FGF activator, IGF activator, TGFβ inhibitor, and gastrin.

[0024] On the other hand, this application provides a culture medium containing the composition of this application.

[0025] In some implementations, the culture medium further comprises basal culture medium components.

[0026] In some implementations, the culture medium is used to culture cells, preserve tissues, culture tissues, prepare organoids, and / or culture organoids.

[0027] In some embodiments, the cells include stem cells. In some embodiments, the cells include Lgr5+ stem cells. In some embodiments, the cells include epithelial stem cells. In some embodiments, the cells include adult stem cells. In some embodiments, the cells include digestive system stem cells. In some embodiments, the tissue includes digestive system tissue. In some embodiments, the cells or tissue are derived from mammals. In some embodiments, the cells or tissue are derived from humans or mice. In some embodiments, the tissue includes stomach and / or intestinal tissue.

[0028] On the other hand, this application provides a method for cell culture, tissue preservation, tissue culture, preparation of organoids and / or culture of organoids, which includes using the composition of this application and / or the culture medium of this application.

[0029] On the other hand, this application provides a method for preparing and / or culturing organoids, comprising the following steps:

[0030] a) Obtaining stem cells; and

[0031] b) Culturing the stem cells obtained in step a) in the presence of the composition of this application or using the culture medium of this application, thereby obtaining the organoid.

[0032] In some embodiments, the stem cells include Lgr5+ stem cells. In some embodiments, the stem cells include epithelial stem cells. In some embodiments, the stem cells include adult stem cells. In some embodiments, the stem cells include digestive system stem cells. In some embodiments, the stem cells include mammalian stem cells. In some embodiments, the stem cells include human or mouse stem cells. In some embodiments, the method of preparing and / or culturing organoids further includes the step of placing the stem cells obtained in step a) on a cell support, embedding them in the cell support, or mixing them with the cell support.

[0033] In some embodiments, the method for preparing and / or culturing organoids further includes the step of inducing the organoids to generate functional cells.

[0034] In some implementations, stem cells include intestinal stem cells, and organoids include intestinal organoids.

[0035] In some embodiments, intestinal stem cells are derived from intestinal crypts. In some embodiments, intestinal crypts include small intestinal or colonic crypts.

[0036] In some embodiments, the method for preparing and / or culturing organoids further includes the step of inducing the organoids to generate intestinal epithelial functional cells.

[0037] In some embodiments, the intestinal epithelial functional cells include Paneth cells. In some embodiments, the method of preparing and / or culturing organoids further includes culturing the organoids obtained in step b) in the presence of a Paneth cell-inducing composition, wherein the Paneth cell-inducing composition comprises a Wnt signaling activator, an inhibitor of Notch or γ-secretase, and a member of the IL-10 family of cytokines. In some embodiments, the Paneth cell-inducing composition further comprises one or more components from the group consisting of: a BMP signaling inhibitor, an EGF activator, an FGF activator, an IGF activator, a TGFβ inhibitor, and vitamin C and its derivatives.

[0038] In some embodiments, the intestinal epithelial functional cells include goblet cells. In some embodiments, the method of preparing and / or culturing organoids further includes culturing the organoids obtained in step b) in the presence of goblet cell induction composition A and goblet cell induction composition B, respectively, wherein goblet cell induction composition A comprises a Wnt signaling activator, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator, and goblet cell induction composition B comprises a Wnt signaling inhibitor, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator. In some embodiments, the method of preparing and / or culturing organoids includes culturing the organoids obtained in step b) for 1-3 days in the presence of goblet cell induction composition A, and then continuing to culture them for 1-5 days in the presence of goblet cell induction composition B.

[0039] In some embodiments, the intestinal epithelial functional cells include intestinal endocrine cells. In some embodiments, the method of preparing and / or culturing organoids includes culturing the organoids obtained in step b) under conditions in the presence of intestinal endocrine cell induction composition A and intestinal endocrine cell induction composition B, wherein intestinal endocrine cell induction composition A comprises a Wnt signaling activator, an inhibitor of Notch or γ-secretase, gastrin, a TGFβ inhibitor, and a BMP signaling inhibitor, and intestinal endocrine cell induction composition B comprises an inhibitor of Notch or γ-secretase, gastrin, a TGFβ inhibitor, a BMP signaling inhibitor, and a MAPK / EGFR signaling inhibitor. In some embodiments, the method of preparing and / or culturing organoids includes culturing the organoids obtained in step b) for 1-3 days in the presence of intestinal endocrine cell induction composition A, and then continuing to culture them for 1-5 days in the presence of intestinal endocrine cell induction composition B.

[0040] In some embodiments, the intestinal epithelial functional cells include intestinal epithelial cells. In some embodiments, the method of preparing and / or culturing organoids includes culturing the organoids obtained in step b) in the presence of an intestinal epithelial cell induction composition, wherein the intestinal epithelial cell induction composition comprises an EGF activator, an FGF activator, an IGF activator, gastrin, a TGFβ inhibitor, a BMP signaling activator, a Wnt signaling inhibitor, and an HDAC inhibitor.

[0041] In some embodiments, stem cells include gastric stem cells, and organoids include gastric organoids. In some embodiments, gastric stem cells are derived from gastric glands. In some embodiments, gastric stem cells are derived from cardia glands, gastric body glands, and / or pyloric glands.

[0042] On the other hand, this application provides an organoid prepared by the method of this application.

[0043] On the other hand, this application provides an intestinal organoid with cell diversity and proliferative capacity, which simultaneously comprises mature intestinal epithelial cells, Paneth cells, stem cells expressing Lgr5, and proliferating cells expressing KI67.

[0044] In some embodiments, the organoid further comprises goblet cells and / or enteroendocrine cells. In some embodiments, the proportion of Paneth cells in the organoid is ≥0.1%. In some embodiments, the proportion of goblet cells in the organoid is ≥1%. In some embodiments, the proportion of enteroendocrine cells in the organoid is ≥1%.

[0045] In some embodiments, organoids comprise cells expressing one or more genes selected from the group consisting of: FABP1, KRT20, ACE2, ALPI, DEFA5, DEFA6, and REG3A.

[0046] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0047] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0048] Figure 1 shows that the human intestinal organoids described in this application exhibit stronger stemness and higher cell diversity. Figure 1A shows a schematic diagram of the screening strategy to optimize the culture of human intestinal organoids. Figure 1B shows a schematic diagram of the targeting strategy for constructing the LGR5-mNeonGreen reporter system (left), the position of PCR primers (top right), and the gel electrophoresis results of the PCR products of the knock-in organoids (bottom right). Figure 1C shows the sequence map of the LGR5 gene and knock-in sequence linkage position (left) and the RT-qPCR quantification of stem cell-specific gene expression levels (right). Two-way ANOVA was performed using the Sidak multiple comparison test. n=3. Figure 1D shows a comparison of the culture medium components of ES, IF, IL, and TpC culture systems. Superscript 1 indicates Noggin or BMP pathway inhibitor DMH1, superscript 2 indicates R-Spondin1 conditioned medium, and superscript 3 indicates WNT3a protein, WNT3a conditioned medium, or WNT substitute. Figure 1E (left) shows representative bright-field and fluorescence images of LGR5-mNeonGreen organoids cultured under IF, IL, or TpC conditions. Scale bar, 200 μm. Figure 1E (right) shows the proportion of LGR5-mNeonGreen-highly expressing cells and the quantification of relative LGR5-mNeonGreen intensity in IF, IL, and TpC organoids cultured for 4 weeks. Figure 1F shows the quantification of colony formation efficiency and cell proliferation efficiency by the number of cells in IF, IL, and TpC organoids cultured for 10 days using single-cell (8000 cells per well) methods. t-test; error bars represent SD; n = 3. Figure 1G (left) shows the quantification of the proportions of secretory cells (Paneth cells, goblet cells, and enteroendocrine cells) in IF and TpC organoids by positive staining with LYZ, MUC2, and CHGA, respectively. Two-tailed unpaired t-test; error bars represent SD; n=3; Figure 1G (right panel) shows representative images of intestinal epithelial cells (EC) (ALPI), goblet cells (GC) (MUC2), enteroendocrine cells (EEC) (CHGA), and Paneth cells (PC) (LYZ) in TpC organoids. Scale bar, 50 μm. Figure 1H shows representative confocal images of LGR5-mNeonGreen and DEFA5-positive Paneth cells. White arrows indicate Paneth cells spaced apart from LGR5 stem cells, and red arrows indicate double-positive cells. Figure 1I shows the growth and mNeonGreen expression of a single LGR5-mNeonGreen+ cell cultured for 13 days under TpC conditions. It also shows Paneth (LYZ), goblet (MUC2), and enteroendocrine (CHGA) cells in a 13-day clone detected by immunofluorescence staining. Scale bar, 50 μm. Arrows indicate mNeonGreen expression at the same location.

[0049] Figure 2 shows the enhanced stemness and cellular diversity of TpC organoids. Figure 2A shows bright-field images of organoids cultured for 21 days under TpC, IF, and IL conditions. Figure 2B shows representative confocal images of Paneth cells (LYZ / DEFA5), goblet cells (MUC2), and enteroendocrine cells (CHGA) in organoids cultured for 21 days under IF conditions. Figure 2C shows representative bright-field and fluorescence images of organoids cultured for 10 and 21 days under TpC conditions; the right image of Figure 2C shows representative images of Paneth-like cells with dark granules (arrows) on day 10 and extensive crypt-like budding structures (arrows) on day 21 in TpC organoids. Figure 2D shows representative confocal images of the stem cell marker OLFM4, the Paneth cell (PC) marker DEFA5, and the enteroendocrine cell (EEC) subtype markers SST and GCG in TpC organoids. Figure 2E shows bright-field images of organoids derived from different donors. Figure 2F shows the secretory lineage markers (LYZ, MUC2, and CHGA) expressed uniformly in each TpC organoid at 10 and 21 days of culture. Scale bar, (A, C, D, and F), 200 μm; (B, E), 50 μm.

[0050] Figure 3 shows other features of TpC organoids. Figure 3A shows the fluorescence images of Paneth cells (LYZ and DEFA5) in TpC organoids cultured for 21 days. The left panel of Figure 3B shows the proportion of DEFA5 and LYZ co-stained cells in DEFA+ and LYZ+ cells; the right panel of Figure 3B shows the proportions shown in the Venn diagram. Figure 3C shows the immunofluorescence staining images of PC, EEC, and GC. The figures show panoramic views of the entire field of view and magnified organoid images, including round and budding organoids. Scale bar, 50 μm.

[0051] Figure 4 shows the analysis of LGR5-positive and LGR5-negative cells derived from TpC organoids. Figure 4A shows the flow cytometry sorting and gating strategy for LGR5-negative, LGR5-high-positive, and LGR5-low-positive cells. The left panel of Figure 4B shows the bright-field and fluorescence images of the organoids after 12 days of culture; the right panel of Figure 4B shows the quantitative analysis of the clonogenic efficiency of LGR5-negative, LGR5-high-positive, and LGR5-low-positive cells.

[0052] Figure 5 illustrates the cell diversity and cell fate dynamics of TpC organoids revealed by scRNA-seq analysis. The left panel of Figure 5A is a UMAP plot showing cell clusters from the scRNA-seq analysis of TpC organoids. Cluster labels indicate cell types. LGR5.high and LGR5.low represent intestinal stem cells with different LGR5 expression levels; TA1, transient expansion cell type 1; TA2, transient expansion cell type 2; SecPre, secretory precursor; SecPre2, secretory precursor type 2; EC, intestinal epithelial cells (EC); EEC, intestinal endocrine cells (EEC). The right panel of Figure 5A shows the proportion of each cell type in the scRNA-seq samples. Figure 5B shows a dot plot displaying the expression levels and percentages of cell type-specific markers for each cell population. The size and color of the dots represent normalized gene expression levels. The left panel of Figure 5C is a UMAP plot showing the subset analysis of secretory cells; the right panel of Figure 5C is a pie chart showing the proportion of each secretory cell type. The total percentage of all secretory cell types equals 100%. The dot plot in Figure 5D shows the expression levels and percentages of classic cell type-specific markers in the cell population shown in Figure 5C. The size and color of the dots represent normalized gene expression levels. Figure 5E shows an RNA rate analysis performed using Dynamo, indicating the inferred transition direction between cell states. The pie chart in Figure 5F (top) shows the proportion of cell types represented in the total cells, and the bar chart (bottom) shows the composition of cell types represented in ES, IF, IL, TpC organoids, and crypts.

[0053] Figure 6 illustrates the diversity and cell fate dynamics of human intestinal tissue cells revealed by scRNA-seq analysis. Figure 6A shows the UMAP and PAGA trajectory analysis of scRNA-seq data from ES, IF, IL, TpC organoids, and Crypt, displaying the inferred developmental trajectories of cell types. The size of the circles represents the cell number, and the line thickness represents the connection strength between cell clusters. Figure 6B's UMAP plot shows a comprehensive analysis of scRNA-seq datasets from ES, IF, IL, TpC organoids, and Crypt cells using the Seurat software package. Clustering shows different cell populations under different conditions. Figure 6C's heatmap shows the predicted PROGENY pathway activity in intestinal stem cells (ISCs) from the ES, IF, IL, TpC organoids, and Crypt datasets.

[0054] Figure 7 shows a comparative analysis of single-cell sequencing data from organoids and in vivo tissues, illustrating the Harmony ensemble analysis results of the organoid and in vivo single-cell datasets. The UMAP plot illustrates the ensemble clustering of all datasets.

[0055] Figure 8 shows the combined effects of TSA, pVc, and CP in inducing increased stemness and cell diversity in organoids. Figure 8A shows bright-field and fluorescence images of organoids cultured for one week under the indicated conditions. Scale bar: 200 μm. Figure 8B shows the percentage of LGR5-mNeonGreen highly expressed cells, relative mean fluorescence intensity, cell colony formation efficiency, and cell number quantification in organoids cultured under the indicated conditions. Figure 8C shows the quantitative detection of cell marker expression levels by RT-qPCR. Two-tailed unpaired t-test between each condition and the TpC condition; error bars represent SD; n = 3. The left panel of Figure 8D shows representative confocal images of Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), and enteroendocrine cells (EEC) (CHGA) in organoids. Scale bar: 50 μm. The right panel of Figure 8D shows the proportion of secretory cells (Paneth cells (PC), goblet cells (GC), and enteroendocrine cells (EEC)) in organoids cultured under the indicated conditions. Two-tailed unpaired t-tests were performed between each condition and the TpC condition; error bars indicate SD; n = 3. Figure 8E (left) shows fluorescence and bright-field images of organoids cultured under the indicated conditions. Scale bar, 200 μm. Figure 8E (right) shows the percentage of LGR5 cells and the quantification of relative LGR5-mNeonGreen mean fluorescence intensity in organoids cultured under the indicated conditions. Two-tailed unpaired t-tests were performed; error bars indicate SD; n = 3. Figure 8F shows EdU and immunofluorescence staining of enteroendocrine cells (EEC) (CHGA), goblet cells (GC) (MUC2), and Paneth cells (PC) (LYZ) in organoids. EdU was added 1 hour before staining and quantified by FACS analysis (8G). Two-tailed unpaired t-tests were performed; error bars indicate SD; n = 3. Figure 8G shows the proportion of EdU, LYZ, MUC2, and CHGA positive cells detected quantitatively. Two-tailed unpaired t-tests were performed; error bars indicate SD; n = 4. Figure 8H shows the quantitative detection of the proportion of LGR5-mNeonGreen cells under the indicated conditions. Two-tailed unpaired t-test; error bars indicate SD; n=6. The left panel of Figure 8I shows the fluorescence spectrum of LGR5-mNeonGreen under the indicated conditions. Scale bar, 50 μm. The right panel of Figure 8I shows the proportion of LGR5-mNeonGreen cells under the indicated conditions. Two-tailed unpaired t-test; error bars indicate SD; n=4.

[0056] Figure 9 shows the increased stemness and cellular diversity of organoids induced by TSA, pVc, and CP. Figure 9A shows representative fluorescence images of Paneth cells (LYZ), goblet cells (MUC2), and enteroendocrine cells (CHGA) in organoids under basal and CP-treated conditions. The left panel of Figure 9B shows bright-field and fluorescence images of organoids cultured for 28 days under the conditions shown in the figure. The right panel of Figure 9B shows the quantitative analysis of the proportion of LGR5-mNeonGreen cells and the mean fluorescence intensity. Figure 9C shows the bright-field and fluorescence images of organoids under CP and its analogues.

[0057] Figure 10 shows bright-field images of organoids cultured under conditions of TSA, pVc, and various signaling pathway inhibitors / activators, indicating that the combination of TSA, pVc, and CP has a proliferative effect.

[0058] Figure 11 illustrates the similar effects of other HDAC inhibitors and TSA in organoids. Figure 11A shows morphological and fluorescence images of organoids cultured from single cells for 10 days, demonstrating the effects of TSA and other HDAC inhibitors. Figure 11B shows the HDAC inhibitors used in Figure 11A. Figure 11C shows the quantitative analysis of the high fluorescence rate and relative fluorescence intensity of LGR5-mNeonGreen under the culture conditions shown in Figure 11B. Two-tailed unpaired t-tests (p.CP) are presented between each experimental condition and the HDAC inhibitor-free condition; error bars represent SD; n = 3. Figure 11D shows representative confocal images of Paneth cells (PCs) and enteroendocrine cells (EECs) in organoids cultured under the conditions shown in Figure 11B.

[0059] Figure 12 shows that the MBD2 inhibitor KCC-7 and TSA exhibit similar effects. Figure 12A (left) shows the morphological image of organoids cultured for 14 days under the illustrated conditions. Figure 12A (right) shows the organoid budding rate statistics. Figure 12B (left) shows the bright-field and fluorescence images of organoids cultured for 28 days under the illustrated conditions. Figure 12B (right) shows the flow cytometry detection of relative fluorescence intensity. Figure 12C (left) shows the fluorescence images of LGR5-mNeonGreen stem cells, Paneth cells (LYZ and DEFA5), goblet cells (MUC2), and enteroendocrine cells (CHGA) in organoids cultured under the illustrated conditions. Figure 12C (right) shows the proportion of Paneth cells under the illustrated conditions. Figure 12D shows the RT-qPCR quantitative detection of stem cell, Paneth cell, goblet cell, and enteroendocrine cell-specific gene expression under TpC, pC, and pC+KCC conditions.

[0060] Figure 13 shows the effect of inhibiting MBD3 on promoting LGR5 stem cell self-renewal. The left panel of Figure 13A shows the targeting strategy for interfering with MBD3 expression. The right panel of Figure 13A shows the Western blotting detection of MBD3 knockdown in clone #14. Figure 13B shows the RT-qPCR quantitative detection of MBD3 and LGR5 gene expression. Figure 13C shows the confocal image (left panel) and relative fluorescence intensity analysis (right panel) of LGR5-mNeonGreen stem cells and Paneth cells (DEFA5) under the illustrated conditions.

[0061] Figure 14 shows the characteristic changes of organoids after TSA removal. The UMAP plot in Figure 14A shows the differences in cell clustering after TSA removal, and the bar chart shows the changes in cell composition after TSA removal. Figure 14B shows the gene expression of different cell types in organoids after TSA removal.

[0062] Figure 15 shows the reversible promotion of organoid proliferation and inhibition of secretory cell differentiation by iBET-151. Figure 15A shows a schematic diagram of the screening strategy. The left image of Figure 15B shows a representative bright-field image and LGR5-mNeonGreen fluorescence image of organoids cultured under the illustrated conditions. Scale bar, 200 μm. The right image of Figure 15B shows the proportion and relative fluorescence intensity of LGR5-mNeonGreen cells in the organoids shown in the left image. Figure 15C shows EdU and immunofluorescence staining of Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), and enteroendocrine cells (EEC) (CHGA) and quantification (right bar chart). EdU was administered 1 hour before staining. Scale bar, 50 μm. Error bars indicate SD; n=3. Figure 15D shows a UMAP plot illustrating single-cell sequencing cell clusters of TpCI organoids (left image) and a comparison of cell types and proportions (right image). The dot plot of Figure 15E shows the specific gene expression and proportion of clustered cell types.

[0063] Figure 16 shows how iBET-151 promotes the proliferation of single cells or established organoids and inhibits differentiation. Figure 16A shows EdU and immunofluorescence staining of Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), and enteroendocrine cells (EEC) (CHGA) cultured under the conditions shown. Scale bar, 200 μm. Figure 16B shows a representative image of the budding crypts of organoids cultured under the conditions shown. Scale bar, 200 μm. The left image of Figure 16C shows a representative bright-field image and LGR5-mNeonGreen fluorescence image of organoids cultured under the conditions shown. Scale bar, 200 μm. The right image of Figure 16C shows the proportion of LGR5-mNeonGreen cells and the quantification of relative fluorescence intensity in organoids under the conditions shown. Figure 16D shows immunofluorescence staining of Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), and enteroendocrine cells (EEC) (CHGA). Scale bar, 50 μm. Figure 16E shows the quantitative proportions of LYZ, MUC2, and CHGA positive cells. Figure 16F shows the immunofluorescence staining of EdU and Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), and enteroendocrine cells (EEC) (CHGA). EdU was administered 1 hour before staining. Scale bar, 50 μm. Figure 16G shows the quantitative proportions of EdU, LYZ, MUC2, and CHGA positive cells in the organoids shown in Figure 16F.

[0064] Figure 17 shows the directed differentiation of various intestinal epithelial cell types induced by microenvironment signal regulation. Figure 17A is a schematic diagram of the small molecule screening strategy for inducing differentiated cell types. Figure 17B, from left to right, shows the differentiation protocols (top) of Paneth cells (PC), goblet cells (GC), enteroendocrine cells (EEC), and intestinal epithelial cells (EC), and representative confocal images of the differentiated organoids, showing Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), enteroendocrine cells (EEC) (CHGA), and intestinal epithelial cells (EC) (ALPI) (middle), as well as the quantified percentage of differentiated cells (bottom). Scale bar, 50 μm. Figure 17C shows representative confocal images of the differentiated organoids, showing Paneth cells (PC) (LYZ), goblet cells (GC) (MUC2), and enteroendocrine cells (EEC) (CHGA) from TpC and TpCI organoids (left), and the quantified percentage of differentiated cells (right). Scale bar, 50 μm. Figure 17D shows a schematic diagram and fluorescence staining image of the experiment inducing M cell differentiation in TpC organoids. Scale bar, 50 μm.

[0065] Figure 18 illustrates the regulation of cell fate by a combination of microenvironment signals. Figure 18A shows a representative confocal image of Paneth cell (PC) differentiation in organoids regulated by IL-22 (labeled with LYZ). Scale bar, 50 μm. Figure 18B shows a representative confocal image of goblet cell (GC) differentiation in organoids regulated by the BMP pathway and a stepwise differentiation protocol (labeled with MUC2). Scale bar, 50 μm. Figure 18C shows a representative confocal image of a stepwise differentiation protocol for enteroendocrine cell (EEC) differentiation (CHGA) in organoids. Scale bar, 50 μm. Figure 18D shows ALPI staining of intestinal epithelial cell (EC) differentiation in organoids, with Wnt, Notch, and BMP signaling regulation as illustrated. Scale bar, 200 μm. Figure 18E shows DEFA5 and LYZ staining patterns 3 days after induction of Paneth cell differentiation in IF and TpC organoids. Scale bar, 50 μm.

[0066] Figure 19 shows the induced directed differentiation of M cells in TpC organoids. It includes representative confocal images illustrating M cell differentiation (GP-2) and E-cadherin staining (left panel), as well as RT-qPCR detection of the expression of M cell-specific genes CCL15 and TNFAIP2 (right panel). Scale bar, 50 μm. Detailed Implementation

[0067] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0068] Terminology Definition

[0069] Unless otherwise stated, the terminology used in this application should be understood in accordance with the common usage of those skilled in the art.

[0070] In this application, the term "inhibitor" generally refers to an agent that causes a reduction in the expression and / or activity of a target gene or protein. An "antagonist" can be an inhibitor, but it is more specifically an agent that binds to a receptor, thereby reducing or eliminating binding to other molecules. The effects of such inhibitors may include, but are not limited to: 1) directly inhibiting protein activity, for example by binding to the active site of an enzyme to prevent substrate entry and inhibit the enzyme's catalytic activity; 2) preventing protein-protein interactions; 3) affecting the subcellular localization of proteins, thereby affecting their function; and 4) promoting protein degradation.

[0071] The inhibitors described in this application can be detected using conventional methods in the art. For example, the inhibitory effect can be demonstrated by detecting the half-maximal inhibitory concentration (IC50), and methods for detecting IC50 are known in the art. The source of the target gene or protein in the inhibitors described in this application is not particularly limited, and target genes or proteins derived from various organisms can be used.

[0072] In this application, the term "agonist" generally refers to an agent that causes an increase in the expression and / or activity of a target gene or protein. An agonist can bind to and activate its homologous receptor in some form, which directly or indirectly leads to physiological effects on the target gene or protein.

[0073] In this application, the term "histone deacetylase (HDAC)" generally refers to a class of enzymes capable of catalyzing the removal of acetyl groups from histone or non-histone substrates. The HDACs described in this application include HDACs derived from various organisms, such as mammalian HDACs. For example, mammalian HDAC family members can be divided into classes I, II, III, and IV. Class I HDACs include HDAC1, HDAC2, HDAC3, and HDAC8; Class II HDACs can include Class IIa and Class IIb HDACs, where Class IIa HDACs include HDAC4, HDAC5, HDAC7, and HDAC9, and Class IIb HDACs include HDAC6 and HDAC10; Class III HDACs are also known as Sirtuins, which include SIRT1-7; and Class IV HDACs include HDAC11. The amino acid sequence of the HDAC described in this application and the base sequence of the gene encoding the HDAC can be obtained from well-known databases such as UniProt and GenBank. For example, the UniProt accession number for human HDAC1 is Q13547, and the UniProt accession number for human HDAC2 is Q92769.

[0074] In this application, the term "HDAC inhibitor" generally refers to an agent that causes a decrease in the expression and / or activity of genes encoding HDACs or HDACs. HDAC inhibitors can include pan-HDAC inhibitors and selective HDAC inhibitors. In this application, the term "pan-HDAC inhibitor" generally refers to a class of compounds capable of inhibiting multiple histone deacetylase (HDAC) subtypes. Some pan-HDAC inhibitors can inhibit almost all HDAC subtypes. For example, the pan-HDAC inhibitors described in this application can have inhibitory activity against multiple subtypes of HDAC1-11. For example, pan-HDAC inhibitors can have activities including, but not limited to, inhibiting members of the class I, II, and IV HDAC families. In this application, the term "pan-HDAC inhibitor" is used in contrast to "selective HDAC inhibitor." Unlike selective HDAC inhibitors, pan-HDAC inhibitors do not have selectivity for a specific HDAC subtype; they act on multiple members of the HDAC family. In this application, the term "selective HDAC inhibitor" generally refers to a compound capable of specifically inhibiting certain HDAC subtypes while having weak or no effect on other subtypes. For example, the selective HDAC inhibitor may have activity against a specific HDAC family member.

[0075] In this application, the term "histone deacetylase 1 / 2 inhibitor" or "HDAC1 / 2 inhibitor" generally refers to an agent capable of causing a decrease in the expression and / or activity of HDAC1 and / or HDAC2. An HDAC1 / 2 inhibitor can be a pan-HDAC inhibitor, also inhibiting other members of the HDAC family. An HDAC1 / 2 inhibitor can be a selective HDAC inhibitor, selectively inhibiting HDAC1 and / or HDAC2. For example, an HDAC1 / 2 inhibitor can include an agent capable of causing a decrease in the expression and / or activity of HDAC1 and HDAC2. For example, an HDAC1 / 2 inhibitor can include an inhibitor that selectively inhibits HDAC1, or an inhibitor that selectively inhibits HDAC2.

[0076] In this application, the term "nucleosome remodeling and deacetylase (NuRD) complex" generally refers to a group of proteins associated with ATP-dependent chromatin remodeling and histone deacetylase activity. The NuRD complex is a large protein complex composed of multiple subunits, including chromatin remodeling enzymes (such as CHD3 / 4), histone deacetylases (such as HDAC1 / 2), methylated CpG-binding proteins (such as MBD2 / 3), transfer-associated proteins (such as MTA1 / 2 / 3), GATA zinc finger binding domain proteins (such as GATAD2A / B), and retinoblastoma-associated proteins (such as RBBP4 / 7). The NuRD complexes described in this application include NuRD complexes derived from various organisms, such as mammalian NuRD complexes.

[0077] In this application, the term "nucleosome remodeling and deacetylase (NuRD) complex inhibitor" or "NuRD complex inhibitor" generally refers to an agent that causes a decrease in the expression and / or activity of the nucleosome remodeling and deacetylase complex. NuRD complex inhibitors may include inhibition of single or multiple subunits of the NuRD complex, or they may function by affecting the assembly and function of the complex. Major subunits of the NuRD complex include CHD family proteins (CHD3 or CHD4), HDAC family proteins (HDAC1 and HDAC2), MTA family proteins (MTA1, MTA2, or MTA3), RBBP family proteins (RBBP4 and RBBP7), GATAD2 family proteins (GATAD2A and GATAD2B), and MBD family proteins (MBD2 or MBD3). For example, a NuRD inhibitor may include an inhibitor of one or more subunits of the NuRD complex. For example, a NuRD complex inhibitor may include a NuRD complex degrader. For example, the NuRD complex inhibitor may include an HDAC1 / 2 inhibitor and / or an MBD2 / 3 inhibitor.

[0078] In this application, the term "methylated CpG-binding protein (MBD)" generally refers to a class of nuclear proteins that can specifically recognize and bind to methylated CpG dinucleotides. MBD2 and MBD3 are members of the MBD protein family. The MBDs described in this application include MBDs derived from various organisms, such as mammalian MBDs. The amino acid sequences of the MBDs described in this application and the base sequences of the genes encoding the MBDs can be obtained from known databases such as UniProt and GenBank. For example, the UniProt accession number for human MBD2 is Q9UBB5, and the UniProt accession number for human MBD3 is Q95983.

[0079] In this application, "methylated CpG-binding protein 2 / 3 inhibitor" or "MBD2 / 3 inhibitor" generally refers to an agent capable of reducing the expression and / or activity of MBD2 and / or MBD3. For example, an MBD2 / 3 inhibitor may include an agent capable of reducing the expression and / or activity of MBD2 and MBD3. For example, an MBD2 / 3 inhibitor may include an inhibitor that selectively inhibits MBD2, or it may include an inhibitor that selectively inhibits MBD3.

[0080] In this application, the term "platelet-derived growth factor receptor (PDGFR)" generally refers to the receptor of the platelet-derived growth factor (PDGF) protein family. The PDGFRs described in this application include PDGFRs derived from various organisms, such as mammalian PDGFRs. PDGFRs mainly comprise two structurally similar receptor subtypes, PDGFR-α and PDGFR-β. The amino acid sequences of the PDGFRs described in this application and the base sequences of the genes encoding the PDGFRs can be obtained from known databases such as UniProt and GenBank. For example, the UniProt accession number for human PDGFR-α is P16234, and the UniProt accession number for human PDGFR-β is P09619.

[0081] In this application, the terms "platelet-derived growth factor receptor inhibitor" or "PDGFR inhibitor" generally refer to agents that can lead to a decrease in the expression and / or activity of platelet-derived growth factor receptor.

[0082] In this application, the term "derivative" generally refers to another chemical substance that is structurally related to a chemical substance, or a chemical substance that can be prepared from another chemical substance (i.e., the chemical substance from which the first chemical substance is derived), for example, through chemical or enzymatic modification. In this application, the term "derivative" can also refer to a structure that is derived from or similar to that of a parent compound. A derivative may exhibit similar functions and / or activities to the parent compound.

[0083] In this application, the term "vitamin C and its derivatives" generally refers to vitamin C (also known as ascorbic acid) and its various modified forms. Vitamin C derivatives may have better stability and / or higher bioavailability than vitamin C.

[0084] In this application, the term "stem cell" generally refers to a pluripotent cell capable of self-renewal and differentiation into multiple cell lineages. Unless otherwise specified, the stem cells described in this application include single stem cells and cell populations containing stem cells.

[0085] In this application, the term "adult stem cell" generally refers to undifferentiated cells present in organs and differentiated tissues, possessing limited self-renewal and differentiation capabilities. In this context, "adult" includes newborns or children, but excludes embryos or fetuses. In some embodiments, the adult stem cells are not derived from embryonic stem cells or embryonic stem cell lines (e.g., human embryonic stem cells or human embryonic stem cell lines).

[0086] In this application, the term "epithelial stem cell" generally refers to a stem cell with the tissue potential to differentiate into multiple cell types of a specific epithelial tissue. Epithelial stem cells include, but are not limited to, epithelial stem cells in organs such as the small intestine, stomach, lung, and colon.

[0087] In this application, the term "Lgr5+ stem cell" or "Lgr5-positive stem cell" generally refers to stem cells that express Lgr5 on their cell surface. The Lgr5+ stem cells described in this application include Lgr5+ stem cells derived from various organisms, such as mammalian Lgr5+ stem cells, such as human Lgr5+ stem cells. Lgr5-positive stem cells are present in multiple tissues, such as the intestine and stomach. For example, in the small intestine and colon, Lgr5-positive stem cells are located at the base of the intestinal crypts and continuously divide to generate different types of intestinal epithelial cells.

[0088] In this application, the term "Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)" or "LGR5" generally refers to G protein-coupled receptor 5 rich in leucine repeat sequences or the gene encoding it. Lgr5 is also known as G protein-coupled receptor 49 (GPR49) or G protein-coupled receptor 67 (GPR67). Lgr5 is a downstream target gene of the Wnt signaling pathway, which is essential for maintaining stem cell self-renewal and tissue regeneration. Lgr5-positive stem cells can have high proliferative capacity and pluripotency, capable of self-renewal and differentiation into various functional cells, participating in tissue repair and regeneration. The Lgr5 described in this application includes Lgr5 derived from various organisms. The amino acid sequence of the Lgr5 described in this application and the base sequence of the gene encoding the PDGFR can be obtained from known databases such as UniProt and GenBank; for example, the UniProt accession number for human Lgr5 is O75473.

[0089] In this application, the term "culture" generally refers to the maintenance and promotion of the survival, function, and / or proliferation of cells, tissues, or biological samples by providing suitable environmental conditions. For example, cell culture may include maintaining the normal survival and / or function of cells, or it may include increasing the number of cells. For example, tissue culture enables tissues to continue to survive, differentiate, or regenerate in vitro.

[0090] In this application, the term "preservation" generally refers to the treatment of cells, tissues, or biological samples under specific conditions to maintain their function, structure, and biological activity. The purpose of preservation is to maintain the biological integrity of the sample for as long as possible, ensuring that the sample does not degenerate, denature, or lose its activity over the extended period.

[0091] In this application, the term "separated" generally refers to the absence, to varying degrees, of the components typically associated with it in its natural state. "Separated" refers to the degree of separation from its original source or environment.

[0092] In this application, the term "composition" generally refers to a product comprising a specified amount of a specified ingredient, and any product produced directly or indirectly from a combination of the specified amounts of the specified ingredients. In this application, the composition may also contain other active ingredients, such as carriers, excipients, adjuvants, stabilizers, etc.

[0093] In this application, the term "organoid" generally refers to a cluster or aggregate of cells that self-organizes by instructing cells to accumulate in a controlled space at high density. An organoid may be a similar organ or part of an organ and possesses a cell type associated with that particular organ.

[0094] As used herein, "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the described structure, as well as derivatives that retain their original function, such as pharmaceutically acceptable salts. The term is also intended to refer to the compounds of this application, regardless of their manner of preparation, such as synthesis, by biological processes (e.g., metabolism or enzymatic conversion), or combinations thereof. All compounds and their pharmaceutically acceptable salts may be found together with other substances, such as water and solvents (e.g., hydrates and solvates), or may be separated. When in the solid state, the compounds and their salts described herein may exist in a variety of forms and may be, for example, in the form of solvates (including hydrates). The compounds may be in any solid form, such as polymorphs or solvates, and therefore, unless otherwise clearly indicated, the reference to compounds and their derivatives in this specification should be understood to cover any solid form of the compound.

[0095] As used herein, macromolecules, such as proteins or polypeptides, are intended to include recombinant analogs of the described molecules, as well as derivatives that retain their original functions. The term is also intended to refer to the macromolecules of this application, regardless of their manner of preparation, such as synthesis, by biological processes (e.g., recombinant DNA expression), or a combination thereof. Recombinant analogs are typically obtained by expression and purification of recombinant DNA technology in a suitable host, having an amino acid sequence identical to or similar to that of the reference, as understood by those skilled in the art, while retaining the key functions of the reference. For example, the growth factors described herein include recombinant analogs of them.

[0096] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0097] In this application, the term "and / or" should be understood to mean any one, two, or more of the alternatives or any combination thereof.

[0098] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0099] Invention Details

[0100] Compositions and their uses

[0101] On the one hand, this application provides a first composition and its use, wherein the first composition can be used to prepare organoids that simultaneously have strong proliferative capacity and cell diversity.

[0102] In some embodiments, the first composition comprises a histone deacetylase (HDAC) inhibitor, vitamin C and its derivatives, and a platelet-derived growth factor receptor (PDGFR) inhibitor.

[0103] The HDAC inhibitors described in this application may be those known in the art, and may include, but are not limited to, pan-HDAC inhibitors including valproic acid (VPA) and trichostatin A, HDAC6 inhibitor Tubastatin A, HDAC4 / 5 inhibitor LMK235, and HDAC2 inhibitor CAY10683 (Santacruzamate A).

[0104] In this application, the concentration of the HDAC inhibitor used can be from about 1 nM to about 50 mM.

[0105] For example, the concentration of the HDAC inhibitor can be approximately 1 nM to approximately 50 mM, approximately 1 nM to approximately 40 mM, approximately 1 nM to approximately 30 mM, approximately 1 nM to approximately 20 mM, approximately 1 nM to approximately 10 mM, approximately 1 nM to approximately 5 mM, approximately 1 nM to approximately 4 mM, approximately 1 nM to approximately 3 mM, approximately 1 nM to approximately 2 mM, approximately 1 nM to approximately 1 mM, approximately 1 nM to approximately 900 μM, approximately 1 nM to approximately 800 μM, approximately 1 nM to approximately 70 μM. 0 μM, approximately 1 nM to approximately 600 μM, approximately 1 nM to approximately 500 μM, approximately 1 nM to approximately 400 μM, approximately 1 nM to approximately 300 μM, approximately 1 nM to approximately 200 μM, approximately 1 nM to approximately 100 μM, approximately 1 nM to approximately 90 μM, approximately 1 nM to approximately 80 μM, approximately 1 nM to approximately 70 μM, approximately 1 nM to approximately 60 μM, approximately 1 nM to approximately 50 μM, approximately 1 nM to approximately 40 μM, approximately 1 nM to approximately 30 μM, approximately 1 nM - Approximately 20 μM, approximately 1 nM - approximately 10 μM, approximately 1 nM - approximately 9 μM, approximately 1 nM - approximately 8 μM, approximately 1 nM - approximately 7 μM, approximately 1 nM - approximately 6 μM, approximately 1 nM - approximately 5 μM, approximately 1 nM - approximately 4 μM, approximately 1 nM - approximately 3 μM, approximately 1 nM - approximately 1 μM, approximately 1 nM - approximately 1 μM, approximately 1 nM - approximately 900 nM, approximately 1 nM - approximately 800 nM, approximately 1 nM - approximately 700 nM, approximately 1 nM - approximately 600 nM, approximately 1 nM - approximately 500 nM, approximately 1 nM - approximately 400 nM, approximately 1 nM - approximately 300 nM, approximately 1 nM - approximately 200 nM, approximately 1 nM - approximately 100 nM, approximately 1 nM - approximately 90 nM, approximately 1 nM - approximately 80 nM, approximately 1 nM - approximately 70 nM, approximately 1 nM - approximately 60 nM, approximately 1 nM - approximately 50 nM, approximately 1 nM - approximately 40 nM, approximately 1 nM - approximately 30 nM, approximately 1 nM - approximately 20 nM, or approximately 1 nM - approximately 10 nM.

[0106] In some embodiments, the HDAC inhibitor may include valproic acid. The CAS Registry Number for valproic acid described in this application is 99-66-1, and its molecular formula is C8H2O. 16 O2, also known as 2-propylvaleric acid, 2-n-propylvaleric acid, 2,2-di-n-propylacetic acid, A-propylvaleric acid, and α-propylvaleric acid.

[0107] In some embodiments, the concentration of valproic acid used can be about 0.01 mM to 50 mM. For example, the concentration of valproic acid used can be about 0.01 mM to 50 mM, about 0.01 mM to 40 mM, about 0.01 mM to 30 mM, about 0.01 mM to 20 mM, about 0.1 mM to 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.9 mM, or about 0.1 mM to about 0.8 mM. For example, the concentration of valproic acid used can be about 0.2 mM to about 1 mM, about 0.3 mM to about 1 mM, about 0.4 mM to about 1 mM, about 0.5 mM to about 1 mM, about 0.6 mM to about 1 mM, or about 0.7 mM to about 1 mM. For example, the concentration of valproic acid used can be about 0.75 mM.

[0108] In some embodiments, the HDAC inhibitor may include trichostatin A (TSA). The CAS Registry Number for trichostatin A described in this application is 58880-19-6, and its molecular formula is C2. 17 H 22 N2O3, also known as qugulijunin A, qugulijunin A, [R-(E,E)]-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide.

[0109] In some embodiments, the concentration of nystatin A used can be from about 1 nM to about 2000 nM. For example, the concentration of nystatin A used can be from about 1 nM to about 5 nM, from about 1 nM to about 10 nM, from about 1 nM to about 20 nM, from about 1 nM to about 30 nM, from about 1 nM to about 40 nM, from about 1 nM to about 50 nM, from about 1 nM to about 60 nM, from about 1 nM to about 70 nM, from about 1 nM to about 80 nM, from about 1 nM to about 90 nM, from about 1 nM to about 100 nM, from about 1 nM to about 250 nM, from about 1 nM to about 500 nM, from about 1 nM to about 1000 nM, from about 1 nM to about 1500 nM, or from about 1 nM to about 2000 nM. For example, the concentration of nystatin A used can be about 10 nM to about 100 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, or about 10 nM to about 20 nM. For example, the concentration of nystatin A used can be about 20 nM.

[0110] In some embodiments, the HDAC inhibitor may include Tubastatin A. The CAS Registry Number for Tubastatin A described in this application is 1252003-15-8, and its molecular formula is C0.05. 20 H 21 N3O2, also known as N-hydroxy-4-(2-methyl-1,2,3,4-tetrahydro-pyrido[4,3-b]indol-5-ylmethyl)benzamide.

[0111] In some embodiments, the concentration of trimethostatin A used can be from about 0.05 μM to about 50 μM. For example, the concentration of trimethostatin A used can be from about 0.05 μM to about 50 μM, from about 0.05 μM to about 25 μM, from about 0.05 μM to about 10 μM, from about 0.05 μM to about 15 μM, from about 0.05 μM to about 10 μM, from about 0.05 μM to about 5 μM, from about 0.05 μM to about 4 μM, from about 0.05 μM to about 3 μM, from about 0.05 μM to about 2.5 μM, from about 0.05 μM to about 2 μM, or from about 0.05 μM to about 1.5 μM. For example, the concentration of nystatin A used may be about 0.1 μM to about 20 μM, about 0.2 μM to about 20 μM, about 0.3 μM to about 20 μM, about 0.4 μM to about 20 μM, about 0.5 μM to about 20 μM, or about 1 μM to about 20 μM. For example, the concentration of trimethoprim A used may be about 1 μM.

[0112] In some embodiments, the HDAC inhibitor may include LMK235. The CAS Registry Number for LMK235 described in this application is 1418033-25-6, and its molecular formula is C2. 15 H 22 N2O4, also known as N-[[6-(hydroxyamino)-6-oxohexyl]oxy]-3,5-dimethylbenzamide.

[0113] In some embodiments, the concentration of LML235 used can be from about 5 nM to about 5000 nM. For example, the concentration of LMK235 used can be from about 5 nM to about 5000 nM, from about 5 nM to about 2500 nM, from about 5 nM to about 1000 nM, from about 5 nM to about 900 nM, from about 5 nM to about 800 nM, from about 5 nM to about 700 nM, from about 5 nM to about 600 nM, from about 5 nM to about 500 nM, from about 5 nM to about 400 nM, from about 5 nM to about 300 nM, from about 5 nM to about 200 nM, or from about 5 nM to about 100 nM. For example, the concentration of LMK235 used can be approximately 10 nM to approximately 1000 nM, approximately 20 nM to approximately 1000 nM, approximately 30 nM to approximately 1000 nM, approximately 40 nM to approximately 1000 nM, approximately 50 nM to approximately 1000 nM, approximately 60 nM to approximately 1000 nM, approximately 70 nM to approximately 1000 nM, approximately 80 nM to approximately 1000 nM, approximately 90 nM to approximately 1000 nM, or approximately 100 nM to approximately 1000 nM. For example, the concentration of LMK235 used can be approximately 100 nM.

[0114] In some embodiments, the HDAC inhibitor may include CAY10683. The CAS Registry Number for CAY10683 described in this application is 1477949-42-0, and its molecular formula is C2. 15 H 22 N2O3, also known as Santacruzamate A, is an ethyl carbamate of N-[4-oxo-4-[(2-phenylethyl)amino]butyl]carbamate.

[0115] In some embodiments, the concentration of CAY10683 used can be from about 0.05 μM to about 50 μM. For example, the concentration of CAY10683 used can be from about 0.05 μM to about 50 μM, from about 0.05 μM to about 25 μM, from about 0.05 μM to about 10 μM, from about 0.05 μM to about 15 μM, from about 0.05 μM to about 10 μM, from about 0.05 μM to about 5 μM, from about 0.05 μM to about 4 μM, from about 0.05 μM to about 3 μM, from about 0.05 μM to about 2.5 μM, from about 0.05 μM to about 2 μM, or from about 0.05 μM to about 1.5 μM. For example, the concentration of CAY10683 used can be about 0.1 μM to about 20 μM, about 0.2 μM to about 20 μM, about 0.3 μM to about 20 μM, about 0.4 μM to about 20 μM, about 0.5 μM to about 20 μM, or about 1 μM to about 20 μM. For example, the concentration of CAY10683 used can be about 1 μM.

[0116] The PDGFR inhibitors described in this application may be those known in the art, and may include, but are not limited to, CP673451 and crenolanib.

[0117] In this application, the concentration of the PDGFR inhibitor can be from about 0.05 μM to about 50 μM.

[0118] For example, the concentration of the PDGFR inhibitor can be about 0.05 μM to about 50 μM, about 0.05 μM to about 40 μM, about 0.05 μM to about 30 μM, about 0.05 μM to about 20 μM, about 0.05 μM to about 10 μM, about 0.05 μM to about 5 μM, about 0.05 μM to about 4 μM, about 0.05 μM to about 3 μM, about 0.05 μM to about 2 μM, about 0.05 μM to about 1 μM, about 0.05 μM to about 1 μM, about 0.05 μM to about 0.9 μM, about 0.05 μM to about 0.8 μM, about 0.05 μM to about 0.7 μM, or about 0.05 μM to about 0.5 μM. For example, the concentration of the PDGFR inhibitor can be about 0.1 μM to about 50 μM, about 0.1 μM to about 50 μM, about 0.2 μM to about 50 μM, about 0.3 μM to about 50 μM, about 0.4 μM to about 50 μM, or about 0.5 μM to about 50 μM. For example, the concentration of the PDGFR inhibitor can be about 0.1 to about 1 μM. For example, the concentration of the PDGFR inhibitor can be about 0.5 μM.

[0119] In some embodiments, the PDGFR inhibitor may include CP673451. The CAS Registry Number for CP673451 described in this application is 343787-29-1, and its molecular formula is C6. 24 H 27 N5O2, which can also be called 1-[2-[5-(2-methoxyethoxy)-1H-benzimidazol-1-yl]-8-quinolinyl]-4-piperidineamine or 1-[2-[5-(2-methoxyethoxy)benzimidazol-1-yl]quinolin-8-yl]piperidine-4-ylamine.

[0120] In some embodiments, the concentration of CP673451 used may be about 0.05 μM to about 50 μM, about 0.05 μM to about 40 μM, about 0.05 μM to about 30 μM, about 0.05 μM to about 20 μM, about 0.05 μM to about 10 μM, about 0.05 μM to about 5 μM, about 0.05 μM to about 4 μM, about 0.05 μM to about 3 μM, about 0.05 μM to about 2 μM, about 0.05 μM to about 1 μM, about 0.05 μM to about 1 μM, about 0.05 μM to about 0.9 μM, about 0.05 μM to about 0.8 μM, about 0.05 μM to about 0.7 μM, or about 0.05 μM to about 0.5 μM. For example, the concentration of CP673451 can be approximately 0.1 μM to approximately 50 μM, approximately 0.1 μM to approximately 50 μM, approximately 0.2 μM to approximately 50 μM, approximately 0.3 μM to approximately 50 μM, approximately 0.4 μM to approximately 50 μM, or approximately 0.5 μM to approximately 50 μM. For example, the concentration of CP673451 can be approximately 0.1 to approximately 1 μM. For example, the concentration of CP673451 can be approximately 0.5 μM.

[0121] In some embodiments, the PDGFR inhibitor may include crenolanib. The CAS Registry Number for crenolanib described in this application is 670220-88-9, and its molecular formula is C2. 26 H 29 N5O2, also known as CP868596, ARO 002, 4-piperidinamine, 1-[2-[5-(3-methyl-3-oxecyclobutane)methoxy]-1-hydro-benzimidazol-1-yl]-8-quinolinyl].

[0122] In some embodiments, the concentration of claranil used may be about 0.05 μM to about 50 μM, about 0.05 μM to about 40 μM, about 0.05 μM to about 30 μM, about 0.05 μM to about 20 μM, about 0.05 μM to about 10 μM, about 0.05 μM to about 5 μM, about 0.05 μM to about 4 μM, about 0.05 μM to about 3 μM, about 0.05 μM to about 2 μM, about 0.05 μM to about 1 μM, about 0.05 μM to about 1 μM, about 0.05 μM to about 0.9 μM, about 0.05 μM to about 0.8 μM, about 0.05 μM to about 0.7 μM, or about 0.05 μM to about 0.5 μM. For example, the concentration of claranil used can be about 0.1 μM to about 50 μM, about 0.1 μM to about 50 μM, about 0.2 μM to about 50 μM, about 0.3 μM to about 50 μM, about 0.4 μM to about 50 μM, or about 0.5 μM to about 50 μM. For example, the concentration of claranil used can be about 0.1 to about 1 μM. For example, the concentration of CP673451 used can be about 0.5 μM.

[0123] The vitamin C and its derivatives described in this application may be those known in the art, and may include, but are not limited to, magnesium ascorbate phosphate, sodium ascorbate phosphate, tetraisopalmitate ascorbate, ascorbate glucoside, ascorbate palmitate, ethyl ascorbic acid, and ascorbate acetate. In some embodiments, vitamin C and its derivatives may include L-ascorbic acid-2-phosphate trisodium salt. The CAS Registry number for L-ascorbic acid-2-phosphate trisodium salt is 66170-10-3, and its molecular formula is C6H6Na3O9P.

[0124] In this application, the concentration of vitamin C and its derivatives used can be from about 10 μg / mL to about 1000 μg / mL.

[0125] For example, the concentration of vitamin C and its derivatives used may be from about 10 μg / mL to about 900 μg / mL, from about 10 μg / mL to about 800 μg / mL, from about 10 μg / mL to about 700 μg / mL, from about 10 μg / mL to about 600 μg / mL, from about 10 μg / mL to about 500 μg / mL, from about 10 μg / mL to about 400 μg / mL, from about 10 μg / mL to about 300 μg / mL, from about 10 μg / mL to about 200 μg / mL, from about 10 μg / mL to about 150 μg / mL, or from about 10 μg / mL to about 100 μg / mL. For example, the concentration of vitamin C and its derivatives used may be about 20 μg / mL to about 200 μg / mL, about 30 μg / mL to about 200 μg / mL, about 40 μg / mL to about 200 μg / mL, about 50 μg / mL to about 200 μg / mL, about 60 μg / mL to about 200 μg / mL, about 70 μg / mL to about 200 μg / mL, about 80 μg / mL to about 200 μg / mL, about 90 μg / mL to about 200 μg / mL, or about 100 μg / mL to about 200 μg / mL. For example, the concentration of vitamin C and its derivatives used may be about 50 μg / mL to about 150 μg / mL. For example, the concentration of vitamin C and its derivatives used may be about 100 μg / mL.

[0126] The NuRD inhibitor described in this application may be known in the art, and may include, but is not limited to, HDAC1 / 2 inhibitors including trichostatin A and MBD2 / 3 inhibitors including KCC-07.

[0127] In this application, the concentration of the NuRD inhibitor can be from about 1 nM to about 5 mM.

[0128] For example, the concentration of the NuRD inhibitor can be approximately 1 nM to approximately 5 mM, approximately 1 nM to approximately 4 mM, approximately 1 nM to approximately 3 mM, approximately 1 nM to approximately 2 mM, approximately 1 nM to approximately 1 mM, approximately 1 nM to approximately 900 μM, approximately 1 nM to approximately 800 μM, approximately 1 nM to approximately 700 μM, approximately 1 nM to approximately 600 μM, approximately 1 nM to approximately 500 μM, approximately 1 nM to approximately 400 μM, approximately... 1 nM - approximately 300 μM, approximately 1 nM - approximately 200 μM, approximately 1 nM - approximately 100 μM, approximately 1 nM - approximately 90 μM, approximately 1 nM - approximately 80 μM, approximately 1 nM - approximately 70 μM, approximately 1 nM - approximately 60 μM, approximately 1 nM - approximately 50 μM, approximately 1 nM - approximately 40 μM, approximately 1 nM - approximately 30 μM, approximately 1 nM - approximately 20 μM, approximately 1 nM - approximately 10 μM, approximately 1 nM - approximately 9 μM Approximately 1 nM to approximately 8 μM, approximately 1 nM to approximately 7 μM, approximately 1 nM to approximately 6 μM, approximately 1 nM to approximately 5 μM, approximately 1 nM to approximately 4 μM, approximately 1 nM to approximately 3 μM, approximately 1 nM to approximately 1 μM, approximately 1 nM to approximately 1 μM, approximately 1 nM to approximately 900 nM, approximately 1 nM to approximately 800 nM, approximately 1 nM to approximately 700 nM, approximately 1 nM to approximately 600 nM, approximately 1 nM to approximately 500 nM, approximately 1 n M - approximately 400 nM, approximately 1 nM - approximately 300 nM, approximately 1 nM - approximately 200 nM, approximately 1 nM - approximately 100 nM, approximately 1 nM - approximately 90 nM, approximately 1 nM - approximately 80 nM, approximately 1 nM - approximately 70 nM, approximately 1 nM - approximately 60 nM, approximately 1 nM - approximately 50 nM, approximately 1 nM - approximately 40 nM, approximately 1 nM - approximately 30 nM, approximately 1 nM - approximately 20 nM, or approximately 1 nM - approximately 10 nM.

[0129] In some embodiments, the NuRD inhibitor may include an HDAC1 / 2 inhibitor. In this application, the HDAC1 / 2 inhibitor may be known in the art and may include, but is not limited to, trichostatin A.

[0130] In some embodiments, the concentration of the HDAC1 / 2 inhibitor can be from about 1 nM to about 100 nM. For example, the concentration of the HDAC1 / 2 inhibitor can be from about 1 nM to about 5 nM, from about 1 nM to about 10 nM, from about 1 nM to about 20 nM, from about 1 nM to about 30 nM, from about 1 nM to about 40 nM, from about 1 nM to about 50 nM, from about 1 nM to about 60 nM, from about 1 nM to about 70 nM, from about 1 nM to about 80 nM, from about 1 nM to about 90 nM, or from about 1 nM to about 100 nM. For example, the concentration of the HDAC1 / 2 inhibitor can be from about 10 nM to about 100 nM, from about 10 nM to about 50 nM, from about 10 nM to about 40 nM, from about 10 nM to about 30 nM, or from about 10 nM to about 20 nM. For example, the concentration of the HDAC1 / 2 inhibitor can be approximately 20 nM.

[0131] In some embodiments, the NuRD inhibitor may include an MBD2 / 3 inhibitor. In this application, the MBD2 / 3 inhibitor may be known in the art and may include, but is not limited to, KCC-07. KCC-07 has a CAS Registry Number of 315702-75-1 and its molecular formula is C2. 14 H 11 N3OS, also known as 3-[[4-(2-pyridyl)-2-thiazolyl]amino]phenol.

[0132] In some embodiments, the concentration of the MBD2 / 3 inhibitor can be from about 0.05 μM to about 100 μM. For example, the concentration of the MBD2 / 3 inhibitor can be from about 0.05 μM to about 100 μM, from about 0.05 μM to about 50 μM, from about 0.05 μM to about 40 μM, from about 0.05 μM to about 30 μM, from about 0.05 μM to about 20 μM, from about 0.05 μM to about 15 μM, or from about 0.05 μM to about 10 μM. For example, the concentration of the MBD2 / 3 inhibitor can be from about 0.1 μM to about 10 μM, from about 0.5 μM to about 10 μM, from about 1 μM to about 10 μM, from about 2 μM to about 10 μM, from about 3 μM to about 10 μM, from about 4 μM to about 10 μM, or from about 5 μM to about 10 μM. For example, the concentration of the MBD2 / 3 inhibitor can be approximately 5 μM.

[0133] In some embodiments, the concentration of KCC-07 used can be from about 0.05 μM to about 100 μM. For example, the concentration of KCC-07 used can be from about 0.05 μM to about 100 μM, from about 0.1 μM to about 100 μM, from about 0.1 μM to about 40 μM, from about 0.1 μM to about 30 μM, from about 0.1 μM to about 20 μM, from about 0.1 μM to about 15 μM, or from about 0.1 μM to about 10 μM. For example, the concentration of KCC-07 used can be from about 0.1 μM to about 10 μM, from about 0.5 μM to about 10 μM, from about 1 μM to about 10 μM, from about 2 μM to about 10 μM, from about 3 μM to about 10 μM, from about 4 μM to about 10 μM, or from about 5 μM to about 10 μM. For example, the concentration of KCC-07 used can be about 5 μM.

[0134] In some embodiments, the first composition further comprises a BET inhibitor (iBET). In some embodiments, the first composition may reversibly comprise a BET inhibitor. In some embodiments, the first composition comprising a BET inhibitor may reversibly promote the enrichment of intestinal epithelial precursor cells in organoids and inhibit the differentiation of endocrine lineage cells in organoids; removing the BET inhibitor from the composition may allow the organoids to re-emerge with multiple differentiated functional cell types.

[0135] In this application, the BET inhibitor may be known in the art and may include, but is not limited to, iBET-151. The CAS Registry Number for iBET-151 is 1300031-49-5, and its molecular formula is C1. 23 H 21 N5O3.

[0136] In this application, the concentration of the BET inhibitor can be from about 0.01 μM to about 50 μM. For example, the concentration of the BET inhibitor can be from about 0.01 μM to about 50 μM, from about 0.01 μM to about 25 μM, from about 0.01 μM to about 10 μM, from about 0.01 μM to about 9 μM, from about 0.01 μM to about 8 μM, from about 0.01 μM to about 7 μM, from about 0.01 μM to about 6 μM, from about 0.01 μM to about 5 μM, from about 0.01 μM to about 4 μM, from about 0.01 μM to about 3 μM, from about 0.01 μM to about 2 μM, from about 0.01 μM to about 1 μM, from about 0.01 μM to about 0.5 μM, from about 0.01 μM to about 0.25 μM, or from about 0.01 μM to about 0.1 μM. For example, the concentration of the BET inhibitor can be about 0.02 μM to about 10 μM, about 0.03 μM to about 10 μM, about 0.04 μM to about 10 μM, about 0.05 μM to about 10 μM, about 0.06 μM to about 10 μM, about 0.07 μM to about 10 μM, about 0.08 μM to about 10 μM, or about 0.09 μM to about 10 μM. For example, the concentration of the BET inhibitor can be about 0.1 μM.

[0137] In some embodiments, the concentration of iBET-151 used can be from about 0.01 μM to about 10 μM. For example, the concentration of the BET inhibitor used can be from about 0.01 μM to about 9 μM, from about 0.01 μM to about 8 μM, from about 0.01 μM to about 7 μM, from about 0.01 μM to about 6 μM, from about 0.01 μM to about 5 μM, from about 0.01 μM to about 4 μM, from about 0.01 μM to about 3 μM, from about 0.01 μM to about 2 μM, from about 0.01 μM to about 1 μM, from about 0.01 μM to about 0.5 μM, from about 0.01 μM to about 0.25 μM, or from about 0.01 μM to about 0.1 μM. For example, the concentration of iBET-151 used can be about 0.02 μM to about 10 μM, about 0.03 μM to about 10 μM, about 0.04 μM to about 10 μM, about 0.05 μM to about 10 μM, about 0.06 μM to about 10 μM, about 0.07 μM to about 10 μM, about 0.08 μM to about 10 μM, or about 0.09 μM to about 10 μM. For example, the concentration of iBET-151 used can be about 0.1 μM.

[0138] On the other hand, this application provides a second composition and its use, the second composition being capable of inducing the production of intestinal epithelial functional cells.

[0139] In some embodiments, the second composition includes a Paneth cell-inducing composition comprising a Wnt signaling activator, an inhibitor of Notch or γ-secretase, and a member of the IL-10 family of cytokines.

[0140] In some embodiments, the Pane cell induction composition may further comprise one or more components from the group consisting of: BMP signaling inhibitors, EGF activators, FGF activators, IGF activators, TGFβ inhibitors, and vitamin C and its derivatives.

[0141] In some embodiments, the second composition includes a goblet cell inducing composition comprising goblet cell inducing composition A and goblet cell inducing composition B, wherein goblet cell inducing composition A comprises a Wnt signaling activator, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator, and goblet cell inducing composition B comprises a Wnt signaling inhibitor, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator.

[0142] In some embodiments, the second composition includes an intestinal endocrine cell inducing composition comprising intestinal endocrine cell inducing composition A and intestinal endocrine cell inducing composition B, wherein intestinal endocrine cell inducing composition A comprises a Wnt signaling activator, an inhibitor of Notch or γ-secretase, gastrin, a TGFβ inhibitor, and a BMP signaling inhibitor, and wherein intestinal endocrine cell inducing composition B comprises an inhibitor of Notch or γ-secretase, gastrin, a TGFβ inhibitor, a BMP signaling inhibitor, and a MAPK / EGFR signaling inhibitor.

[0143] In some embodiments, the second composition includes an intestinal epithelial cell inducing composition comprising an EGF activator, an FGF activator, an IGF activator, gastrin, a TGFβ inhibitor, a BMP signaling activator, a Wnt signaling inhibitor, and an HDAC inhibitor.

[0144] In some embodiments, the second composition includes an M-cell inducing composition comprising tumor necrosis factor-α (TNF-α) and receptor activator for nuclear factor-κB (RANKL). In some embodiments, the concentration of TNF-α used may be about 1 ng / mL to about 500 ng / mL, about 1 ng / mL to about 250 ng / mL, about 1 ng / mL to about 100 ng / mL, about 10 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 30 ng / mL to about 60 ng / mL, or about 50 ng / mL. In some embodiments, the concentration of RANKL used may be about 1 ng / mL to about 500 ng / mL, about 1 ng / mL to about 250 ng / mL, about 1 ng / mL to about 100 ng / mL, about 10 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 30 ng / mL to about 60 ng / mL, or about 50 ng / mL.

[0145] In some embodiments, the first composition and / or second composition described in this application comprise a Wnt signaling activator. In this application, the Wnt signaling activator may be known in the art, and may include, but is not limited to, Wnt-1 / Int-1, Wnt-2 / Irp (Int-I-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, R-spondin 5, R-spondin 6, R-spondin 7, R-spondin 8, R-spondin 9, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 9, R-spondin 1, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 1 ... 4. Norrin, CHIR99021, LiCl, BIO((2'Z,3E)-6-bromoindigo-3'-oxime), CHIR98014, SB216763, SB415286, 3F8, kenparone, 1-azakenparone, TC-G24, TCS2002, AR-A 014418, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, IQ 1, DCA, QS11, WAY-316606, (hetero)arylpyrimidine, 10Z-hemendexa, TCS 21311, TWS119, GSK-3 inhibitor IX, GSK-3 inhibitor IV, GSK-3β inhibitor II, GSK-3β inhibitor I, GSK-3β inhibitor XXVII, GSK-3β inhibitor XXVI, FRATTide, CdK1 / 5 inhibitors, Bikinin, and 1-azakenparonone. One or more Wnt signaling activators may be used, such as 2, 3, 4, or more.

[0146] The appropriate concentration can be selected depending on the type of Wnt signaling activator. In some embodiments, the concentration of the Wnt signaling activator can be about 0.01 μM to about 150 μM, about 0.1 μM to about 150 μM, about 0.5 μM to about 100 μM, about 0.1 μM to about 50 μM, about 0.2 μM to about 50 μM, about 1 μM to about 100 μM, about 0.5 μM to about 20 μM, about 10 μM to about 80 μM, about 1 μM to about 20 μM, or about 1 μM to about 5 μM. For example, the Wnt signaling activator includes CHIR99021, which is used at a concentration of about 4 μM.

[0147] In some embodiments, the first composition and / or the second composition described in this application comprise a Wnt signaling inhibitor. In this application, the Wnt signaling inhibitor may be known in the art, and may include, but is not limited to, IWP-2, XAV-939, ICG-001, LGK-974, IWR-1, KY02111, Wnt-C59, DKK-1, FH-535, Box5, peptide Pen-N3, anti-SFRP antibody, and anti-LRP6 antibody. One or more Wnt signaling inhibitors may be used, such as two, three, four, or more.

[0148] The appropriate concentration can be used depending on the type of Wnt signaling inhibitor. In some embodiments, the concentration of the Wnt inhibitor used can be about 0.01 μM to about 150 μM, about 0.1 μM to about 150 μM, about 0.5 μM to about 100 μM, about 0.1 μM to about 100 μM, about 0.2 μM to about 50 μM, about 1 μM to about 100 μM, or about 10 μM to about 80 μM, about 1 μM to about 20 μM, or about 1 μM to about 5 μM. For example, the Wnt signaling inhibitor includes IWR-1, which is used at a concentration of about 2 μM.

[0149] In some embodiments, the first composition and / or the second composition described in this application comprise a BMP signaling activator. In some embodiments, the BMP signaling activator includes a BMP pathway activator. In this application, the BMP signaling activator can be known in the art, and may include, but is not limited to, BMP2, BMP4, SB4, ventromorphins (including SJ000291942, SJ000063181 and SJ000370178), isoliquiritigenin, diosmetin, apigenin, and biochanin. One or more BMP signaling activators may be used, such as two, three, four, or more.

[0150] The appropriate concentration can be selected depending on the type of BMP signaling activator. In some embodiments, the concentration of the BMP signaling activator can be about 0.5 μM-500 μM, about 1 μM-500 μM, about 1 μM-250 μM, about 1 μM-100 μM, about 1 μM-50 μM, about 1 μM-25 μM, about 1 μM-20 μM, or about 5 μM-15 μM. For example, the BMP signaling activator includes SJ000291942, which is used at a concentration of about 10 μM.

[0151] In some embodiments, the first composition and / or the second composition described in this application comprise a BMP signaling inhibitor. In some embodiments, the BMP signaling inhibitor comprises a BMP pathway inhibitor. In this application, the BMP signaling inhibitor may be known in the art, and may include, but is not limited to, Noggin, Chordin, Follistatin, DAN, proteins containing the cysteine ​​domain of DAN, osteoscleroprotein, gastrulation protein, uterine sensitivity-related gene-1, connective tissue growth factor, inhibin, BMP-3, Dorsomorphin, and their derivatives including DMH1 and LDN-193189. One or more BMP signaling inhibitors may be used, such as two, three, four, or more.

[0152] The appropriate concentration can be selected depending on the type of BMP signaling inhibitor. In some embodiments, the concentration of the BMP signaling inhibitor can be about 0.1 μM to about 50 μM, about 0.1 μM to about 20 μM, about 0.5 μM to about 10 μM, or about 1 μM to about 5 μM. For example, the BMP signaling inhibitor includes DMH1, which is used at a concentration of about 2 μM.

[0153] In some embodiments, the first and / or second compositions described in this application comprise an EGF (epidermal growth factor) activator. The EGF activator described in this application may include, for example, EGF or molecules that activate EGF signaling pathways (e.g., bind to and activate EGF receptors). Examples of EGF activators include purified natural or recombinant EGF proteins, functional derivatives of EGF, and EGF analogs and mimics. In this application, the EGF activator may be known in the art and may include, but is not limited to, EGF, HB-EGF, amphiregulin, betacellulin, Epigen, epithelial regulatory protein, neuroregulatory protein 1, neuroregulatory protein 2, neuroregulatory protein 3, and neuroregulatory protein 4. One or more EGF activators may be used, such as two, three, four, or more.

[0154] The appropriate concentration can be selected depending on the type of EGF activator. In some embodiments, the concentration of the EGF activator can be about 1-1000 ng / mL, about 1-500 ng / mL, about 1-250 ng / mL, about 5 ng / mL to about 200 ng / mL, about 10 ng / mL to about 150 ng / mL, about 15 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 40 ng / mL to about 60 ng / mL, or about 50 ng / mL. For example, the FGF activator includes EGF, and its concentration is about 50 ng / mL.

[0155] In some embodiments, the first and / or second compositions described in this application comprise an FGF (fibroblast growth factor) activator. The FGF activators described in this application include, for example, FGF or molecules that activate FGF signaling pathways (e.g., bind and activate FGF receptors). Examples of FGF activators include purified natural or recombinant FGF proteins, functional derivatives of FGF, and FGF analogs and mimics. In this application, the FGF activator may be known in the art and may include, but is not limited to, bFGF (FGF-2), FGF4, FGF7, FGF9, and FGF10. One or more FGF activators may be used, such as two, three, four, or more.

[0156] The appropriate concentration can be selected depending on the type of FGF activator. In some embodiments, the concentration of the FGF activator can be about 1 ng / mL to about 1000 ng / mL, about 1 ng / mL to about 500 ng / mL, about 5 ng / mL to about 250 ng / mL, about 5 ng / mL to about 200 ng / mL, about 10 ng / mL to about 150 ng / mL, about 15 ng / mL to about 100 ng / mL, about 20 ng / mL to about 80 ng / mL, about 40 ng / mL to about 60 ng / mL, or about 50 ng / mL. For example, the FGF activator includes bFGF (FGF-2), and its concentration is about 50 ng / mL.

[0157] In some embodiments, the first and / or second compositions of this application comprise an IGF (insulin-like growth factor) activator. The IGF activators of this application include, for example, IGF or molecules that activate IGF signaling pathways (e.g., bind and activate IGF receptors). Examples of IGF activators include purified natural or recombinant IGF proteins, functional derivatives of IGF, and IGF analogs and mimics. In this application, the IGF activator may be known in the art and may include, but is not limited to, IGF-I and IGF-II. One or more IGF activators may be used, such as two, three, four, or more.

[0158] The appropriate concentration can be selected depending on the type of IGF activator. In some embodiments, the concentration of the IGF activator can be about 1 ng / mL to about 1000 ng / mL, about 1 ng / mL to about 500 ng / mL, about 5 ng / mL to about 250 ng / mL, about 10 ng / mL to about 200 ng / mL, about 20 ng / mL to about 200 ng / mL, about 50 ng / mL to about 150 ng / mL, about 75 ng / mL to about 150 ng / mL, about 75 ng / mL to about 125 ng / mL, or about 100 ng / mL. For example, the IGF activator includes IGF-I, and its concentration is about 100 ng / mL.

[0159] In some embodiments, the first and / or second compositions of this application comprise a TGFβ inhibitor. The TGFβ superfamily ligands include bone morphogenetic protein (BMP), growth and differentiation factor (GDF), anti-Müllerian hormone (AMH), activin, nodal, and TGFβ. Typically, Smad2 and Smad3 are phosphorylated by ALK4, 5, and 7 receptors in the TGF-β / activin pathway. In contrast, Smad1, Smad5, and Smad8 are phosphorylated as part of the bone morphogenetic protein (BMP) pathway. Although there is some overlap between pathways, in this application, a “TGFβ inhibitor” or “inhibitor of TGF-β signaling” is preferably a TGFβ pathway inhibitor that acts via Smad2 and Smad3 and / or via ALK4, ALK5, or ALK7. Therefore, in some embodiments, the TGFβ inhibitor is not a BMP signaling inhibitor. In some embodiments, in addition to a TGFβ inhibitor, the composition may also contain a BMP signaling inhibitor. In this application, the TGFβ inhibitor may be known in the art, and may include, but is not limited to, A83-01, SB505124, GW 788388, SB 525334 and dorsomorphine.

[0160] The appropriate concentration can be selected depending on the type of TGFβ inhibitor. In some embodiments, the concentration of the TGFβ inhibitor (e.g., A83-01) can be about 10 nM to about 50 μM, about 50 nM to about 100 μM, about 50 nM to about 10 μM, about 100 nM to about 1 μM, about 200 nM to about 800 nM, about 350 nM to about 650 nM, or about 500 nM. For example, the TGFβ inhibitor includes A83-01, which is used at a concentration of about 500 nM.

[0161] In some embodiments, the first and / or second compositions of this application comprise an inhibitor of Notch or a γ-secretase. In some embodiments, the Notch inhibitor is an inhibitor capable of reducing ligand-mediated Notch activation (e.g., via a dominant-negative ligand of Notch, or via a dominant-negative Notch, or via an antibody capable of at least partially blocking the interaction between the Notch ligand and Notch) or an inhibitor of the ADAM protease. In some embodiments, the Notch inhibitor is a γ-secretase inhibitor, such as DAPT, dibenzodiazepines (DBZ), benzodiazepines (BZ), or LY-411575. One or more Notch inhibitors or γ-secretase inhibitors may be used, such as two, three, four, or more.

[0162] The appropriate concentration can be selected depending on the type of Notch or γ-secretase inhibitor. In some embodiments, the concentration of the Notch or γ-secretase inhibitor (e.g., DAPT) can be about 0.5 μM to about 500 μM, about 0.5 μM to about 100 μM, about 1 μM to about 50 μM, about 1 μM to about 25 μM, about 1 μM to about 20 μM, or about 5 μM to about 15 μM. For example, Notch or γ-secretase inhibitors include DAPT, which is used at a concentration of about 10 μM.

[0163] In some embodiments, the first and / or second compositions of this application comprise a MAPK / EGFR signaling inhibitor. EGFR-mediated downstream pathways include the RAS-RAF-MAPK pathway, wherein phosphorylated EGFR recruits guanine-nucleotide exchange factors via the GRB2 and Shc adaptor proteins, thereby activating RAS and subsequently stimulating RAF and MAP kinase pathways to influence cell proliferation, tumor invasion, and metastasis. Activated RAS activates the protein kinase activity of RAF kinase. RAF kinase phosphorylates and activates MEK (also known as MAP2K or MAPKK), which in turn phosphorylates and activates MAP kinase (also known as ERK, an extracellular signal-regulated kinase). In some embodiments, the MAPK / EGFR signaling inhibitor comprises an EGFR inhibitor. The EGFR inhibitor may be known in the art and may include, but is not limited to, gefitinib, erlotinib, and lapatinib. In some embodiments, the MAPK / EGFR signaling inhibitor includes a RAS-RAF-MAPK pathway inhibitor. In some embodiments, the MAPK / EGFR signaling inhibitor includes a RAF inhibitor. In some embodiments, the MAPK / EGFR signaling inhibitor includes a MEK inhibitor. One or more MAPK / EGFR signaling inhibitors may be used, such as two, three, four, or more.

[0164] The appropriate concentration can be selected depending on the type of MAPK / EGFR signaling inhibitor. In some embodiments, the concentration of the MAPK / EGFR signaling inhibitor can be about 0.01-about 200 μM, about 0.01-about 100 μM, about 0.1-about 100 μM, about 0.1-about 50 μM, about 0.1-about 20 μM, about 1-about 100 μM, about 1-about 50 μM, about 1-about 30 μM, about 5-about 100 μM, about 5-about 50 μM, or about 5-about 20 μM. For example, the MAPK / EGFR signaling inhibitor includes gefitinib, which is used at a concentration of about 5 μM.

[0165] In some embodiments, the first composition and / or the second composition described in this application comprise gastrin. For example, the gastrin may include gastrin I. In some embodiments, the concentration of the gastrin used may be about 0.1 to about 500 nM, about 0.1 to about 100 nM, about 1 to about 100 nM, about 1 to about 20 nM, or about 5 to about 15 nM. For example, the concentration of the gastrin used is about 10 nM.

[0166] In some embodiments, the first and / or second compositions described in this application comprise members of the IL-10 family of cytokines. Examples of members of the IL-10 family of cytokines include purified natural or recombinant members of the IL-10 family of cytokines, functional derivatives of members of the IL-10 family of cytokines, and analogues and mimics of members of the IL-10 family of cytokines. The members of the IL-10 family of cytokines can be those known in the art and may include, but are not limited to, IL-10, IL-22, and IL-26. One or more members of the IL-10 family of cytokines may be used, such as two, three, four, or more.

[0167] The appropriate concentration can be selected based on the type of IL-10 family member. In some embodiments, the concentration of the IL-10 family member can be approximately 0.1 ng / mL to approximately 50 ng / mL, approximately 0.2 ng / mL to approximately 25 ng / mL, approximately 0.5 ng / mL to approximately 15 ng / mL, approximately 0.5 ng / mL to approximately 10 ng / mL, approximately 0.5 ng / mL to approximately 5 ng / mL, approximately 1 ng / mL to approximately 5 ng / mL, approximately 1 ng / mL to approximately 4 ng / mL, approximately 1 ng / mL to approximately 3 ng / mL, or approximately 2 ng / mL. For example, the IL-10 family member includes IL-22, which is used at a concentration of approximately 2 ng / mL.

[0168] On the other hand, this application also provides the use of the compositions described in this application and methods of using the compositions described in this application.

[0169] This application provides the first composition and its uses. The first composition described in this application can be used for culturing cells, preserving tissues, culturing tissues, preparing organoids, and / or culturing organoids.

[0170] For example, this application provides the use of a composition comprising the histone deacetylase (HDAC) inhibitor, the vitamin C and its derivatives and the platelet-derived growth factor receptor (PDGFR) inhibitor in culturing cells, preserving tissues, culturing tissues, preparing organoids and / or culturing organoids.

[0171] For example, this application provides the use of a composition comprising the histone deacetylase (HDAC) inhibitor, the vitamin C and its derivatives, the platelet-derived growth factor receptor (PDGFR) inhibitor and the BET inhibitor in culturing cells, preserving tissues, culturing tissues, preparing organoids and / or culturing organoids.

[0172] For example, this application provides the use of a composition comprising the NuRD complex inhibitor, the vitamin C and its derivatives and the platelet-derived growth factor receptor (PDGFR) inhibitor in culturing cells, preserving tissues, culturing tissues, preparing organoids and / or culturing organoids.

[0173] For example, this application provides the use of a composition comprising the NuRD complex inhibitor, the vitamin C and its derivatives, the platelet-derived growth factor receptor (PDGFR) inhibitor and the BET inhibitor in culturing cells, preserving tissues, culturing tissues, preparing organoids and / or culturing organoids.

[0174] This application provides the second composition and its uses. The second composition described in this application can be used for differentiating stem cells, inducing the generation of functional cells, preparing organoids, and / or culturing organoids.

[0175] For example, the second composition can be used to induce stem cells to differentiate into intestinal epithelial functional cells, culture intestinal epithelial functional cells, prepare organoids containing intestinal epithelial functional cells, and / or culture organoids containing intestinal epithelial functional cells.

[0176] For example, the second composition described in this application includes a Paneth cell induction composition, which can be used to induce intestinal stem cells to differentiate into Paneth cells, culture Paneth cells, prepare organoids containing Paneth cells, and / or culture organoids containing Paneth cells. For example, this application provides a method for inducing stem cells to differentiate into Paneth cells, culturing Paneth cells, preparing organoids containing Paneth cells, and / or cultured organoids containing Paneth cells, which includes using the second composition described in this application comprising the Paneth cell induction composition. On the other hand, this application also provides a kit for inducing stem cells to differentiate into Paneth cells, which comprises the second composition described in this application comprising the Paneth cell induction composition.

[0177] For example, the second composition described in this application includes a goblet cell induction composition, which can be used to induce intestinal stem cells to differentiate into goblet cells, culture goblet cells, prepare organoids containing goblet cells, and / or culture organoids containing goblet cells. For example, this application provides a method for inducing stem cells to differentiate into goblet cells, culturing goblet cells, preparing organoids containing goblet cells, and / or cultured organoids containing goblet cells, which includes using the second composition described in this application that includes the goblet cell induction composition. On the other hand, this application also provides a kit for inducing stem cells to differentiate into goblet cells, which comprises the second composition described in this application that includes the goblet cell induction composition.

[0178] For example, the second composition described in this application includes an intestinal endocrine cell inducing composition, which can be used to induce intestinal stem cells to differentiate into intestinal endocrine cells, culture intestinal endocrine cells, prepare organoids containing intestinal endocrine cells, and / or culture organoids containing intestinal endocrine cells. For example, this application provides a method for inducing stem cells to differentiate into intestinal endocrine cells, culturing intestinal endocrine cells, preparing organoids containing intestinal endocrine cells, and / or cultured organoids containing intestinal endocrine cells, which includes using the second composition described in this application comprising the intestinal endocrine cell inducing composition. On the other hand, this application also provides a kit for inducing stem cells to differentiate into intestinal endocrine cells, which comprises the second composition described in this application comprising the intestinal endocrine cell inducing composition.

[0179] For example, the second composition described in this application includes an intestinal epithelial cell inducing composition, which can be used to induce intestinal stem cells to differentiate into intestinal epithelial cells, culture intestinal epithelial cells, prepare organoids containing intestinal epithelial cells, and / or culture organoids containing intestinal epithelial cells. For example, this application provides a method for inducing stem cells to differentiate into intestinal epithelial cells, culturing intestinal epithelial cells, preparing organoids containing intestinal epithelial cells, and / or cultured organoids containing intestinal epithelial cells, comprising using the second composition described in this application that includes the intestinal endocrine cell inducing composition. On the other hand, this application also provides a kit for inducing stem cells to differentiate into intestinal endocrine cells, which comprises the second composition described in this application that includes the intestinal endocrine cell inducing composition.

[0180] In some embodiments, the stem cells include Lgr5+ stem cells. In some embodiments, the stem cells include epithelial stem cells. In some embodiments, the stem cells include intestinal stem cells. For example, the stem cells include Lgr5+ intestinal stem cells. In some embodiments, the stem cells include adult stem cells. In some embodiments, the stem cells include mammalian stem cells, such as human or mouse stem cells. For example, the stem cells include adult human or mouse stem cells. In some embodiments, the organoids include intestinal organoids. In some embodiments, the organoids include gastric organoids.

[0181] The first composition and the second composition described in this application can be used independently or in combination. In some embodiments, the combined use of the first composition and the second composition includes using them sequentially in a certain order. For example, the second composition may be used after the first composition is applied. The first composition and / or the second composition described in this application can be used in the preparation of culture media or added as additional ingredients to existing culture media to meet specific culture requirements. The first composition and / or the second composition described in this application can be directly added to a culture medium containing basic components to achieve its function. The first composition and / or the second composition described in this application can also be mixed with other components to form a customized culture medium to meet specific culture conditions or application scenarios.

[0182] Culture media and their uses

[0183] On the other hand, this application also provides a culture medium comprising the composition provided in this application.

[0184] In some embodiments, the culture medium described in this application may also contain basal culture medium components. Those skilled in the art will understand from common general knowledge the types of culture media that can be used as basal media in the culture medium described in this application. Potentially suitable cell culture media are commercially available, including but not limited to Durbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM / Ham's F12, Modified DMEM / Ham's F12, Iscove Modified Durbecco Media and Minimal Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI 1640.

[0185] For example, the basal medium can be selected from DMEM / F12 and RPMI 1640 supplemented with glutamine, insulin, penicillin / streptomycin, and transferrin. In some embodiments, a modified DMEM / F12 or modified RPMI optimized for serum-free culture is used and already contains insulin. In this case, the modified DMEM / F12 or modified RPMI medium is preferably supplemented with glutamine and penicillin / streptomycin. AdDMEM / F12 supplemented with N2 and B27 is also preferred. Preferably, the basal medium is modified DMEM / F12. More preferably, the basal medium contains modified DMEM / F12, glutamine, and B27.

[0186] In some implementations, the basal culture medium composition includes modified DMEM / F12, HEPES, penicillin / streptomycin, glutamine, N-acetylcysteine, and B27.

[0187] In some embodiments, the culture medium described in this application contains N-acetylcysteine. For example, N-acetylcysteine ​​may be present in the culture medium described in this application at the following concentrations: about 0.1 mM to about 200 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.5 mM to about 200 mM, about 0.5 mM to about 100 mM, about 0.5 mM to about 50 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM. In some embodiments, N-acetylcysteine ​​is present in the culture medium at a concentration of about 1 mM.

[0188] In some embodiments, the culture medium described in this application may contain one or more amino acids. Those skilled in the art will understand the appropriate types and amounts of amino acids used in the differentiation medium. Amino acids that may be present include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof. Some differentiation media will contain all of these amino acids. Typically, when present, each amino acid is present at about 0.001 to about 1 g / L of the medium (typically about 0.01 to about 0.15 g / L), except for L-glutamine, which is present at about 0.05 to about 1 g / L (typically about 0.1 to about 0.75 g / L). The amino acids may be of synthetic origin. In some embodiments, the culture medium described in this application may contain amino acid substitutes. For example, the culture medium described in this application may contain L-alanyl-glutamine, a substitute for L-glutamine.

[0189] In some embodiments, the culture medium described in this application is supplemented with purified, natural, semi-synthetic, and / or synthetic growth factors and does not contain unspecified components such as fetal bovine serum or calf serum. For example, supplements such as B27, N-acetylcysteine, and N2 stimulate the proliferation of some cells. In some embodiments, the culture medium is supplemented with one or more of these supplements, such as one, any two, or all three of these supplements.

[0190] In some embodiments, the culture medium described in this application contains B27. The B27 supplement described in this application is 'B27 supplement minus Vitamin A' (also referred to as 'B27 without Vitamin A' or 'B27 without Vitamin A'). In some embodiments, a generic formulation containing one or more components selected from the following can be used instead of the B27 supplement: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (Vitamin E), albumin, insulin, and transferrin. The components of the B27 supplement are known to those skilled in the art, and it is also contemplated that some or all of the B27 components can be added to the culture medium separately, instead of using the B27 supplement.

[0191] The culture medium described in this application is typically prepared in deionized distilled water. Before use, the culture medium is usually sterilized to prevent contamination, for example, by ultraviolet light, heating, radiation, or filtration. The culture medium may be frozen (e.g., at -20°C or -80°C) for storage or transport. The culture medium described in this application may contain one or more antibiotics to prevent contamination.

[0192] The culture medium described in this application can use any suitable pH. For example, the pH of the culture medium can be in the range of about 7.0 to 7.8, about 7.2 to 7.6, or about 7.4. A buffer can be used to maintain the pH. Those skilled in the art can readily select a suitable buffer. Buffers that can be used include, but are not limited to, carbonate buffers (e.g., NaHCO3) and phosphate buffers (e.g., NaH2PO4). These buffers are typically used at concentrations of about 50 to about 500 mg / L. Other buffers such as N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES) and 3-[N-morpholino]propanesulfonic acid (MOPS) can also be used, typically at concentrations of about 1000 to about 10,000 mg / L. In some embodiments, the buffer is selected from one or more of the following: phosphate buffers (e.g., KH₂PO₄, K₂HPO₄, Na₂HPO₄, NaCl, NaH₂PO₄), acetate buffers (e.g., HOAc or NaOAc), citrate buffers (e.g., citric acid or sodium citrate), or TRIS buffers (e.g., TRIS, TRIS-HCl), or organic buffers. In some embodiments, the organic buffer is a zwitterionic buffer, such as Good's buffers, for example selected from HEPES, MOPS, MES, ADA, PIPES, ACES, MOPSO, choline chloride, BES, TES, DIPSO, acetamindoglycine, TAPSO, POPSO, HEPPSO, HEPPS, N-tris(hydroxymethyl)methylglycine (Tricine), glycineamide, N-dihydroxyethylglycine (Bicine), TAPS, AMPSO, CABS, CHES, CAPS, and CAPSO. Preferred buffers are HEPES, such as HEPES at concentrations of 0.1-100 mM, 0.1-50 mM, 0.5-50 mM, 1-50 mM, 1-20 mM, or 5-15 mM. In some embodiments, HEPES is added to the culture medium at about 10 mM. The culture medium may also contain a pH indicator (such as phenol red) to allow easy monitoring of the pH status of the medium (e.g., at about 5 to about 50 mg / L).

[0193] The culture medium described in this application can be liquid, or it can be prepared into solid forms such as gel or agar medium as needed.

[0194] The isotonicity of the culture medium can be in the range of about 200 to about 400 mOsm / kg, about 290 to about 350 mOsm / kg, or about 280 to about 310 mOsm / kg. The isotonicity of the culture medium can be less than about 300 mOsm / kg (e.g., about 280 mOsm / kg).

[0195] The culture medium used in this application may contain carbon energy in one or more sugar forms. Those skilled in the art will understand the appropriate type and amount of sugar for use in the differentiation medium. Sugars that may be present include glucose, galactose, maltose, and fructose. The sugar is preferably glucose, especially D-glucose (dextrose). The carbon energy will typically be present at a concentration of about 1 to about 10 g / L.

[0196] In some embodiments, the culture medium described herein may contain serum. Serum obtained from any suitable source may be used, including fetal bovine serum (FBS), goat serum, or human serum. Serum may be used at about 1% to about 30% of the culture medium volume according to conventional techniques. In some embodiments, the culture medium described herein may contain serum substitutes. A variety of different serum substitute formulations are commercially available and are known to those skilled in the art. When using serum substitutes, they may be used at about 1% to about 30% of the culture medium volume according to conventional techniques. In some embodiments, the culture medium described herein may be serum-free and / or serum-free. Serum-free culture medium is a culture medium that does not contain any type of animal serum. Serum-free culture medium may be preferred to avoid potential xenogeneic contamination of stem cells. Serum-free substitute culture medium is a culture medium for which no commercial serum substitute formulation has been added.

[0197] In some embodiments, the culture medium described in this application may also contain cell supports. The cell supports described in this application may include, but are not limited to, matrix gelatin, gelatin, methylcellulose, collagen, alginate, alginate beads, agarose, fibrin, fibrin glue, fibrinogen, plasma fibrin beads, whole plasma or a component thereof, laminin, fibronectin, proteoglycans, HSP, chitosan, heparin, and materials of synthetic polymers or polymer scaffolds.

[0198] On the other hand, this application also provides an airtight container containing the culture medium provided in this application. The airtight container is preferably used for transporting or storing the culture medium to prevent contamination. The container can be any suitable container, such as a narrow-necked flask, plate, bottle, jar, vial, or bag.

[0199] On the other hand, this application also provides the use of the culture medium described in this application and methods including using the culture medium described in this application.

[0200] In some embodiments, the culture medium described in this application can be used to culture cells, preserve tissues, culture tissues, prepare organoids, and / or culture organoids. In some embodiments, the culture medium described in this application can be used to differentiate stem cells, induce the generation of functional cells, prepare organoids containing said functional cells, and / or culture organoids containing said functional cells.

[0201] In some embodiments, the culture medium comprises the first composition provided in this application. For example, the culture medium comprising the first composition can be used for culturing cells, preserving tissues, culturing tissues, preparing organoids, and / or culturing organoids.

[0202] In some embodiments, the culture medium comprises the second composition provided in this application. For example, the culture medium comprising the second composition can be used to differentiate stem cells, induce the generation of functional cells, prepare organoids containing said functional cells, and / or culture organoids containing said functional cells.

[0203] In some embodiments, the stem cells comprise Lgr5+ stem cells. In some embodiments, the stem cells comprise epithelial stem cells. In some embodiments, the stem cells comprise intestinal stem cells. For example, the stem cells comprise Lgr5+ intestinal stem cells.

[0204] In some embodiments, the stem cells include adult stem cells. In some embodiments, the stem cells include mammalian stem cells, such as human or mouse stem cells. For example, the stem cells include adult stem cells from humans or mice.

[0205] In some embodiments, the organoids include intestinal organoids. In some embodiments, the organoids include gastric organoids.

[0206] Organoids and their uses, methods for culturing organoids

[0207] On the other hand, this application also provides a method for preparing and / or culturing organoids.

[0208] In some embodiments, the method includes using the composition described in this application and / or the culture medium described in this application.

[0209] In some embodiments, the method includes the steps of: a) obtaining stem cells; and b) culturing the stem cells obtained in step a) in the presence of the composition described in this application or using the culture medium described in this application, thereby obtaining the organoid.

[0210] In some embodiments, the method includes the steps of: a) obtaining stem cells; and b) culturing the stem cells obtained in step a) in the presence of the first composition of this application or using a culture medium containing the first composition of this application, thereby obtaining the organoid.

[0211] In some embodiments, the stem cells include Lgr5+ stem cells. In some embodiments, the stem cells include epithelial stem cells. In some embodiments, the stem cells include adult stem cells.

[0212] In this application, the stem cells may be derived from epithelial tissue, more preferably adult epithelial tissue. Epithelial tissue includes the liver, intestine, stomach, prostate, lung, mammary gland, ovary, salivary gland, esophagus, bladder, or thyroid gland. Therefore, in some embodiments, the stem cells are obtained from the liver, intestine, stomach, prostate, lung, mammary gland, ovary, salivary gland, esophagus, bladder, or thyroid gland. In some embodiments, the stem cells are obtained from the stomach, lung, or intestine. In a preferred embodiment, the stem cells are obtained from the intestine or stomach.

[0213] In some embodiments, the stem cells include mammalian stem cells, such as human or mouse stem cells. For example, the stem cells include adult human or mouse stem cells.

[0214] In some embodiments, the stem cells are derived from a subject. The stem cells may be obtained directly from living tissue, or they may be cultured and / or passaged cells, or expanded cells. The term "expanded" means that the cells have been cultured in vitro in a medium that promotes cell expansion (e.g., proliferation) prior to their use in preparing organoids or differentiation.

[0215] In a preferred embodiment of this application, the stem cells are obtained from expanded epithelial stem cell cultures, preferably expanded organoids, which have been expanded and / or passaged without immortalization or transformation. In some embodiments, these expanded epithelial cultures or organoids may be genetically heterogeneous (different from conventional cell lines). Therefore, in some embodiments, the stem cells are not immortalized or transformed cells, or are not derived from immortalized or transformed cell lines.

[0216] The stem cells described in this application can be obtained by any suitable method. In some embodiments, cells can be isolated by collagenase digestion. In some embodiments, collagenase digestion is performed on tissue biopsy sections. In some embodiments, collagenase and accutase are used to digest and obtain the stem cells for use in this application.

[0217] In some implementations, the culture time in step b) is, for example, 3 days to 10 weeks, 1 to 10 weeks, 1 to 4 weeks, or 10 days to 3 weeks.

[0218] In some embodiments, the method further includes placing the stem cells obtained in step a) on a cell support, embedding them in the cell support, or mixing them with the cell support.

[0219] In some embodiments, the method further includes the step of inducing the organoid to produce functional cells.

[0220] In some embodiments, the organoids include intestinal organoids. In some embodiments, the method includes the steps of: a) obtaining stem cells; b) culturing the stem cells obtained in step a) in the presence of the first composition of this application or using a culture medium containing the first composition of this application; and c) culturing the stem cell culture obtained in step b) in the presence of the second composition of this application or using a culture medium containing the second composition of this application, thereby obtaining the organoids.

[0221] In some implementations, the culture time in step c) is, for example, 3 days to 10 weeks, 1 to 10 weeks, 1 to 4 weeks, or 10 days to 3 weeks.

[0222] In some embodiments, the stem cells include intestinal stem cells. For example, the stem cells include Lgr5+ intestinal stem cells. In some embodiments, the intestinal stem cells are derived from intestinal crypts. For example, the intestinal crypts include small intestinal or colonic crypts. In this application, the small intestine can refer to a segment of the digestive tract connecting the stomach and large intestine, which may be located between the stomach and the small intestine, and may include the duodenum, jejunum, and ileum; the colon can refer to a part of the large intestine, which may be located between the cecum and rectum, and may include the ascending colon, transverse colon, descending colon, and sigmoid colon.

[0223] In some embodiments, the stem cells include gastric stem cells. In some embodiments, the organoids include gastric organoids. The culture protocol for gastric organoids is similar to that for small intestinal organoids (see Sina Bartfeld, et al. In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection. Gastrienterology. 148(1):126-136(2015).). Therefore, the compositions, culture media, or methods for culturing organoids described in this application can also be used for culturing gastric organoids. This application also provides a gastric organoid and a method for preparing and / or culturing gastric organoids. In some embodiments, the gastric stem cells include Lgr5+ gastric stem cells. In some embodiments, the gastric stem cells are derived from gastric glands. In some embodiments, the gastric stem cells are derived from cardia glands, gastric body glands, and / or pyloric glands. In some embodiments, the organoid is an intestinal organoid, and the method further includes the step of inducing the organoid to generate intestinal epithelial functional cells.

[0224] For example, the intestinal epithelial functional cells include Paneth cells, and the method further includes culturing the organoids obtained in step b) in the presence of a Paneth cell-inducing composition, wherein the Paneth cell-inducing composition comprises a Wnt signaling activator, an inhibitor of Notch or γ-secretase, and a member of the IL-10 family of cytokines. In some embodiments, the Paneth cell-inducing composition further comprises one or more components from the group consisting of: a BMP signaling inhibitor, an EGF activator, an FGF activator, an IGF activator, a TGFβ inhibitor, and vitamin C and its derivatives.

[0225] For example, the intestinal epithelial functional cells include goblet cells, and the method further includes culturing the organoids obtained in step b) under conditions in the presence of goblet cell induction composition A and goblet cell induction composition B, respectively, wherein goblet cell induction composition A comprises a Wnt signaling activator, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator, and goblet cell induction composition B comprises a Wnt signaling inhibitor, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator. In some embodiments, the method includes culturing the organoids obtained in step b) for 1-3 days in the presence of the intestinal endocrine cell induction composition A, and then continuing to culture them for 1-5 days in the presence of the intestinal endocrine cell induction composition B.

[0226] For example, the intestinal epithelial functional cells include intestinal epithelial cells, and the method further includes culturing the organoids obtained in step b) in the presence of an intestinal epithelial cell induction composition, wherein the intestinal epithelial cell induction composition comprises an EGF activator, an FGF activator, an IGF activator, gastrin, a TGFβ inhibitor, a BMP signaling activator, a Wnt signaling inhibitor, and an HDAC inhibitor.

[0227] For example, the intestinal epithelial functional cells include M cells, and the method further includes culturing the organoids obtained in step b) in the presence of an M cell induction composition, wherein the M cell induction composition comprises TNF-α and RANKL. In some embodiments, the method includes culturing the organoids obtained in step b) for 4 hours in the presence of the M cell induction composition, followed by culturing for 3 days in the absence of TNF-α.

[0228] The method described in this application can be used in combination with methods known in the art for culturing stem cells, improving stem cell survival rate, enhancing stem cell proliferation capacity, or improving stem cell differentiation capacity. For example, it includes a step of culturing stem cells in the presence of a ROCK inhibitor. For example, the ROCK inhibitor may include Y27632. For example, the concentration of the ROCK inhibitor used may be about 0.1 μM to about 1000 μM, about 0.1 μM to about 1000 μM, about 1 μM to about 500 μM, about 1 μM to about 100 μM, about 1 μM to about 50 μM, about 5 μM to about 25 μM, or about 10 μM.

[0229] For example, the compositions or culture media described in this application may not contain certain components that are detrimental to organoid formation. For example, components detrimental to organoid formation can be found in Sato, T., et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011), and Fujii, M., et al. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell 23, 787-793e786 (2018). In some embodiments, the compositions or culture media described in this application do not contain p38 MAPK inhibitors, nicotinamide, and prostaglandins. In some embodiments, the compositions or culture media described in this application do not contain SB202190, nicotinamide, and PGE2.

[0230] On the other hand, this application also provides an organoid and its uses.

[0231] The organoids described in this application may comprise cell populations having the following cell numbers: at least 10 cells, at least 50 cells, at least 100 cells, at least 500 cells, at least 1 × 10⁻⁶ cells, or at least 1 × 10⁻⁶ cells. 3 1 × 10 cells, at least 1 × 10 4 1 × 10 cells, at least 1 × 10 5 Cells, at least 1 × 10 6 1 × 10 cells, at least 1 × 10 7 One or more cells. In some implementations, each organoid contains approximately 1 × 10⁻⁶ cells. 3 5 × 10 cells 3 One cell; typically, 10-20 organoids can grow together in one well (e.g., one well in a 24-well plate).

[0232] The organoids described in this application preferably comprise at least 50% live cells, more preferably at least 60% live cells, more preferably at least 70% live cells, more preferably at least 80% live cells, and more preferably at least 90% live cells. Cell viability can be assessed using Hoechst staining or propidium iodide staining in FACS. The live cells preferably possess corresponding in vivo functions or characteristics. For example, live enteroendocrine cells preferably possess enteroendocrine function or characteristics of enteroendocrine cells.

[0233] In some embodiments, the organoids described in this application have morphological features similar to organs or tissues in vivo. It will be clear to those skilled in the art that the organoids of this application are not naturally occurring tissue fragments and / or do not contain blood vessels.

[0234] In some embodiments, the organoids are prepared and / or cultured using the methods described in this application.

[0235] In some embodiments, the organoids possess cellular diversity and proliferative capacity, and simultaneously comprise mature intestinal epithelial cells, Paneth cells, Lgr5-expressing stem cells, and KI67-expressing proliferating cells. In some embodiments, the organoids are capable of long-term single-cell passage expansion and are rich in budding structures, containing little or no vesicular structures.

[0236] In some embodiments, the organoids further comprise goblet cells and / or enteroendocrine cells.

[0237] Methods for distinguishing the cell types present in organoids and detecting the presence of each cell type are known in the art. In some embodiments, the proportion of mature intestinal epithelial cells in the organoids may be ≥ about 0.1%. For example, the proportion of mature intestinal epithelial cells in the organoids may be ≥ about 0.1%, ≥ about 0.15%, ≥ about 0.2%, ≥ about 0.25%, ≥ about 0.3%, ≥ about 0.4%, ≥ about 0.5%, ≥ about 1%, ≥ about 2%, ≥ about 3%, ≥ about 4%, ≥ about 5%, or a higher proportion. Markers of mature intestinal epithelial cells may include, but are not limited to, ALPI and ACE2.

[0238] In some embodiments, the proportion of Lgr5-expressing stem cells in the organoid can be ≥ about 0.1%. For example, the proportion of Lgr5-expressing stem cells in the organoid can be ≥ about 0.1%, ≥ about 0.15%, ≥ about 0.2%, ≥ about 0.25%, ≥ about 0.3%, ≥ about 0.4%, ≥ about 0.5%, ≥ about 1%, ≥ about 2%, ≥ about 3%, ≥ about 4%, ≥ about 5%, or a higher proportion.

[0239] In some embodiments, the proportion of proliferating cells expressing KI67 in the organoids can be ≥ about 0.1%. For example, the proportion of stem cells expressing Lgr5 in the organoids can be ≥ about 0.1%, ≥ about 0.15%, ≥ about 0.2%, ≥ about 0.25%, ≥ about 0.3%, ≥ about 0.4%, ≥ about 0.5%, ≥ about 1%, ≥ about 2%, ≥ about 3%, ≥ about 4%, ≥ about 5%, or a higher proportion. In some embodiments, the proportion of Paneth cells in the organoids can be ≥ about 0.1%. For example, the proportion of Paneth cells in the organoids can be ≥ about 0.1%, ≥ about 0.15%, ≥ about 0.2%, ≥ about 0.25%, ≥ about 0.3%, ≥ about 0.4%, ≥ about 0.5%, ≥ about 1%, ≥ about 2%, ≥ about 3%, ≥ about 4%, ≥ about 5%, or a higher proportion. Markers for Paneth cells may include, but are not limited to, DEFA5, DEFA6, and LYZ.

[0240] In some embodiments, the proportion of goblet cells in the organoids can be ≥1%. For example, the proportion of goblet cells in the organoids can be ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3%, ≥3.5%, ≥4%, ≥4.5%, ≥5%, or higher. Markers of goblet cells may include, but are not limited to, MUC2 and SPINK4.

[0241] In some embodiments, the proportion of enteroendocrine cells in the organoid may be ≥1%. For example, the proportion of enteroendocrine cells in the organoid may be ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3%, ≥3.5%, ≥4%, ≥4.5%, ≥5%, or higher. Markers for enteroendocrine cells may include, but are not limited to, CHGA and NEUROD1.

[0242] In some embodiments, the organoids comprise cells expressing one or more genes selected from the group consisting of: FABP1, KRT20, ACE2, ALPI, DEFA5, DEFA6, and REG3A.

[0243] Cell types in organoids can be determined by detecting the presence of specific markers associated with a particular cell type. The term "expression" is used to describe the presence of intracellular markers. For a marker to be considered expressed, it must be present at a detectable level. "Detectable level" means that the marker can be detected using one of the standard laboratory methods (such as PCR, blot, or FACS analysis). If expression can be reasonably detected after 30 PCR cycles (which corresponds to an expression level in cells with at least about 100 copies per cell), the gene is considered to be expressed by cells of the population of the present invention. The terms "expression" and "expression" have the same meaning. Below this threshold, the marker is considered not to be expressed. A comparison of the expression level of the marker in the cells of the present invention with the expression level of the same marker in another cell type (e.g., embryonic stem cells) can preferably be made by comparing two cell types that have been isolated from the same species. Preferably, the species is a mammal, and more preferably, a human. Such comparisons can be conveniently performed using reverse transcriptase polymerase chain reaction (RT-PCR) experiments. In some implementations, intracellular marker mRNA expression is measured by single-cell RNA sequencing analysis.

[0244] In this application, the cell proportion generally refers to the proportion of a specific cell type in the total cell population. This proportion can be determined in various ways, including but not limited to: 1) using flow cytometry (FACS) or fluorescence activated cell sorting technology to label and sort cells using specific markers and count the number of target cells; 2) using immunohistochemistry or immunofluorescence methods to observe and count the proportion of a specific cell type under a microscope; 3) using gene expression analysis, such as using quantitative PCR (qPCR) or RNA sequencing technology, to indirectly estimate the proportion of a specific cell type in the cell population based on the expression level of a marker gene for that cell type.

[0245] This application also provides for the use of the organoids of this application and the cells derived from said organoids. When organoids are referred to in this section, these are differentiated organoids according to the invention. Those skilled in the art will understand that their use is also applicable to cell populations obtained and / or available by the methods of this application. Such uses of cell populations obtained and / or available by the methods of this application are also provided.

[0246] For example, this application provides the use of the organoids or cells derived from the organoids in the following: drug discovery screening; toxicity assays; histological, embryological, cell lineage and differentiation pathway studies; studies to identify chemical and / or neuronal signals that lead to the release of their respective hormones; gene expression studies, including recombinant gene expression; studies of mechanisms involved in tissue damage and repair; inflammatory and infectious disease studies; pathogenesis studies; or studies of cell transformation and cancer etiology mechanisms.

[0247] This application also provides the organoids of the present invention or cells derived from said organoids for use in medicine.

[0248] This application also provides the use of organoids or cells derived from said organoids in the treatment of symptoms, conditions or diseases.

[0249] This application also provides the use of organoids or cells derived from said organoids in regenerative medicine, for example, said use involves transplanting organoids or cells into a patient.

[0250] This application provides the use of organoids or cells derived from said organoids in drug screening, (drug) target validation, (drug) target discovery, toxicology and toxicology screening, personalized medicine, regenerative medicine and / or as ex vivo cell / organ models (such as disease models).

[0251] Cells and organoids cultured according to the culture medium and method of this application are considered to faithfully represent the in vivo situation. This is true for both differentiated cell populations and organoids grown from normal tissues and differentiated cell populations and organoids grown from diseased tissues. Therefore, in addition to providing normal ex vivo cell / organ models, the organoids of this application can be used as ex vivo disease models.

[0252] The organoids of this application can also be used to culture pathogens and therefore can be used as in vitro infection models. Examples of pathogens that can be cultured using the organoids of this application include viruses, bacteria, prions, or fungi that cause disease in their animal hosts. Therefore, the organoids of this application can be used as disease models representing infection states. In some embodiments of this application, the organoids can be used for vaccine development and / or production.

[0253] Therefore, diseases that can be studied through the organoids of this application include genetic diseases, metabolic diseases, pathogenic diseases, inflammatory diseases, etc., such as but not limited to: diabetes (e.g., type I or type II), cystic fibrosis, cancer, adenocarcinoma, adenoma, gastrointestinal neuroendocrine tumors, and inflammatory bowel diseases (e.g., Crohn's disease).

[0254] Example

[0255] The present application is further illustrated by the following specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods not specifically described in the following embodiments are performed according to conventional conditions in the art, such as those described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001), CSHL Press, or according to the manufacturer's recommendations.

[0256] The actual chemicals, peptides, and recombinant proteins used in the examples are as follows: Advanced DMEM / F-12 (Gibco, Cat#12634010), B-27 supplement (without Vitamin A, Gibco, Cat#12587010), N-acetyl-L-cysteine ​​(Sigma, Cat#A9165), HEPES buffer (Corning, Cat#25-060-CI), GlutaMAX™ supplement (Gibco, Cat#35050061), penicillin-streptomycin (Gibco, Cat#15140122), and Opti-MEM. TM I-reduced serum medium (Gibco, Cat#51985034), TrypLE TMExpress enzyme (1×, Gibco, Cat#12605028), recombinant mouse EGF (Peprotech, Cat#315-09), recombinant human IGF-I (Peprotech, Cat#100-11), recombinant human FGF (basic fibroblast growth factor, Peprotech, Cat#100-18C), DMH1 (ApexBio, Cat#B3686), gastrin I (MCE, Cat#HY-P1097), A83-01 (ApexBio, Cat#A3133), CHIR-99021 (ApexBio, Cat#A3011), Trichostatin A (TSA, ApexBio, Cat#A8183), trisodium 2-phosphate-L-ascorbic acid (pVc, Sigma, Cat#49752), CP673451 (TargetMol, Cat#T6091), Crenolanib (TargetMol, Cat#T2677), Y-27632 dihydrochloride (TargetMol, Cat#T1725), sodium valproate (VPA, Sigma, Cat#P4543), turostatin hydrochloride (Tubastatin A) HCl, ApexBio, Cat#A8547), LMK235 (TargetMol, Cat#T6061), CAY10683 (Selleck, Cat#S7595), Gefitinib (TargetMol, Cat#T1181), DAPT (TargetMol, Cat#T6202), IWR-1 (TargetMol, Cat#T2651), SJ000291942 (Tar getMol (Cat#T4662), I-BET151 (Selleck, Cat#S2780), KCC-07 (TargetMol, Cat#T8554), recombinant human IL-22 (PrimeGene, Cat#101-22), recombinant human RANKL (Peprotech, Cat#310-01), recombinant human TNF-α (Peprotech, Cat#300-01A), and basement membrane extract (type 2, R&D Systems, Cat#3533-005-02).

[0257] Unless otherwise stated, the cell culture reagents, small molecule compounds, antibodies and molecular biology reagents used in this application are all commercially available products in the field, and those skilled in the art may choose equivalent substitutes as needed.

[0258] The antibodies used for immunostaining and flow cytometry were commercially available antibodies targeting common intestinal cell markers, including antibodies for detecting lysozyme (LYZ) and α-defensin 5 (DEFA5) in Paneth cells, MUC2 antibody for detecting goblet cells, antibodies for detecting chromogranin A (CHGA), somatostatin (SST), and glucagon (GCG) in enteroendocrine cells, OLFM4 antibody for detecting intestinal stem cells, GP2 antibody for detecting M cells, and E-cadherin antibody for epithelial cell junction labeling, etc., along with corresponding fluorescently labeled secondary antibodies. Nuclear staining used DAPI or functionally equivalent nuclear dyes.

[0259] Molecular cloning and nucleic acid manipulation employed standard molecular biology reagents, including restriction endonucleases, high-fidelity DNA polymerases, DNA ligases, alkaline phosphatases, chemocompetent cells, and commercially available kits for genomic DNA extraction, gel extraction, plasmid preparation, and PCR purification. RNA extraction used standard phenol / chloroform reagents, reverse transcription employed a commercial cDNA synthesis kit, and quantitative PCR was performed using the SYBR Green system. Cell proliferation was detected using an EdU probe-based cell proliferation assay kit, and alkaline phosphatase activity was detected using standard alkaline phosphatase chromogenic reagents.

[0260] method

[0261] 1. Constructing an LGR5-mNeonGreen reporter system and an MBD3 knockdown system for human small intestinal organoids.

[0262] To construct the fluorescent reporter gene, the LGR5-P2A-mNeonGreen reporter gene (SEQ ID NO: 4) was introduced into the last exon of LGR5 via electroporation, using the previously published sequence for sgRNA and homologous arms (Shimokawa, M., et al. Visualization and targeting of LGR5(+)human colon cancer stem cells. Nature 545, 187-192 (2017).). Two LGR5-targeting sgRNAs (sgRNA sequences shown in SEQ ID NO:1 and SEQ ID NO:2) were cloned into the HP180 plasmid containing SpCas9 (the HP180 plasmid backbone is plasmid pX330(Addgene, #42230), with an EGFP sequence inserted after the Cas9 gene; the HP180 plasmid map can be found in Huo Mengfei, Meng Fanming, Wang Sutian, et al. Construction and functional verification of CRISPR / Cas9 vector targeting porcine Y chromosome [J]. Journal of South China Agricultural University, 2023, 44(02):187-196.), generating LGR5 sgRNA plasmids. Fast-Fusion was used to... TM The cloning kit (GeneCopoeia) replaced the CMV promoter in pBluescript II SK(+) with the LGR5-P2A-mNeonGreen sequence, flanked by LGR5 homologous arms. Using a NEPA21 electroporator (Nepa Gene), the LGR5 sgRNA plasmid and HDR template were co-electroplated into human intestinal organoids according to published methods (Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10, 1474-1485 (2015).). Positive clones were then manually selected, amplified, and genotyped.

[0263] To interfere with MBD3 expression, the sgRNA targeting MBD3 (the sequence of the sgRNA is shown in SEQ ID NO: 3) was cloned into the SpCas9-equipped HP180 plasmid and spot-transferred into LGR5-mNeonGreen cells. The clones that grew on single cells were selected, amplified, and screened using Western blotting.

[0264] 2. Crypt isolation and organoid culture

[0265] The small intestinal tissue used in this application was obtained from a patient's biopsy sample. The study was approved by the Ethics Committee of Shanghai Oriental Hospital, and the patient provided written informed consent (ethics approval number: 2023-064). The obtained biopsy tissue samples were transferred to Dulbecco phosphate-buffered saline (DPBS) and placed on ice until the next step of processing. Cell isolation was performed according to the published protocol (Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10, 1474-1485 (2015).). Briefly, the tissue was washed with DPBS to remove debris. The tissue was treated with 2.5 mM EDTA at 4°C for 40 min to release crypts. The supernatant containing crypts was collected and embedded in Cultrex RGF BME (R&D Systems) droplets (approximately 50 crypts / well) in 48-well plates. After solidification, the droplets were covered with medium containing 10 μM Y-27632 (TargetMol). The medium was changed every 3 days. For cell passage, the established organoids were dissociated into single cells using TrypLE (Gibco) and re-embedded in fresh BME at 8,000 cells per well. The basal medium consisted of Advanced DMEM / F12 (Gibco), supplemented with B-27 (Gibco), 10 mM HEPES (Corning), 2 mM GlutaMAX (Gibco), penicillin-streptomycin (Gibco), and 1 mM N-acetyl-L-cysteine ​​(Sigma).

[0266] The organoid TpC culture medium consisted of: basal medium supplemented with 50 ng / mL EGF, 100 ng / mL human IGF-I (Peprotech), 50 ng / mL FGF-2 (Peprotech), 2 μM DMH1, 10% R-spondin1 conditioned medium, 10 nM gastrin I (MCE), 0.5 μM A83-01, 4 μM CHIR, 20 nM TSA (ApexBio), 100 μg / mL pVc (Sigma), and 0.5 μM CP673451 (TargetMol).

[0267] 3. Direct differentiation in organoids

[0268] The culture medium was directly replaced with differentiation medium to induce organoid differentiation. The differentiation protocol is as follows:

[0269] Paneth cells (PC): EGF (50 ng / mL), IGF-I (100 ng / mL), FGF-2 (50 ng / mL), DMH1 (2 μM), R-spondin1 CM (10%), gastrin I (10 nM), A83-01 (0.5 μM), CHIR (4 μM), DAPT (10 μM), pVc (100 μg / mL), and IL-22 (2 ng / mL). Cultured for 3 days.

[0270] Goblet cells (GC): SJ000291942 (10 μM), R-spondin 1 CM (10%), gastrin I (10 nM), A83-01 (0.5 μM), CHIR (4 μM) and DAPT (10 μM) were treated for 2 days, and then treated with IWR-1 (2 μM) instead of CHIR for 3 days.

[0271] Enteroendocrine cells (EECs): treated with DMH1 (2 μM), R-spondin1 CM (10%), gastrin I (10 nM), A83-01 (0.5 μM), CHIR (4 μM), DAPT (10 μM), and CP673451 (0.5 μM) for 2 days, followed by treatment with DMH1 (2 μM), gastrin I (10 nM), A83-01 (0.5 μM), DAPT (10 μM), CP673451 (0.5 μM), and Gefenitib (5 μM) for 3 days.

[0272] Intestinal epithelial cells (ECs) were treated with EGF (50 ng / mL), IGF-I (100 ng / mL), FGF-2 (50 ng / mL), SJ000291942 (10 μM), gastrin I (10 nM), A83-01 (0.5 μM), IWR-1 (2 μM), and VPA (0.75 mM) for 3 days.

[0273] M cells: TNF-α (50 ng / mL) and RANKL (100 ng / mL) were treated for 4 hours, then TNF-α was withdrawn, for a total of 3 days.

[0274] 4. EdU cell proliferation assay: 10 μM EdU (5-ethynyl-2'-deoxyuridine, Beyotime) was added to organoid culture medium and cultured for 1 hour. Then, organoids were collected, fixed, permeabilized, and EdU was detected according to the manufacturer's instructions (Beyotime).

[0275] 5. Flow Cytometry Detection: Organoids were dissociated from the BME using TrypLE and separated into single cells. Cells were washed with ice-cold DPBS and filtered through a 40 μm Falcon cell filter into fluorescence-activated cell sorting (FACS) tubes. Cells were stained with Hoechst (Beyotime) to identify live cells before FACS analysis. Flow cytometry was performed on a BD FACSVerse (BD Biosciences), and data analysis was conducted in FlowJo. The gate-gating strategy for the mNeonGreen fluorescent reporter gene was consistent throughout this application.

[0276] 6. Immunostaining: Immunofluorescence was performed as described previously (Yin, X., et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat Methods 11, 106-112 (2014).). In short, after releasing the organoids from the substrate gel, they were washed with buffer and fixed with approximately 4% paraformaldehyde at room temperature, followed by permeabilization and blocking in a buffer containing nonionic surfactants and bovine serum albumin. Samples were incubated overnight at 4°C in blocking buffer containing primary antibodies, including antibodies against lysozyme (LYZ), mucin 2 (MUC2), chromogranin A (CHGA), α-defensin 5 (DEFA5), OLFM4, somatostatin (SST), glucagon (GCG), glycoprotein 2 (GP2), and E-cadherin. After washing, the corresponding fluorescently labeled secondary antibody was added and incubated at room temperature. After DAPI staining of the nuclei, images were taken under a confocal or inverted fluorescence microscope, and quantitative analysis was performed using standard image analysis software. Alkaline phosphatase activity (ALPI) was detected using a commercially available alkaline phosphatase colorimetric kit according to the manufacturer's instructions.

[0277] 7. RNA extraction and quantitative reverse transcription PCR (RT-qPCR): Organoids were collected before lysis and washed with ice-cold DPBS. They were then lysed with a phenol-containing total RNA extraction reagent (e.g., Trizol-based reagent) and total RNA was extracted according to the instructions. No more than 1 μg of total RNA was used to synthesize cDNA using a standard reverse transcription kit. Real-time quantitative PCR was performed on a real-time PCR instrument using the SYBR Green system, and relative gene expression levels were calculated using the ΔΔCt method. The qPCR primer sequences are shown in SEQ ID NO:5-10.

[0278] 8. Clonogenesis Assay: Organoids were dissociated into single cells and seeded into 48-well plates at a density of 8,000 cells / well. After 7 days, the clones were fixed, and the colony formation efficiency was calculated using DAPI staining. Clonogenesis efficiency = Number of clones / Initial number of single cells in culture (8000) × 100%.

[0279] 9. Single-cell RNA sequencing data processing: Cell and library preparation: TpC organoids were digested into single cells (37℃ for 10 min), filtered, counted, and then used to construct a library using a 10x Genomics Chromium 3' kit, and sequenced on a NovaSeq6000. Clustering and annotation: The raw data were aligned and counted using CellRanger. After removing double-cell and low-quality cells, Seurat was used for normalization, dimensionality reduction, and clustering. Cell types were annotated according to classical marker genes, including stem cells (LGR5, OLFM4, ASCL2), TA1 (PCNA, HELLS, MCM6), TA2 (MKI67, TOP2A), early intestinal cells (Early EC) (FABP1, KRT20), mature intestinal cells (EC) (ALPI, ACE2), secretory precursors (SecPre) (HES6, DLL1, DLL4), goblet cells (GC) (MUC2, SPINK4, FCGBP), Paneth cells (PC) (DEFA5, DEFA6, PRSS2), enteroendocrine cells (EEC) (CHGA, NEUROD1), and tuft cells (ALOX5, AVIL). Trajectory Analysis: Developmental trajectory and RNA velocity analysis were performed using Scanpy's PAGA and Dynamo tools, with hypervariable gene sets visualized along Seurat's UMAP coordinates. Data Integration: Publicly available data on ES, IF, IL22 organoids and in vivo crypts were processed using the same workflow, integrated using Seurat, and inferred from the signaling pathway activity of each cell population using PROGENy. TA2 cells in the IL22 dataset were renamed TA1 and TA2 based on the expression of the aforementioned TA markers. TA1 cells were renamed early intestinal cells because they expressed early EC markers instead of TA markers. Intestinal stem cell (ISC) populations were divided into LGR5-high expression and LGR5-low expression subpopulations. In the IL22 dataset, since no LGR5-high expression subpopulation was identified, all intestinal stem cells (ISCs) were labeled as the LGR5-low expression subpopulation.

[0280] Example 1: Establishment of a stem-enhanced human intestinal organoid system

[0281] To enhance cellular diversity in human intestinal organoids, our initial goal was to increase the proportion of LGR5+ stem cells in the organoids, rather than directly driving stem cell differentiation. To achieve this, we attempted to replicate the in vivo stem cell microenvironment using a combination of small molecules and growth factors (Fig. 1A). We developed the LGR5-mNeonGreen reporter system to visualize LGR5+ stem cells in the organoids (Figs. 1B-1C). We compared the components used in previously determined culture conditions (Fig. 1D). ES conditions promoted the expansion of intestinal progenitor cells but inhibited their differentiation, resulting in the absence of secretory cell types in the organoids (Sato, T., et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).). While modified IF conditions enable organoids to differentiate into multiple lineages and self-renew, organoids under IF conditions still exhibit limited cellular diversity and lack key cell types, including mature intestinal epithelial cells (ECs) and paneth cells (PCs) (Fujii, M., et al. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell 23, 787-793e786 (2018)). Recently developed optimized conditions use IL22 to induce the generation of paneth cells (PCs) based on ES conditions, but this comes at the cost of inhibiting organoid growth and proliferation (He, GW, et al. Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation. Cell Stem Cell 29, 1333-1345e1336 (2022)). Organoids cultured under IL22 conditions have very low proliferative capacity, which is not conducive to organoid expansion.

[0282] Using the LGR5-mNeonGreen reporter gene, we found that LGR5-mNeonGreen expression was extremely low in stem cells under IF and IL culture conditions (He, GW, et al. Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation. Cell Stem Cell 29, 1333-1345e1336 (2022).) (Fig. 1E), but under IF conditions, various secretory cell types were generated (Fig. 2B), which is consistent with previous studies. To promote the maintenance of LGR5+ stem cells while preserving their differentiation process, we added key factors used in mouse intestinal organoid culture to the organoid culture medium, including EGF, the BMP inhibitor Noggin (or small molecule DMH1), and R-Spondin1. We removed factors such as SB202190, nicotinamide, and PGE2, combined with intestinal stem cell (ISC) microenvironment factors such as IGF1 and FGF2, and used CHIR99021 as a substitute for Wnt protein. We also added the ALK inhibitor A83-01, which promotes cell growth. We used this condition as the basis for screening small molecule drugs (Figures 1A and 1D). Through screening, we found that a combination of three small molecules (TpCs), including Trichostatin A (abbreviated as TSA or T, an HDAC inhibitor), 2-phospho-L-ascorbic acid (abbreviated as pVc or p, vitamin C), and CP673451 (abbreviated as CP or C, a PDGFR inhibitor), significantly increased the proportion of LGR5-mNeonGreen positive cells and their mNeonGreen expression level in the obtained organoids (Figure 1F). Furthermore, the colony formation efficiency of ex vivo single cells was significantly improved, and the total number of cells in the culture was also significantly increased (Figures 1F and 2A). These results indicate that the combination of TpCs improved the proportion of LGR5 stem cells, cloning efficiency, and organoid proliferation rate.

[0283] Example 2: Generating diverse and fate-plastic cell types under TpC conditions

[0284] Under TpC conditions, dissociated single cells efficiently generate organoids, with scattered LGR5-mNeonGreen expression observed in each clone (Fig. 2C). Long-term culture leads to the formation of extensive crypt-like budding structures within the organoids, containing Paneth-like cells with dark granules (Fig. 2C), indicating cell differentiation.

[0285] TpC conditions, while promoting organoid proliferation, can induce the generation of various intestinal cells, as evidenced by positive staining for markers such as mature intestinal cells (alkaline phosphatase, ALPI), goblet cells (GC) (mucin 2, MUC2), enteroendocrine cells (EEC) (chromogranin A, CHGA), and Paneth cells (PC) (defensin α5, DEFA5, and lysozyme, LYZ) (Fig. 1G and 1H). In the organoids, spaced-alternating DEFA5-negative / LGR5-positive cells and DEFA5-positive Paneth cells (PCs) were observed, closely resembling the structural distribution of stem cells and Paneth cells in in vivo in intestinal crypts. We also observed the coexistence of DEFA5-positive / LGR5-positive cells in the organoids, which may reflect the retention of LGR5-mNeonGreen expression when LGR5-positive stem cells differentiate into Paneth cells (PCs) (Fig. 1H). The budding region also exhibited extensive OLFM4 expression (Fig. 2D), a highly specific stem cell marker (Schuijers, J., van der Flier, LG, van Es, J. & Clevers, H. Robust cre-mediated recombination in small intestinal stem cells utilizing the olfm4 locus. Stem Cell Reports 3, 234-241 (2014).). Furthermore, the organoids contained various EEC cell subtypes, including SST and GCG (Fig. 2D) positive cells. Notably, regardless of whether the culture was short-term (7–10 days) or long-term (3–4 weeks), LGR5-positive stem cell and secretory cell progeny, such as Paneth cells (PC), goblet cells (GC), and enteroendocrine cells (EEC), were uniformly distributed in organoids cultured under TpC conditions (Fig. 2C, Fig. 2F, and Fig. 3C). This indicates that TpC-derived organoids exhibit high homogeneity. Furthermore, the TpC condition supported the generation and long-term maintenance of hSIOs from multiple donors (Fig. 2E), demonstrating the robustness of the culture system. Most organoids under the TpC condition were budding, but a small number were round or vesicular (Fig. 3C). The LYZ gene is widely expressed in human small intestinal organoids, but at the protein level, lysozyme can specifically label Paneth cells and co-stain with another Paneth cell-specific gene, DEFA5 (Fig. 3A).

[0286] To investigate cell growth dynamics, we tracked the proliferation of single LGR5-positive stem cells under TpC conditions. We observed that single LGR5-positive stem cells could generate organoids of various secretory cell types, including Paneth cells (PCs), goblet cells (GCs), and enteroendocrine cells (EECs) (Figure 1I). We also detected the disappearance and reappearance of LGR5-mNeonGreen expression in organoids, indicating the presence of dynamic differentiation and dedifferentiation processes of stem cells within organoids (Figure 1I). These phenomena suggest that TpC conditions support the generation of dynamic and fate-plastic intestinal cell populations in organoids, and that TpC organoids have the ability to generate multiple cell types and support plastic fate transitions among these cells.

[0287] Intestinal cells exhibit strong plasticity (Tetteh, PW, et al. Replacement of Lost-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters. Cell Stem Cell 18, 203-213 (2016). van Es, JH, et al. Dll1 secretory progenitor cells revert to stem cells upon crypt damage. Nat Cell Biol 14, 1099-+ (2012). Buczacki, SJ, et al. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 495, 65-69 (2013).). To detect the cloning ability of various cells in organoids, we used flow cytometry to sort LGR5-negative, LGR5-low (LGR5-low), and LGR5-high (LGR5-high) cells, and cultured them at the same cell density under TpC conditions (Figure 4A). We found that LGR5-negative cells exhibited the highest clonogenic efficiency, and LGR5-positive cells also appeared during organoid formation (Figure 4B). We hypothesize that some progenitor cells or other types of stem cells also possess organoid formation capabilities. These results indicate that organoids cultured under TpC conditions contain dynamic and plastic intestinal cells, possessing the ability to generate multi-lineage cells and exhibiting extremely high plasticity.

[0288] Example 3: Single-cell sequencing analysis revealed cellular diversity and cell fate dynamics in TpC organoids.

[0289] To gain a deeper understanding of the cellular diversity of organoids cultured from TpC, we performed single-cell RNA sequencing (scRNA-seq). We annotated the cells based on the expression of maturation markers and identified 12 distinct cell populations (Figure 5A). Within these cell populations, we found that intestinal stem cells (ISCs) could be further divided into two subpopulations based on their LGR5 and OLFM4 expression levels (Figures 5A-5B). Specifically, we identified ISCs with high and low LGR5 expression, two transiently expanding (TA) cell populations expressing low levels of LGR5 but high levels of cell cycle genes such as MKI67 and PCNA, a population of FABP1-positive early intestinal epithelial cells (early ECs) lacking LGR5 expression, mature intestinal epithelial cells (ECs) expressing ALPI and ACE2, and various secretory cell types, including Paneth cells (expressing DEFA5 and DEFA6), goblet cells (GCs) (expressing MUC2 and SPINK4), enteroendocrine cells (EECs) (expressing CHGA and NEUROD1), and cluster cells (expressing ALOX5 and AVIL) (Figure 5B). We discovered two distinct secretory precursor cell types. The first type (SecPre) expresses classic secretory precursor markers such as HES6, DLL1, and DLL4, representing typical secretory precursor cells. The second type (SecPre2) expresses OLMF4 as well as markers for secretory cells (goblet cells (GC), Paneth cells (PC), and enteroendocrine cells (EEC)) and absorptive cells (FABP1 and KRT20) (Figure 5B). We infer that these cells represent cells that transdifferentiate from absorptive cells to secretory cells.

[0290] Differential gene expression analysis confirmed that, across all functional cell subpopulations, classical markers for each cell type were upregulated relative to ISCs with high LGR5 expression. Notably, DEFA5, DEFA6, and PRSS2 showed the most significant expression changes in Paneth cells (PCs). To better understand the secretory cell subpopulations, we performed a subpopulation analysis of secretory cells (Figs. 5C-5D). Among secretory cells, goblet cells (GCs) were the most numerous, accounting for 32.3% of all secretory cells, consistent with their composition in vivo. Paneth cells (PCs) were less numerous, accounting for 8.6% of secretory cells, and we also detected a small number of cluster cells (1.4%) (Fig. 5C). These cells expressed classical markers for various cell types (Fig. 5D). Intestinal endocrine cells accounted for 24.7% of all secretory cells and expressed multiple subtype markers, including NEUROG3 (endocrine progenitor cells). Among these subtypes, TPH1-positive enterochromaffin cells (ECs) and CCK-positive I cells were the most abundant, consistent with their abundance in vivo (Gehart, H., et al. Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping. Cell 176, 1158-1173e1116 (2019). Beumer, J., et al. High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells. Cell 181, 1291-1306e1219 (2020).).We also observed expression patterns similar to those previously reported, namely, GHRL-positive cells co-expressing MLN (Fujii, M., et al. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell 23, 787-793e786 (2018). Wierup, N., et al. Ghrelin and motilin are cosecreted from a prominent endocrine cell population in the small intestine. J Clin Endocrinol Metab 92, 3573-3581 (2007).), and mutually exclusive expression of MLN and GAST, GHRL and CHGA, and TPH1 and GCG (Beumer, J., et al. High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells. Cell 181, 1291-1306e1219 (2020).). This indicates that our organoids are highly similar to the gut in vivo.

[0291] Notably, the UMAP plots of cells in TpC organoids revealed a differentiation pathway for secretory cells from LGR5-highly expressing ISCs to secretory precursors (Sec Pre). This pathway was evident in both the entire organoid dataset (Fig. 5A) and the secretory subset (Fig. 5C). We also observed a differentiation pathway from partially differentiated LGR5-low-expressing cells and early EC cells to second-generation secretory precursors (Sec Pre2) cells, and a third pathway from more differentiated early / mature EC cells to goblet cells (GCs). Force atlas and PAGA trajectory analyses also captured these pathways. The primary source of secretory cells was the pathway from LGR5-highly expressing ISCs to secretory precursors (Sec Pre), which was confirmed by RNA rate analysis using the Dynamo software package (Fig. 5E). This observation is consistent with scRNA-seq data from freshly isolated intestinal biopsy tissues (Triana, S., et al. Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection. Mol Syst Biol 17, e9833 (2021).). This data also includes Paneth cells (PCs) and secretory cells from LGR5. +Stem cells. The pathways we observed are also consistent with those observed in mouse organoids cultured under ENR conditions (Qu, M., et al. Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration. Cell Res 31, 259-271 (2021).). These trajectories are also present in single-cell sequencing data from freshly isolated crypts (Fig. 6A), but not in IF (Fig. 6A) or IL22 organoid data (Fig. 6A). In both datasets, the likely origin of secretory cells is partially differentiated early EC cells (Fig. 6A). Notably, when we compared pathway activity of ISCs in different samples, we found that the signaling pathway activity of ISCs under TpC conditions was more similar to that of ISCs in freshly isolated crypt samples (Fig. 6C). To compare the cellular composition of organoids cultured under different conditions, we integrated scRNA-seq datasets of cells cultured under various organoid conditions using the Seurat software package (Fig. 6B). This analysis confirms our previous observation that a population with high LGR5 expression connects absorptive and secretory cells. This pathway was present in freshly isolated Crypt data, but not in IF and IL22 samples (Figure 6B).

[0292] Furthermore, comparative analysis of cell composition confirmed that TpC organoids contain the main secretory cell types, including Paneth cells (PCs), goblet cells (GCs), enteroendocrine cells (EECs), and cluster cells (Figure 5F). We further compared organoid data with in vivo data, and the integrated analysis showed that most cell types in the organoids exhibited high similarity to in vivo cells. Comparison of intestinal epithelial lineage cells revealed that ECs obtained under different in vitro conditions still differed from in vivo ECs in some transcriptional features, but cell subpopulations highly similar to in vivo ECs were detected under TpC conditions, and the overall transcriptional features showed better similarity to in vivo samples than under other conditions (Figure 7). In addition, TpC organoids also showed the highest similarity to in vivo cells in gene expression. These results demonstrate the high biological relevance of the TpC organoid system in mimicking the diversity of intestinal epithelial cells.

[0293] Example 4: Small molecules play different roles in supporting stemness maintenance and inducing cell differentiation.

[0294] Our TpC condition maintained cell diversity and self-renewal in LGR5-positive stem cells, providing a platform to elucidate the roles of individual small chemical molecules in the regulation of human intestinal cell fate. We focused on the roles of TSA, pVc, and CP.

[0295] The results showed that the addition of TSA, pVc, and CP, individually or in combination, to the basal condition significantly increased the proportion of LGR5-mNeonGreen, with CP exhibiting the most significant effect in promoting cell proliferation, colony formation, and LGR5-mNeonGreen expression (Figures 8A-8B). TSA and CP promoted the expression of Paneth cell-specific genes DEFA5, DEFA6, and LYZ, as well as stem cell-specific genes LGR5 and SMOC2 (Figure 8C). Adding CP alone to the basal condition also promoted the differentiation of secretory cells (Figure 9A). Removal of each of these three small molecules from the TpC condition led to a significant decrease in ISC self-renewal, as evidenced by the percentage of cells highly expressing LGR5-mNeonGreen and their average fluorescence intensity (Figures 9B-9C). While the removal of TSA and pVc did not affect cell growth, the absence of CP significantly affected organoid growth (Figure 9B). We further investigated the effect of TpC on cell differentiation. We found that removing TSA significantly reduced Paneth and EEC cells but increased goblet cells, while removing pVc reduced Paneth cells (PCs) but had little effect on EECs and goblet cells. Conversely, removing CP increased Paneth and EEC cells but reduced proliferative capacity (Fig. 8D). The TpC combination showed the best overall performance in organoid growth, colony formation, LGR5-mNeonGreen expression, and secretory cell diversity (Figs. 8A-8D and 9B-9C).

[0296] We examined the effects of removing small molecules alone under TpC conditions. We found that removal of CP significantly decreased cell proliferation (Fig. 8G), reduced LGR5 expression and the proportion of LGR5-positive cells (Fig. 8E), while increasing the differentiation of secretory cells (Fig. 8F, 8G), indicating the role of CP in inhibiting ISC differentiation. Furthermore, we found that other inhibitors of the PDGFR signaling pathway, such as crenolanib, could effectively replace the role of CP (Fig. 9C).

[0297] We examined the effects of replacing CP in TpC with other small molecules, including the Hedgehog inhibitor SANT1, the B-Raf inhibitor SB590885, the p38 MAPK inhibitor SB202190, the AKT inhibitor AT7867, the JAK1 / 2 inhibitor Ruxolitinib, the AMPK activator Metformin, the RAR activator AM580, and the mTOR inhibitor Rapamycin. The results are shown in Figure 10. The activation / inhibition of other signaling pathways did not achieve similar effects to the PDGFR inhibitor (CP).

[0298] Example 5: TSA maintains LGR5 stem cells by targeting HDAC-MBD-NURD

[0299] We further analyzed the mechanism of action of TSA. TSA is a pan-HDAC inhibitor. We tested various HDAC inhibitors and found that they showed similar effects in promoting LGR5-mNeonGreen expression and increasing cell diversity, although their potency differed (Figs. 11A-11C). Another pan-HDAC inhibitor, VPA, also significantly increased the proportion of LGR5-mNeonGreen, but this proportion was not associated with organoid growth (Fig. 11A). The HDAC6 inhibitor Tubastatin A and the HDAC2 inhibitor CAY10683 also increased the proportion of LGR5-mNeonGreen, similar to TSA (Fig. 11D), while Tubastatin A was detrimental to the production of Paneth cells (Fig. 11D).

[0300] To further investigate the TSA-targeted chromatin complex, we used small molecules to regulate the complex containing the HDAC1 / 2 subunits. We found that the MBD2 inhibitor KCC-07 could reverse the decrease in budding rate and LGR5-mNeonGreen expression caused by TSA deficiency (Fig. 8C and Fig. 12A-12B). Organoids treated with KCC-07 showed significantly increased Paneth cell abundance compared to the -TSA condition (Fig. 12C), and gene expression was similar to that under the +TSA condition (Fig. 12D). MBD2 is one of the core subunits of the nucleosome remodeling and deacetylation complex (NuRD). The experimental results suggest that TSA promotes LGR5-mNeonGreen expression and Paneth cell abundance by targeting the MBD2-NuRD complex.

[0301] The methylated CpG-binding domain protein family includes two members, MBD2 and MBD3. Previous studies have shown that the MBD3-NuRD complex inhibits LGR5 expression and precursor cell proliferation in the mouse small intestine (Aguilera, C., et al. c-Jun N-terminal phosphorylation antagonises recruitment of the Mbd3 / NuRD repressor complex. Nature 469, 231-235 (2011)). We hypothesized that MBD3-NuRD also mediates TSA-induced gene expression in the human small intestine. Therefore, in the absence of MBD3-specific inhibitors, we used CRISPR / Cas9 technology with guide RNA targeting the third exon of the human MBD3 gene to interfere with MBD3 expression in human small intestinal cells (Figure 13A). We screened for cell lines with knocked-down MBD3 expression and validated this using Western blotting and qPCR experiments (Figures 13A-13B). Knockdown of MBD3 significantly increased the expression of LGR5-mNeonGreen (Fig. 13B) and the proportion of LGR5 stem cells (Fig. 8I, Fig. 13C). Simultaneous inhibition of MBD2 and MBD3 (MBD3-KD+KCC-07) significantly enriched LGR5 stem cells. These data indicate that in human small intestinal organoids, TSA, as an inhibitor of HDAC1 / 2, enriches LGR5 stem cells and increases Paneth cell abundance by targeting the MBD2 and MBD3 NuRD complex.

[0302] We further investigated the effects of TSA on different cell types using single-cell sequencing. We found that removing TSA from the established TpC organoid culture system induced immature differentiation and death of Paneth cells. This conclusion was supported by increased expression of Paneth cell-specific genes (DEFA5, DEFA6, and PRSS2), as well as low UMI values ​​and eigenvalues. These cells also co-expressed the precursor cell-specific genes OLFM4, FABP2, and FGFBP1 (Figures 14A-14B). Cell type preference analysis showed that Paneth cells, secretory precursor cells, and LGR5-highly expressing cells were all significantly affected by TSA withdrawal. These data indicate that TSA plays a crucial role in maintaining LGR5 stem cells and inhibiting the differentiation of ISCs into secretory cell lineages.

[0303] Example 6: iBET-151 reversibly promotes self-renewal and inhibits secretory cell differentiation.

[0304] In vivo intestinal epithelial cell (EC) fate transitions are highly dynamic, involving a variety of concurrent cellular events, including self-renewal, differentiation, and dedifferentiation. Since TpC organoids exhibit balanced self-renewal and differentiation, our next question was whether processes including self-renewal, differentiation, and dedifferentiation could be similarly regulated in our TpC organoids. To this end, we further screened the effects of various small molecule pathway regulators based on TpC conditions (Figure 15A). We found that the BET bromide domain protein inhibitor iBET-151 (iBET or I) effectively promoted organoid proliferation and reduced the number of LGR5-overexpressing cells (Figures 15B-15C). Simultaneously, differentiation of secretory cells, including Paneth cells (PCs), goblet cells (GCs), and EECs, was significantly reduced (Figures 15C and 16A), with almost no Paneth cells (PCs) generated in organoids treated with iBET (Figures 15C and 16A-16B). Furthermore, the presence of iBET induces significant morphological changes in organoid crypts, resulting in a more homogeneous cell population rather than a mixed arrangement of Paneth cells (PCs) and ISC-like cells (Fig. 16B). These phenomena suggest that the TpCI ensemble partially maintains organoids in a more stem state by inhibiting the differentiation of secretory cell types.

[0305] To investigate the reversibility of iBET action and assess the function of TpCI-cultured cells, we removed iBET from TpCI-cultured organoids. After culturing for another 7 days under TpC conditions, LGR5-mNeonGreen expression increased to levels comparable to those observed in TpC-cultured organoids (Fig. 16C), while secretory cells reappeared in the organoids (Figs. 16D-16E).

[0306] To further investigate the effects of iBET on organoids cultured under TpC conditions, we added iBET to TpC-cultured organoids (Fig. 16F). After 12 days of culture under TpCI conditions, we observed significantly enhanced organoid proliferation, evidenced by increased organoid size and a higher number of EdU-positive proliferating cells (Figs. 16F-16G). Conversely, secretory cell differentiation was reduced in the organoids (Figs. 16F-16G), consistent with observations of organoids derived from single intestinal cells. These findings suggest that the effect of iBET is reversible, and that cell fate in TpC or TpCI organoids is dynamic and adjusts with different culture conditions.

[0307] To clarify the cell types and cell fate transition pathways in organoids under TpCI conditions, we performed single-cell sequencing. Cell type annotation results showed the presence of the same cell types under TpCI conditions as under TpC conditions, but with almost no Paneth cells (Fig. 15D), supported by the loss of expression of Paneth cell-related genes (Fig. 15E). Cell composition analysis revealed lower abundance of secretory cells such as EECs and secretory precursor cells, while goblet cell abundance remained comparable, and intestinal epithelial lineage cells and TA cells significantly increased (Fig. 15D). Augur cell type priority analysis showed that iBET had the greatest impact on LGR5-highly expressing cells and early EC cells. This indicates that iBET treatment alters the cell trajectory, shifting it from secretory differentiation to intestinal absorption lineage. Further trajectory analysis and MetaCell analysis revealed the main differentiation pathway from LGR5-linked TA cells to EC cells. These data support the hypothesis that iBET treatment promotes intestinal epithelial cell lineage differentiation and inhibits secretory cell differentiation.

[0308] Example 7: Regulation of cell fate transition in organoids through a defined combination of microenvironment signals

[0309] Our optimized combination of small molecules effectively maintains the balance between stem cell self-renewal and differentiation in organoids and controls the shift of this balance toward enhanced stem cell self-renewal. Building on this, we aim to selectively and unidirectionally redirect this balance toward conditions specific to differentiating cell types, thereby inducing directed cell differentiation and enabling us to generate organoids enriched with Paneth cells (PCs), goblet cells (GCs), enteroendocrine cells (EECs), or intestinal epithelial cells (ECs). Although several protocols for inducing the differentiation of Paneth cells (PCs) or enteroendocrine cells (EECs) have been reported, such as using IL22 in Paneth cells (PCs) (He, GW, et al. Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation. Cell Stem Cell 29, 1333-1345e1336 (2022)) or regulating the endocannabinoid receptor, c-Jun N-terminal kinase (JNK), or forkhead box O1 (FOXO1) pathways to induce EECs (Zeve, D., et al. Robust differentiation of human enteroendocrine cells from intestinal stem cells. Nat Commun 13, 261 (2022)), our goal is to induce differentiation by reproducing the in vivo cell differentiation process under physiological conditions, utilizing normal intestinal stem cell or crypt microenvironmental signals (such as Wnt, Notch, and BMP signaling).

[0310] We systematically screened different combinations of regulators of the Wnt, Notch, BMP, and EGFR pathways (Figure 17A). We found that the reported effects of the Wnt and Notch signaling pathways on the differentiation of mouse ISCs are also applicable to human cells. Specifically, inhibition of the Notch signaling pathway with DAPT promoted the differentiation of stem cells into secretory cells. Further regulation of the Wnt signaling pathway induced cell differentiation into Paneth cells (PCs), goblet cells (GCs), or enteroendocrine cells (EECs), respectively. Continuous activation of Wnt in the presence of DAPT promoted the differentiation of Paneth cells (PCs). The addition of IL-22 further promoted the differentiation of Paneth cells (PCs) (Figure 18A), and using this differentiation condition, 3 days of induction resulted in a differentiation efficiency of 15% for Paneth cells (PCs) (Figures 17B and 17C). In contrast, we found that a two-step differentiation induction process, consisting of an initial step using Wnt activation and Notch inhibition, and a second step using Wnt inhibition and sustained Notch inhibition, promoted the differentiation of goblet cells (GCs) and enteroendocrine cells (EECs). In this process, the initial differentiation induction likely induced the generation of secretory precursor cells. Furthermore, the addition of a BMP signaling inhibitor in the first step and an activator of BMP signaling in the second step further enhanced the differentiation efficiency of goblet cells (GCs) (Fig. 18B), resulting in a nearly 50% differentiation efficiency of goblet cells (GCs) in TpC organoids (Fig. 17B). For EEC differentiation, we found that while CP mildly inhibited EEC generation in TpC organoids, removing CP did not further promote differentiation. Therefore, we added CP and the epidermal growth factor receptor inhibitor gefitinib to our EEC differentiation protocol to prevent the generation of Paneth cells (PCs) or goblet cells (GCs). Under these conditions, we were able to induce 17% CHGA-positive enteroendocrine cells (EECs) in organoids (Fig. 17B). The duration of both the first and second steps significantly affected the differentiation of goblet cells (GCs) and enteroendocrine cells (EECs) (Figs. 18B-18C). Furthermore, the combination of IWR-1, SJ000291942, and TSA rapidly and effectively induced intestinal epithelial cell (EC) differentiation within three days (Fig. 17B). Under these conditions, Paneth cells (PCs), goblet cells (GCs), and enteroendocrine cells (EECs) were not enriched (Fig. 17C). Removal of any single factor from the EC induction conditions reduced the differentiation efficiency of intestinal epithelial cells (ECs) (Fig. 18D).

[0311] We further tested these differentiation conditions in TpCI organoids, and as expected, these conditions induced a higher proportion of EC differentiation in TpCI organoids than in TpC organoids (Fig. 17C), while Paneth cells and EECs showed lower differentiation efficiency. We also examined Paneth cell differentiation in IF organoids and found a very low proportion of Paneth cells (Fig. 18E). In addition, we tested the M cell differentiation conditions obtained in mouse small intestinal cells in previous studies in TpC organoids and observed highly efficient M cell differentiation (Figs. 17D and 19). We also detected transdifferentiation of partially differentiated ECs into GCs.

[0312] These results demonstrate that stem cells in TpC organoids still possess multi-lineage differentiation capacity. The homeostasis of TpC organoids can selectively shift to the major functional cell types of the human gut through a defined combination of signaling pathways. By integrating our differentiation data and single-cell trajectory analysis, we propose a unified model of fate transition for human intestinal epithelial cells (ISCs). In this model, the spatiotemporal gradients of Wnt, BMP, and Notch signaling guide ISC self-renewal and differentiation. A high Wnt, low BMP, and activated Notch signaling environment at the crypt base maintains ISC self-renewal in this region, and pVc and CP can further enhance stem cell self-renewal in this region. Downregulation of Notch signaling at the crypt base induces intestinal stem cell differentiation into secretory progenitor cells in a high Wnt, low BMP environment. Depending on the strength of Wnt and BMP levels, secretory progenitor cells can further differentiate into different secretory cell types in different microenvironments. Conversely, with Notch signaling maintained and activated, the absence of Wnt signaling reduces the self-renewal capacity of ISCs and promotes their differentiation into intestinal epithelial cells (ECs). This balance can be disrupted by small chemical molecules or genetic interference. For example, we found that inhibition of the NuRD complex and iBET have a significant impact on cell trajectory, thereby affecting the cellular composition of organoids.

Claims

1. A composition comprising a histone deacetylase (HDAC) inhibitor, vitamin C and its derivatives, and a platelet-derived growth factor receptor (PDGFR) inhibitor.

2. The composition according to claim 1, wherein the HDAC inhibitor comprises a pan-HDAC inhibitor and a selective HDAC inhibitor.

3. The composition according to any one of claims 1-2, wherein the concentration of the HDAC inhibitor used is about 1 nM to about 50 mM.

4. The composition according to any one of claims 1-3, wherein the HDAC inhibitor comprises trichostatin A, valproic acid (VPA), CAY10683, tubastatin A, and any combination thereof.

5. The composition according to any one of claims 1-4, wherein: a) The HDAC inhibitor is nystatin A, with a concentration of about 1 to about 2000 nM; b) The HDAC inhibitor is valproic acid, with a concentration of approximately 0.01-50 mM; c) The HDAC inhibitor is CAY10683, with a concentration of about 0.05-about 50 μM; d) The HDAC inhibitor is LMK235, at a concentration of approximately 5–approximately 5000 nM; or e) The HDAC inhibitor is Tubastatin A, with a concentration of about 0.05 to about 50 μM.

6. A composition comprising a nucleosome remodeling and deacetylase (NuRD) complex inhibitor, vitamin C and its derivatives, and a platelet-derived growth factor receptor (PDGFR) inhibitor.

7. The composition of claim 6, wherein the NuRD complex inhibitor comprises histone deacetylase 1 / 2 (HDAC1 / 2) inhibitor and / or methylated CpG binding protein 2 / 3 (MBD2 / 3) inhibitor.

8. The composition according to any one of claims 6-7, wherein the HDAC1 / 2 inhibitor comprises trichostatin A.

9. The composition according to claim 8, wherein the concentration of nystatin A is about 1 to about 2000 nM.

10. The composition according to any one of claims 6-9, wherein the MBD2 / 3 inhibitor comprises KCC-07.

11. The composition according to claim 10, wherein the concentration of KCC-07 is about 0.1 to about 100 μM.

12. The composition according to any one of claims 1-11, wherein the concentration of the PDGFR inhibitor used is about 0.05 μM to about 50 μM.

13. The composition according to any one of claims 1-12, wherein the PDGFR inhibitor comprises CP673451 and / or crenolanib.

14. The composition according to any one of claims 1-13, wherein: a) The PDGFR inhibitor is CP673451, with a concentration of approximately 0.05–approximately 50 μM; or b) The PDGFR inhibitor is crenolanib, at a concentration of about 0.05 to about 50 μM.

15. The composition according to any one of claims 1-14, wherein the concentration of the vitamin C and its derivatives used is from about 10 μg / mL to about 1000 μg / mL.

16. The composition according to any one of claims 1-15, wherein the vitamin C and its derivatives comprise L-ascorbic acid-2-phosphate.

17. The composition according to any one of claims 1-16, wherein the vitamin C and its derivatives are L-ascorbic acid-2-phosphate, having a concentration of about 10 to about 1000 μg / mL.

18. The composition according to any one of claims 1-17, comprising nystatin A, L-ascorbic acid-2-phosphate and CP673451.

19. The composition according to claim 18, wherein the concentration of nystatin A is about 1 to about 2000 nM, the concentration of L-ascorbic acid-2-phosphate is 10 to 1000 μg / mL, and the concentration of CP673451 is about 0.05 to about 50 μM.

20. The composition according to any one of claims 1-19, further comprising a BET inhibitor.

21. The composition of claim 20, wherein the BET inhibitor comprises iBET-151.

22. The composition according to any one of claims 20-21, wherein the BET inhibitor is iBET-151 and has a concentration of about 0.01 to about 50 μM.

23. The composition according to any one of claims 1-22, further comprising one or more components from the group consisting of: Wnt signaling activator, BMP signaling inhibitor, EGF activator, FGF activator, IGF activator, TGFβ inhibitor, and gastrin.

24. A culture medium comprising the composition of any one of claims 1-23.

25. The culture medium according to claim 24, further comprising basal culture medium components.

26. The culture medium according to any one of claims 24-25, which is used for culturing cells, preserving tissues, culturing tissues, preparing organoids and / or culturing organoids.

27. The culture medium according to claim 26, wherein the cells comprise stem cells.

28. The culture medium according to any one of claims 26-27, wherein the cells comprise Lgr5+ stem cells.

29. The culture medium according to any one of claims 26-28, wherein the cells comprise epithelial stem cells.

30. The culture medium according to any one of claims 26-29, wherein the cells comprise adult stem cells.

31. The culture medium according to any one of claims 26-30, wherein the cells comprise digestive system stem cells.

32. The culture medium according to any one of claims 26-31, wherein the tissue comprises digestive system tissue.

33. The culture medium according to any one of claims 26-32, wherein the cells or tissues are derived from mammals.

34. The culture medium according to any one of claims 26-33, wherein the cells or tissues are derived from humans or mice.

35. The culture medium according to any one of claims 26-34, wherein the tissue comprises stomach and / or intestinal tissue.

36. A method for cell culture, tissue preservation, tissue culture, preparation of organoids and / or culture of organoids, comprising using the composition of any one of claims 1-23 and / or the culture medium of any one of claims 24-35.

37. A method for preparing and / or culturing organoids, comprising the following steps: a) Obtaining stem cells; and b) Culturing the stem cells obtained in step a) in the presence of the composition of any one of claims 1-23 or using the culture medium of any one of claims 24-35, thereby obtaining the organoid.

38. The method of claim 37, wherein the stem cells comprise Lgr5+ stem cells.

39. The method according to any one of claims 37-38, wherein the stem cells comprise epithelial stem cells.

40. The method according to any one of claims 37-39, wherein the stem cells comprise adult stem cells.

41. The method according to any one of claims 37-40, wherein the stem cells comprise digestive system stem cells.

42. The method according to any one of claims 37-41, wherein the stem cells comprise mammalian stem cells.

43. The method according to any one of claims 37-42, wherein the stem cells comprise human or mouse stem cells.

44. The method according to any one of claims 37-43, further comprising the step of placing the stem cells obtained in step a) on a cell support, embedding them in the cell support, or mixing them with the cell support.

45. The method according to any one of claims 37-44, further comprising the step of inducing the organoid to produce functional cells.

46. ​​The method according to any one of claims 37-45, wherein the stem cells comprise intestinal stem cells, and the organoids comprise intestinal organoids.

47. The method according to claim 46, wherein the intestinal stem cells are derived from the intestinal crypts.

48. The method of claim 47, wherein the intestinal crypts comprise small intestinal or colonic crypts.

49. The method according to any one of claims 46-48, further comprising the step of inducing the organoid to produce intestinal epithelial functional cells.

50. The method of claim 49, wherein the intestinal epithelial functional cells comprise Paneth cells.

51. The method according to any one of claims 49-50, further comprising culturing the organoid obtained in step b) in the presence of the Panthéon-Inducing Composition, wherein the Panthéon-Inducing Composition comprises a Wnt signaling activator, an inhibitor of Notch or γ-secretase, and a member of the IL-10 family of cytokines.

52. The method of claim 51, wherein the Paneth cell-inducing composition further comprises one or more components from the group consisting of: BMP signaling inhibitors, EGF activators, FGF activators, IGF activators, TGFβ inhibitors, and vitamin C and its derivatives.

53. The method according to any one of claims 49-52, wherein the intestinal epithelial functional cells comprise goblet cells.

54. The method according to any one of claims 49-53, further comprising culturing the organoid obtained in step b) under conditions in the presence of goblet cell induction composition A and goblet cell induction composition B, wherein goblet cell induction composition A comprises a Wnt signaling activator, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator, and wherein goblet cell induction composition B comprises a Wnt signaling inhibitor, gastrin, an inhibitor of Notch or γ-secretase, a TGFβ inhibitor, and a BMP signaling activator.

55. The method according to claim 54, comprising culturing the organoids obtained in step b) for 1-3 days in the presence of the goblet cell induction composition A, and then culturing them for another 1-5 days in the presence of the goblet cell induction composition B.

56. The method according to any one of claims 49-55, wherein the intestinal epithelial functional cells comprise intestinal endocrine cells.

57. The method according to any one of claims 49-56, further comprising culturing the organoid obtained in step b) under conditions in the presence of enteroendocrine cell inducing composition A and enteroendocrine cell inducing composition B, wherein enteroendocrine cell inducing composition A comprises a Wnt signaling activator, an inhibitor of Notch or γ-secretase, gastrin, a TGFβ inhibitor, and a BMP signaling inhibitor, and wherein enteroendocrine cell inducing composition B comprises an inhibitor of Notch or γ-secretase, gastrin, a TGFβ inhibitor, a BMP signaling inhibitor, and a MAPK / EGFR signaling inhibitor.

58. The method according to claim 57, further comprising culturing the organoids obtained in step b) for 1-3 days in the presence of the intestinal endocrine cell induction composition A, and then culturing them for another 1-5 days in the presence of the intestinal endocrine cell induction composition B.

59. The method according to any one of claims 49-58, wherein the intestinal epithelial functional cells comprise intestinal epithelial cells.

60. The method according to any one of claims 49-59, further comprising culturing the organoid obtained in step b) in the presence of the intestinal epithelial cell induction composition, wherein the intestinal epithelial cell induction composition comprises an EGF activator, an FGF activator, an IGF activator, gastrin, a TGFβ inhibitor, a BMP signaling activator, a Wnt signaling inhibitor, and an HDAC inhibitor.

61. The method according to any one of claims 37-45, wherein the stem cells comprise gastric stem cells, and the organoids comprise gastric organoids.

62. The method of claim 61, wherein the gastric stem cells are derived from gastric glands.

63. The method according to any one of claims 61-62, wherein the gastric stem cells are derived from the cardia gland, gastric body gland and / or pyloric gland.

64. An organoid prepared by the method of any one of claims 36-63.

65. An intestinal organoid possessing cellular diversity and proliferative capacity, comprising mature intestinal epithelial cells, Paneth cells, Lgr5-expressing stem cells, and KI67-expressing proliferating cells.

66. The intestinal organoid of claim 65, further comprising goblet cells and / or enteroendocrine cells.

67. The intestinal organoid according to any one of claims 65-66, wherein the proportion of Paneth cells is ≥0.1%.

68. The intestinal organoid according to any one of claims 65-67, wherein the proportion of goblet cells is ≥1%.

69. The intestinal organoid according to any one of claims 65-68, wherein the proportion of enteroendocrine cells is ≥1%.

70. The intestinal organoid according to any one of claims 65-69, comprising cells expressing one or more genes selected from the group consisting of: FABP1, KRT20, ACE2, ALPI, DEFA5, DEFA6 and REG3A.

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