Colonic organoids and methods of making and using same

Abstract

To provide human colonic organoids in vitro, usable for determining the efficacy and / or toxicity of potential therapeutics for a disease selected from the group consisting of colitis, colon cancer, polyposis syndrome and / or irritable bowel syndrome.SOLUTION: The disclosure provides a human colonic organoid (HCO), where the human colonic organoid comprises epithelium and mesenchyme and expresses SATB2, MUC2 and / or MUC5B.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 429,948, filed December 5, 2016, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] While the generation of gastric and small intestinal organoids from pluripotent stem cells (PSCs) has revolutionized the study of human gastrointestinal (GI) development and disease, efforts to generate colonic organoids have been slowed, in part, by the lack of a robust understanding of hindgut development. Summary of the Invention

[0003] Disclosed herein is a method for in vitro differentiation of progenitor cells into definitive endoderm, which can be further differentiated into human colon organoid (HCO) through the regulation of signaling pathway.Furthermore, disclosed is HCO and the method of using HCO, for example, HCO can be used to determine the efficacy and / or toxicity of potential therapeutic agents for diseases selected from colitis, colon cancer, polyposis syndrome and / or irritable bowel syndrome. [Brief description of the drawings]

[0004] This application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the fee.

[0005] Those skilled in the art will understand that the drawings, described below, are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0006] [Figure 1]Bmp signaling regulates Satb2 expression in mouse and frog embryos. (A) Whole-mount pSmad158 (red) and Foxa2 (green) staining of embryonic day 8.5 mouse embryos showing nuclear staining around the developing hindgut (n=6). (B) Inset of optical tissue slices from boxed regions in (A) showing pSmad1 / 5 / 8 staining in hindgut mesoderm and endoderm (D: dorsal, V: ventral). (C) Schematic of mouse embryos isolated at the head fold stage and cultured for 2 days with or without Bmp inhibition using DMH-1. (D,E) Whole-mount pSmad1 / 5 / 8 (red) and Foxa2 (green) staining of DMSO-treated embryos (0) and DMH-1-treated embryos (E) after 48 hours of culture. (F) Quantification of pSmad1 / 5 / 8 and pSmad2 / 3 staining for Cdx2 in embryos (n=3 embryos per condition) cultured in DMSO or DMH-1. (G-J) Whole mount immunostaining for Cdx2 (green), Satb2 (red) and Foxa2 (white) in mouse embryos (n=6 per condition) after 2 days of culture in DMSO (G,H) or DMH-1 (I,J). Arrows in H-J indicate the approximate location of the yolk stalk (BA1, first brachial arch). (K) Quantification of Satb2 expression in mouse embryos treated with DMSO or DMH-1. (L) Schematic of Bmp inhibition in Xenopus tropicalis embryos. In situ hybridization of Satb2 in Xenopus tropicalis embryos treated with DMSO (M) or DMH-1 (R). White dotted lines in (M) and (R) delineate the plane of section used for subsequent analysis. Mx and md = maxillary and mandibular processes of the first brachial arch. Cba = caudal brachial arch. Immunofluorescence of Satb2 (red), pSmad1 / 5 / 8 (green), DAPI (blue), and hue merged images from Xenopus tropicalis embryos treated with DMSO (N-Q) or DMH-1 (S-V). Scale bars = 100 μM in G-H, 50 μM in all other panels. **p<0.01 and ***p0.001 for two-tailed t-tests. [Diagram 2]BMP2 induces SATB2 and posterior HOX codes in human intestinal spheroids. (A) Schematic of intestinal spheroid patterning protocol. (B-D) BMP signaling levels measured by pSMAD1 / 5 / 8 (red) staining of spheroids treated with Noggin (B), untreated (C) and BMP2 (D) for 12 hours. (E) pSmad1 / 5 / 8 staining of adult mouse colon showing elevated BMP signaling at the apex of crypts. (F-H) SATB2 expression in spheroids treated with Noggin (F), untreated (G) and BMP2 (H) for 72 hours. (I) Quantification of the percentage of SATB2+CDH1+ epithelium after patterning. (J) Principal component analysis of nascent spheroids and spheroids after 3 days of patterning. (K) Gene ontology analysis of differentially expressed genes between BMP- and NOG-treated spheroids. (L) Graph of TPM (transcripts per million) values ​​of spheroids before and after patterning. Samples analyzed were spheroids before patterning (n=2), and Noggin-, control- and BMP2-treated spheroids 3 days after patterning (n=4 per group). For quantification in I, 20 organoids from at least three experiments were tested. Error bars represent standard deviation. Scale bar=50 microns. ****ps 0.0001 determined by two-tailed t-test. [Diagram 3]Regional patterning is maintained in human intestinal organoids after extended in vitro culture. (A-D) Whole mount immunofluorescence and QPCR analysis with the proximal marker ONECUTI (green) of 28-day-old organoids resulting from the first 3 days of treatment of spheroids with Noggin, control, or BMP2. Staining with CDX2 (red) and DAPI (blue) was also used to detect epithelium and mesenchyme. (E-H) Expression of the posterior marker SATB2 (red) detected by IF and by QPCR. (I-L) Analysis of the pan-goblet cell marker MUC2 (red) by IF and by QPCR. (M-P) Analysis of the colon-specific goblet cell marker MUC5B (red) by IF. The number of MUC5B+ cells was quantified in (P). (Q-S) Analysis of patterning markers in isolated mesenchymal cultures compared to whole organoids. QPCR analysis of CDH1 (Q), the proximal HOX gene HOXD3 (R), and the distal HOX gene HOXA13 (S) in whole organoids and in mesenchymal cultures derived from organoids treated with Noggin, control, or BMP2. CDH1 was observed only in whole organoids containing epithelial cells. Error bars represent standard error of the mean. For IF, a minimum of 10 organoids from at least three different experiments were considered for each condition. For QPCR, a minimum of five biological replicates from two separate experiments were considered. Scale bar = 100 microns. **p 5 0.01 and ****p 5 0.0001 as determined by two-tailed t-test. [Figure 4]HCOs, but not HIOs, gave rise to colon-specific enteroendocrine cells in response to expression of the proendocrine transcription factor Neurogenin3. (A-B) Schematic of the doxycycline-inducible NEUROG3 lentiviral construct used to generate IPSC72.3-inducible NEUROG3 lines, and the doxycycline induction protocol. Whole mount staining with chromagranin A (green), CDX2 (red) and INSL5 (white) of 35-day-old organoids patterned with Noggin (C,F), untreated (D,G) or BMP (E,H). (C-E) Untreated organoids (-Dox) and (F-H) organoids with expressed NEUROG3 (+Dox). Insets in E and H show magnified views of INSL5 staining. (I,J) QPCR analysis of NEUROG3 induction in enteroendocrine cells in HIOs and HCOs as measured by CHGA (I), and for INSL5 (J) expression. Data are representative of two different experiments with Noggin (n=3), control (n=3) or BMP (n=6) treated organoids. Error bars represent standard error of the mean. Scale bar=50 microns. *p<0.05 as determined by two-tailed t-test. [Diagram 5] HIOs and HCOs maintained regional identity after in vivo transplantation. (A-E) H&E staining of biopsies from human jejunum and colon, and of Noggin-derived HIOs, control HIOs, and BMP2-derived HCOs transplanted beneath the mouse kidney capsule and grown in vivo for 8-10 weeks. Samples from the same conditions were stained for the proximal gut marker GATA4 (F-J), the distal gut marker SATB2 (K-0), the Paneth cell marker DEFAS (P-T), and the colon-specific goblet cell marker MUC5B (U-Y). Note that double staining for GATA4 and SATB2 was performed in different channels but on the same slide for panels (F-0), but they are shown as individual pseudocolor (red) images. n=2 for human biopsies; n=12 for transplanted Noggin-treated organoids, n=7 for control organoids, and n=16 for BMP2-treated organoids. Scale bar=50 pm. [Figure 6]Organoids grown in vivo express regionally specific hormones. Expression analysis of regionally expressed hormones (A-D) ghrelin (GHRL), motilin (MLN), (E-H) GIP, (I-L) GLP-1, (M-P) PYY and (Q-T) INSL5 in HIOs and HCOs grown for 8-10 weeks beneath the mouse renal capsule. Proximally enriched hormones GHRL, GIP and MLN were abundant in noggin and control HIOs (A-H). Distally enriched hormones GLP-1 and PYY were abundant in BMP2-derived HCOs (1-0). Colon-specific hormone INSL5 was only present in HCOs (Q-T). Data are representative of a minimum of five transplanted organoids per condition. Insets in (A) and (B) show GHRL and MLN double positive cells. (D, H, L, P, T) FPKM values ​​for GHRL, MLN, GIP, GLP1, PYY, and INSL5 are from RNA-seq data. FPKM values ​​represent three biological replicates per condition. Scale bar = 30 microns. [Figure 7] Global transcriptional analysis of HIO and HCO and comparison with human small intestine and colon. (A) Principal component analysis of human adult and fetal small intestine and colon compared to transplanted HIO and HCO. (B) Hypergeometric median test comparing human adult small intestine with HIO and human adult colon with HCO. (C) Four-way scatter plot comparing differentially expressed transcripts in human small intestine and colon compared to HIO and HCO. [Figure 8]Gata4 and Satb2 mark discrete regional boundaries during small and large intestine development. (A) Whole mount staining for Gata4 (green) and Satb2 (red) in E11.5 mouse embryos showing the expression boundary at the yolk stalk (n=9). (B) Model depicting the expression domains of Gata4 and Satb2 in E11.5 small intestine showing the transition zone of low Gata4 and low Satb2 expression. (C-E) Whole mount staining for Gata4 and Satb2 in E11.5 mouse embryos showing the posterior boundary of Gata4 and the anterior boundary of Satb2 at the yolk stalk (n=3). (F-H) Whole mount staining for Satb2 and Foxa2 in E12.5 mouse embryos showing that the anterior boundary of Satb2 expression is maintained (n=3). (I) Whole mount staining for Gata4 and Satb2 in the proximal intestine isolated from E16.5 mouse embryos (n=6). (J) Whole mount staining for Gata4 and Satb2 in the ileum and large intestine isolated from E16.5 mouse embryos (n=6). (K) Staining for GATA4 and SATB2 in sections of human jejunum (n=2) and (L) colon (n=2). Scale bars = 50 μM (B-D) and 100 μM (E-M). Dotted lines in (C) and (F) label the approximate location of the umbilicus. Abbreviations: ys: yolk stalk, cb: appendix, tz: transition zone, mx: maxillary, md: mandibular portion of the first brachial arch, ti: terminal ileum, icj: ileocecal transition zone. [Figure 9]SATB2 is expressed in GATA4-negative human small and large intestines. SATB2 staining in human adult duodenum, small intestine, appendix, colon and rectum showing that SATB2 expression is present throughout the ileum and large intestine. Analysis of GATA4 and SATB2 from published RNA-seq data from human adult and fetal intestinal samples. Samples plotted include human adult duodenum (HuSI_Duo_A), human adult ileum-duodenum (HuSI_Dist_A), human adult colon (HuColon_A) and human fetal small intestine (HuSI_F). (C) Analysis of GATA4 and SATB2 expression from microarray data generated by Wang et al. 2015 for fetal intestinal stem cells derived from duodenum (Duo), jejunum (Jej), ileum (Ile), ascending colon (AC), transverse colon (TC) and descending colon grown in air-liquid interface (ALI). The r2 values ​​were determined using the CORREL function in Excel. [Figure 10] BMPs mediate SHH activation of posterior HOX genes. (A) Classic model of SHH-mediated activation of posterior HOX genes. (B) New model of SHH-mediated activation of posterior HOX genes and BMP-mediated activation of endodermal HOX genes. (C) QPCR analysis of HOX factors after treatment with Noggin, control, smoothened agonist (SAG), or BMP2. (D) Model of BMP4-dependent activation of HOX13 gene induced by SAG. (E) QPCR analysis of HOXA13 in organoids treated with control, 5 μM SAG, 5 μM SAG+NOG, and BMP2 after 3 days. (F) Model of SHH-independent activation of HOX13 gene induced by exogenous recombinant human BMP2. (G) QPCR analysis of HOXA13 in organoids treated with control, BMP, and BMP+cyclopamine after 3 days (n=6 per condition). [Figure 11]Extended in vivo culture allows maturation of goblet cells. (A) Quantification of the percentage of CDX2+SATB2+ cells in patterned and then re-patterned organoids. QPCR analysis of HOXB13 (B) and HOXD13 (C) in 28-day-old organoids. (D-F) Whole mount staining and (G-I) transverse section staining with CDH1 (green), CDX2 (red), and MUC2 (white) from 44-day-old Noggin, control, and BMP-treated organoids. (J-L) Staining of sections from 44-day-old BMP2-treated organoids. White arrows point to goblet cells that were in the process of secreting mucin 2. For QPCR, a minimum of five biological replicates from two separate experiments were considered. For IF, a minimum of 10 organoids per condition were considered. Scale bar = 50 pm. [Figure 12] BMP patterning of organoids is stable in vitro and in vivo. (A) Organoid engraftment efficiency of Noggin, control, and BMP patterned organoids. Quantification of the percentage of GATA4+CDX2+ cells (B) and SATB2+CDX2+ cells (C) in transplanted patterned organoids. FPKM values ​​from RNA-seq data for GATA4 (D), SATB2 (E), DEFAS (F), and MUCSB (G) in transplanted organoids. (H-I) MUC2 (red) staining of human jejunal and colonic biopsies (n=2 per area) and (J-L) transplanted organoids (n=5 per condition). Scale bars = 50 microns. [Figure 13]Organoids grown in vitro and in vivo contain intestinal progenitor cells. Representative whole mount images (A,F,K) and tissue slice images (B,G,L) of CDH1 and GFP from H9-LGR5-GFP-derived organoids treated with Noggin, control, or BMP. CDX2 staining (red) and SOX9 staining (green) on sections from organoids treated with (C-E) Noggin, (H-J) control, or (M-O) BMP2. Representative images of in vivo organoids derived from H9-LGR5-GFP organoids treated with Noggin, control, or BMP stained with CDX2 and LGR5-GFP (P,S,V), CDX2 and SOX9 (Q,T,W), and CDH1 and KI67 (R,U,X). (Y-A') Stereomicrographs showing enteroids derived from Noggin, control, or BMP explants, respectively. (B'-D') QPCR analysis of proximal and distal genes in control enteroids (>100 pooled enteroids from two explants) and BMP2-treated colonoids (>50 colonoids from one explant). Scale bar = 50 μm. [Figure 14] Ribosomal and immune cell signatures are differentially expressed between transplanted organoids and primary human tissues. (A) Principal component analysis of patterned transplanted organoids and human adult and fetal small intestine and colon. (B) Gene ontology analysis of genes upregulated in transplants vs. human primary tissues. (C) Gene ontology analysis of genes upregulated in human primary tissues vs. transplants. [Figure 15](A) Whole mount immunofluorescence staining of HCOs after 15 days of growth in Matrigel. HCO cultures were stained for the endothelial marker CD31 (green) and the hindgut epithelial marker CDX2 (red). Cultures were also stained for the hematopoietic cell marker PU.1 (right panel in red). (B) Schematic of the hematopoietic progenitor cell assay. Cells were collected from HCOs, centrifuged and stained using Giemsa-Wright stain or plated in Methocult medium and assayed for hematopoietic cell differentiation. (C) Representative images of Giemsa-Wright stained cells with morphological features consistent with differentiation into macrophages, neutrophils, basophils and eosinophils. (D) Representative images of colonies formed after 14 days in Methocult. Erythroid, macrophage and granulocytic colonies were present in HCO-induced cells but not in Noggin-treated HIO-induced cells. [Figure 16] (A) Immunofluorescence staining of human colon biopsies or HCOs grown in Matrigel for 28 days. Staining was performed for CD68, a marker for macrophages. (B) Plots of CYTOF analysis of CD14 and CD16 in HIOs and HCOs. A small percentage of CD14+ / CD16+ cells are present in HCOs (blue squares) but not in HIOs. In addition, CD16 single positive cells were present in HCOs, suggesting that monocytes were present in the cultures. (C) Luminex array analysis of supernatants collected from 14- and 28-day-old HIOs and HCOs. IL6 and IL8 were detected in 28-day-old HCOs (BMPs) but not in HIOs. (D) Luminex array analysis of supernatants collected from 14- and 28-day-old HIOs and HCOs. The macrophage-specific cytokines MIP1A and MIP1B were detected in 14- and 28-day-old HCOs (BMPs) but not in 14- or 28-day-old HIOs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] definition Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the art.

[0008] The term "about" or "approximately" means within an acceptable error range for a particular value, depending on how the value is measured or determined, for example, of the limitations of the measurement system, as determined by one of ordinary skill in the art. For example, "about" can mean within one or more standard deviations, as per practice in the art. Alternatively, "about" can mean within 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within 10-fold, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise indicated.

[0009] As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) are stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells can build complete viable organisms. These cells are produced from the fusion of an egg cell and a sperm cell. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0010] As used herein, the term "pluripotent stem cells (PSCs)," also commonly known as PS cells, encompasses any cell that can differentiate into nearly any cell, i.e., cells derived from any of the endoderm (inner lining of the stomach, digestive tract, lungs), mesoderm (muscle, bone, blood, kidney, urinary tract, and renal tract), and ectoderm (epidermal tissue and nervous system). PSCs can be derived from embryonic stem cells (including embryonic germ cells) or the progeny of totipotent cells obtained through the induction of non-pluripotent cells, such as adult somatic cells, by forcing the expression of certain genes.

[0011] As used herein, the term "induced pluripotent stem cells (iPSCs)," commonly abbreviated as iPS cells, refers to a type of pluripotent stem cell that is artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing "forced" expression of certain genes.

[0012] As used herein, the term "embryonic stem cells (ESCs)" is also commonly abbreviated as ES cells, and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For the purposes of the present invention, the term "ESCs" is used broadly to optionally include embryonic germ cells.

[0013] As used herein, the term "progenitor cell" encompasses any cell that may be used in the methods described herein, where one or more progenitor cells will acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with exogenous factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells may be totipotent (or omnipotent) stem cells, pluripotent stem cells (induced or non-induced), multipotent stem cells, oligopotent stem cells and unipotent stem cells. In some embodiments, the progenitor cells may be from embryos, infants, children, or adults. In some embodiments, the progenitor cells may be somatic cells that undergo treatment such that pluripotency is imparted via genetic engineering or protein / peptide treatment.

[0014] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" refers to the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or change the fate of initial cells. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0015] As used herein, the term "cellular constituent" refers to individual genes, proteins, mRNA expression genes, and / or any other variety of cellular components or protein activities, such as protein modification (e.g., phosphorylation), typically measured by those skilled in the art in biological experiments (e.g., by microarray or immunohistochemical characterization). Important discoveries about biological systems, common human diseases, and the complex network of biochemical processes underlying gene discovery and structure determination can now result from the application of cellular constituent abundance data as part of the research process. Cellular constituent abundance data can help identify biomarkers, distinguish disease subtypes, and identify mechanisms of toxicity.

[0016] As described herein, methods and systems are established using a time sequence of growth factor manipulation to mimic embryonic gut development in culture. In particular, methods and systems are established for generating in vitro differentiation of PSCs, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), into gut tissue.

[0017] The generation of gastric and small intestinal organoids from pluripotent stem cells (PSCs) has revolutionized the study of human gastrointestinal (GI) development and disease. However, efforts to generate colonic organoids have lagged behind, due in part to a robust molecular understanding of posterior gut development. Herein, we discovered that the intestinal epithelium posterior to the umbilical cord expresses Satb2 throughout development and postnatally. We further discovered that BMP signaling establishes Satb2+ domains in frog and mouse embryos, and that brief activation of BMP signaling is sufficient to activate the posterior HOX code and direct human PSC-derived intestinal cultures toward colonic organoids (HCOs). HCOs grown in vitro had marker characteristics and unique cell types consistent with colonic identity. After transplantation into mice, HCOs underwent morphogenesis and maturation to form tissues with molecular, cellular, and morphological characteristics of the human colon. The disclosed colon organoids may be used in future studies of colitis and colon cancer.

[0018] In one embodiment, disclosed is the method for inducing the formation of human colon organoid.This method can include: (a) contacting definitive endoderm (DE) with FGF signaling pathway activator and WNT signaling pathway activator (for example, CHIRON / GSK2 inhibitor) for a sufficient period of time for said DE to form mid-hindgut spheroid; (b) contacting mid-hindgut spheroid of step (a) with BMP activator and EGF signaling pathway activator for a sufficient period of time for said human colon organoid to form, wherein said human colon organoid expresses SATB2.

[0019] In one embodiment, the DE may be derived from progenitor cells selected from embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesoderm cells, definitive endoderm cells, posterior endoderm cells, hindgut cells, or combinations thereof.

[0020] In one embodiment, the FGF signaling pathway activator may be selected from a small molecule or protein FGF signaling pathway activator, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, or a combination thereof. The WNT signaling pathway activator may be selected from a small molecule or protein Wnt signaling pathway activator, preferably lithium chloride, 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidine, IQ1, QS11, NSC668036, DCA beta-catenin, 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, a GSK3 inhibitor, preferably CHIRON, or a combination thereof. In one embodiment, the BMP activator may be selected from BMP2, BMP4, BMP7, BMP9, small molecules that activate the BMP pathway, proteins that activate the BMP pathway, and may include the following: noggin, dorsomorphin, LDN189, DMH-1, ventromophin, and combinations thereof.

[0021] In one embodiment, the period of time sufficient for the DE to form mid-hindgut spheroids can be determined by the expression of CDX2 by the mid-hindgut spheroids of step (a). Such measurements are within the ability of a person skilled in the art using routine methods.

[0022] In one embodiment, the sufficient period that mid-hindgut spheroid forms human colon organoid is determined by the expression of SATB2 and CDX2 by the cells of said human colon organoid, and when SATB2 and CDX2 are expressed, mid-hindgut spheroid has already formed human colon organoid.This measurement can be used instead of time measurement, in that the expression of the genes listed above indicates that step (a) and step (b) have been carried out for a sufficient period.

[0023] In one embodiment, HCO obtained by the method described herein is disclosed.HCO of the present invention can be characterized in various different ways.In one embodiment, HCO can be characterized by the presence of colon enteroendocrine cells (EEC).In one embodiment, HCO can be characterized by the presence of crypts and substantially no villi.In one embodiment, HCO can be characterized by the presence of colon-specific goblet cells.In one embodiment, HCO can be characterized by the substantial absence of Paneth cells.In one embodiment, HCO can be characterized by the ability to secrete colon-specific hormone INSL5.Intestinal organoid can be free of one or more of immune function, innervation, blood vessels, villi, and Paneth cells.

[0024] In one embodiment, a method of forming colon tissue is disclosed, wherein the HCO of the present invention described herein can be engrafted under the kidney capsule of a mammal, preferably a rodent, preferably an immunocompromised rodent, preferably an immunocompromised mouse.

[0025] In one embodiment, the HCOs disclosed herein can be used to determine the efficacy and / or toxicity of potential therapeutics for a disease selected from colitis, colon cancer, polyposis syndrome, and / or irritable bowel syndrome. The method can include contacting the potential therapeutic with an HCO described herein for a period of time sufficient to determine the efficacy and / or toxicity of the potential therapeutic.

[0026] In one embodiment, an intestinal coronoid derived from the HCO of any preceding claim is contemplated.

[0027] In some embodiments, stem cells that are pluripotent or can be induced to become pluripotent can be used. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early stage embryo. Methods for deriving embryonic stem cells from blastocysts are well known in the art. For example, three cell lines (H1, H13, and H14) had normal XY karyotype, and two cell lines (H7 and H9) had normal XX karyotype. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in this application, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0028] Additional stem cells that can be used in embodiments according to the present invention include, but are not limited to, those provided by or described in the National Stem Cell Bank (NSCB), databases maintained by the University of California, San Francisco (UCSF) Center for Human Embryonic Stem Cell Research, Wi Cell Institute WISC Cell Bank, University of Wisconsin Center for Stem Cell and Regenerative Medicine (UW-SCRMC), Novocell (San Diego, CA), Cellartis AB (Gothenburg, Sweden), ES Cell International Pte Ltd (Singapore), Technion, Israel Institute of Technology (Haifa, Israel), and stem cell databases maintained by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (I3), TE04 (I4), TE06 (I6), UC01 (HSF1), UC06 (HSF6), WA01 (H1), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14).

[0029] In some embodiments, the stem cells are further modified to incorporate additional characteristics. Exemplary modified cell lines include, but are not limited to, H1 OCT4-EGFP, H9 Cre-LoxP, H9 hNanog-pGZ, H9 hOct4-pGZ, H9 inGFPhES, and H9 Syn-GFP.

[0030] Further details regarding embryonic stem cells can be found in, for example, Thomson et al., 1998, "Embryonic Stem Cell Lines Derived from Human Blastocysts", Science 282(5391):1145-1147; Andrews et al., 2005, "Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin", Biochem Soc Trans 33:1526-1530; Martin 1980, "Teratocarcinomas and mammalian embryogenesis", Science 209(4458):768-776; Evans and Kaufman, 1981, "Establishment in culture of pluripotent cells from mouse embryos", Nature 292(5819):154-156; Klimanskaya et al., 2005, "Human embryonic stem cells derived without feeder cells", Lancet 365(9471):1636-1641, each of which is incorporated herein by reference in its entirety.

[0031] Alternatively, pluripotent stem cells can be derived from embryonic germ cells (EGCs), which are cells that give rise to gametes in sexually reproducing organisms. EGCs are derived from primordial germ cells found in the gonadal ridges of late embryos, which have many of the characteristics of embryonic stem cells. Primordial germ cells in the embryo develop into stem cells that create reproductive gametes (sperm or eggs) in adults. In mice and humans, embryonic germ cells can be grown in tissue culture under appropriate conditions. Both EGCs and ESCs are pluripotent. For the purposes of the present invention, the term "ESCs" is sometimes used broadly to encompass EGCs.

[0032] Induced pluripotent stem cells (iPSC) In some embodiments, iPSCs are derived by transfection of certain stem cell-associated genes into non-pluripotent cells such as adult fibroblasts. Transfection can be achieved via viral vectors such as retroviruses. Transfected genes include master transcriptional regulators Oct-3 / 4 (Pouf51) and Sox2, although other genes have been suggested to increase the efficiency of induction. After 3-4 weeks, a small number of transfected cells become morphologically and biochemically similar to pluripotent stem cells and are usually isolated through morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include, but are not limited to, first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells.

[0033] In some embodiments, non-viral techniques can be employed to generate iPSCs. In some embodiments, adenovirus can be used to deliver the four required genes into the DNA of mouse skin and liver cells, resulting in cells identical to embryonic stem cells. Adenovirus does not integrate any of its own genes into the target host, eliminating the risk of creating tumors. In some embodiments, reprogramming can be achieved via plasmids without any viral transfection system, albeit with very low efficiency. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiments, generation of mouse iPSC cells is possible using a similar methodology: repeated treatment of cells with specific proteins introduced into the cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, expression of pluripotency-inducing genes can also be elevated by treating somatic cells with FGF2 under hypoxic conditions.

[0034] For further details regarding embryonic stem cells, see Kaji et al., 2009, "Virus free induction of pluripotency and subsequent excision of reprogramming factors," Nature 458:771-775; Woltjen et al., 2009, "piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells," Nature 458:766-770; Okita et al., 2008, "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors," Science 322(5903):949-953; Stadtfeld et al., 2008, "Induced Pluripotent Stem Cells Generated without Viral Integration," Science 322(5903):945-949; and Zhou et al., 2009, "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cell 4(5):381-384; each of which is incorporated herein by reference in its entirety.

[0035] In some embodiments, exemplary iPS cell lines include, but are not limited to, iPS-DF19-9, iPS-DF19-9, iPS-DF4-3, iPS-DF6-9, iPS(foreskin), iPS(IMR90), and iPS(IMR90).

[0036] Definitive endoderm The HCO of the present disclosure may be derived from a simple cell sheet called definitive endoderm (DE). Methods for inducing definitive endoderm from progenitor cells are well known in the art, as taught by D'Armour et al. 2005 and Spence et al. The anterior DE forms the foregut and its associated organs, including the liver and pancreas, while the posterior DE forms the small and large intestines, as well as the midgut and hindgut, which form part of the renal urogenital system. Studies with mouse, chicken and frog embryos suggest that establishing an anterior-posterior pattern in the DE at the gastrula stage is a prerequisite for subsequent development of the foregut and hindgut. The Wnt and FGF signaling pathways are believed to be important for this process, acting to promote posterior endoderm and hindgut fates, and to suppress anterior endoderm and foregut fates. The simple cuboidal epithelium of the hindgut develops first into a pseudostratified columnar epithelium and then into villi containing polarized columnar epithelium and a proliferative region at the base of the villi, which corresponds to the putative progenitor cell domain.

[0037] We herein describe a robust and efficient method for directed differentiation of DE into intestinal tissue, particularly human colonic tissue, in vitro. Directed differentiation can be achieved by selectively activating certain signaling pathways in iPSCs and / or DE cells.

[0038] Additional details regarding pathways related to intestinal development generally can be found, for example, in Sancho et al., 2004, "Signaling Pathways in Intestinal Development and Cancer," Annual Review of Cell and Developmental Biology 20:695-723; Logan and Nusse, 2004, "The Wnt Signaling Pathway in Development and Disease," Annual Review of Cell and Developmental Biology 20:781-810; Taipale1 and Beachy1, 2001, "The Hedgehog and Wnt signalling pathways in cancer," Nature 411:349-354; Gregorieff and Clevers, 2005, "Wnt signaling in the intestinal epithelium: from endoderm to cancer," Genes & Dev. 19:877-890, each of which is hereby incorporated by reference in its entirety.More details on the function of signaling pathways in the development of DE can be found, for example, in Zorn and Wells, 2009, "Vertebrate endoderm development and organ formation," Annu Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, "FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo," Mech Dev 123:42-55; McLin et al., 2007, "Repression of Wnt / {beta}-catenin signaling in the anterior endoderm is essential for liver and pancreas development," Development 134:2207-2217; Wells and Melton, 2000, Development 127:1563-1572; de Santa Barbara et al., 2003, "Development and differentiation of the intestinal epithelium," Cell Mol Life Sci. 60(7):1322-1332, each of which is hereby incorporated in its entirety.

[0039] Any method for generating definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. In some embodiments, the pluripotent cells are derived from morulae. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods can include, but are not limited to, embryonic stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or the gonadal crest of an embryo. Embryonic stem cells or germ cells can be derived from various animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to generate definitive endoderm. In some embodiments, human embryonic germ cells are used to generate definitive endoderm. In some embodiments, iPSCs are used to generate definitive endoderm.

[0040] In some embodiments, one or more growth factors are used in the differentiation process from pluripotent stem cells to DE cells. The one or more growth factors used in the differentiation process may include growth factors from the TGF-beta superfamily. In such embodiments, the one or more growth factors may include the Nodal / Activin and / or BMP subgroups of the TGF-beta superfamily of growth factors. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, Wnt3a, or any combination of these growth factors. In some embodiments, the embryonic stem cells or germ cells and iPSCs are treated with one or more growth factors for 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 120 hours or more, 150 hours or more, 180 hours or more, or 240 hours or more. In some embodiments, the embryonic stem cells or germ cells and iPSCs are treated with one or more growth factors at a concentration of 10ng / ml or more, 20ng / ml or more, 50ng / ml or more, 75ng / ml or more, 100ng / ml or more, 120ng / ml or more, 150ng / ml or more, 200ng / ml or more, 500ng / ml or more, 1,000ng / ml or more, 1,200ng / ml or more, 1,500ng / ml or more, 2,000ng / ml or more, 5,000ng / ml or more, 7,000ng / ml or more, 10,000ng / ml or more, or 15,000ng / ml or more. In some embodiments, the concentration of the growth factor is maintained at a constant level throughout the treatment. In other embodiments, the concentration of the growth factor is changed during the course of the treatment. In some embodiments, the growth factor is suspended in a medium containing various HyClone concentrations of fetal bovine serine (FBS). Those skilled in the art will appreciate that the dosing regimens described herein are applicable to any known growth factors, either alone or in combination. When more than one growth factor is used, the concentration of each growth factor may be varied independently.

[0041] In some embodiments, a cell population enriched in definitive endoderm cells is used. In some embodiments, definitive endoderm cells are isolated or substantially purified. In some embodiments, isolated or substantially purified definitive endoderm cells express SOX17, FOXA2, and / or CXRC4 markers to a greater extent than OCT4, AFP, TM, SPARC and / or SOX7 markers. Methods for enriching cell populations with definitive endoderm are also contemplated. In some embodiments, definitive endoderm cells can be isolated or substantially purified from a mixed cell population by contacting cells with a reagent that binds to a molecule present on the surface of definitive endoderm cells but not on the surface of other cells in the mixed cell population, and then isolating the cells that bind to the reagent. In certain embodiments, the cellular constituent present on the surface of definitive endoderm cells is CXCR4.

[0042] Additional methods for obtaining or creating DE cells that can be used in the present invention include, but are not limited to, those described in U.S. Pat. No. 7,510,876 to D'Amour et al., U.S. Pat. No. 7,326,572 to Fisk et al., Kubol et al., 2004, "Development of definitive endoderm from embryonic stem cells in culture," Development 131:1651-1662, D'Amour et al., 2005, "Efficient differentiation of human embryonic stem cells to definitive endoderm," Nature Biotechnology 23:1534-1541, and Ang et al., 1993, "The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3 / forkhead proteins," Development 119:1301-1315, each of which is hereby incorporated by reference in its entirety.

[0043] Transition from definitive endoderm to mid / hindgut spheroids In some embodiments, the posteriorized endoderm cells of the DE further develop into one or more specialized cell types. The Activin-induced definitive endoderm (DE) can further undergo FGF / Wnt-induced posterior endoderm culmination, hindgut specification and morphogenesis, and ultimately a foregut culture system that promotes intestinal tube growth and cell differentiation into functional intestinal cell types, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. In some embodiments, human PSCs are effectively directed to differentiate in vitro into intestinal epithelium, which may include secretory, endocrine, and absorptive cell types. It will be appreciated that molecules such as growth factors may be added at any developmental stage to promote specific types of intestinal tissue formation.

[0044] PSCs, such as ESCs and iPSCs, undergo directed differentiation in a stepwise or non-stepwise manner, first to definitive endoderm (DE), then to mid / hindgut epithelium and mesenchyme (e.g., hindgut spheroids), and then to intestinal tissue. In some embodiments, definitive endoderm cells and hESCs are treated with one or more growth factors.

[0045] In some embodiments, soluble FGF and Wnt ligand are used to mimic early hindgut specification in culture, and through directed differentiation, DE developed from iPSC or ESC is converted into hindgut epithelium that efficiently generates all major intestinal cell types.In humans, directed differentiation of DE is achieved by selectively activating certain signaling pathways that are important for intestinal development.Those skilled in the art will understand that altering the expression of any Wnt signaling protein in combination with any FGF ligand can cause directed differentiation as described herein.

[0046] For more details, see, for example, Liu et al., "A small-molecule agonist of the Wnt signaling pathway," Angew Chem Int Ed Engl. 44(13):1987-1990(2005); Miyabayashi et al., "Wnt / beta-catenin / CBP signaling maintains long-term murine embryonic stem cell pluripotency," Proc Natl Acad Sci US A. 104(13):5668-5673(2007); Zhang et al., "Small-molecule synergist of the Wnt / beta-catenin signaling pathway," Proc Natl Acad Sci US A. 104(18):7444-7448(2007); Neiiendam et al., "An NCAM-derived FGF-receptor agonist, the FGL-peptide,induces neurite outgrowth and neuronal survival in primary rat neurons," J. Neurochem.91(4):920-935(2004), Shan et al.,"Identification of a specific inhibitor of the dishevelled PDZ domain,"Biochemistry 44(47):15495-15503(2005), Coghlan et al.,"Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription," Chem Biol.7(10):793-803 (2000), Coghlan et al., "Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription," Chemistry & Biology 7(10):793-803, and Pai et al., "Deoxycholic acid activates beta-catenin signaling pathway and increases colon cell cancer growth and invasiveness," Mol Biol Cell. 15(5):2156-2163 (2004), each of which is hereby incorporated by reference in its entirety.

[0047] In some embodiments, siRNAs and / or shRNAs targeting cellular constituents associated with the Wnt and / or FGF signaling pathways are used to activate these pathways.

[0048] Modulators / activators of Wnt signaling pathway include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In some embodiments, the regulation of pathway can be through the use of small molecule modulators or protein modulators that activate the above-mentioned pathways, or proteins that activate the above-mentioned pathways. For example, small molecule modulators of Wnt pathway include, but are not limited to, lithium chloride, 2-amino-4,6-disubstituted pyrimidine (hetero)arylpyrimidine, IQ1, QS11, NSC668036, DCA beta-catenin, 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. Exemplary natural inhibitors of Wnt signaling include, but are not limited to, Dkk1, SFRP proteins, and FrzB. In some embodiments, exogenous molecules include, but are not limited to, small molecules such as WAY-316606, SB-216763, or BIO (6-bromoindirubin-3'-oxime). In some embodiments, siRNA and / or shRNA targeting cellular components related to Wnt and / or FGF signaling pathways may be used to activate these pathways. It will be understood by those skilled in the art that the target cellular components include, but are not limited to, SFRP proteins, GSK3, Dkk1, and FrzB. Additional modulators include molecules or proteins that inhibit GSK3, which activates the Wnt signaling pathway. Exemplary GSK3 inhibitors include, but are not limited to, Chiron / CHIR99021, which inhibits GSK3β, for example. Those skilled in the art will recognize suitable GSK3 inhibitors for carrying out the disclosed methods. The GSK3 inhibitor may be administered in an amount of about 1 μM to about 100 μM, or about 2 μM to about 50 μM, or about 3 μM to about 25 μM. One of skill in the art will readily recognize appropriate amounts and durations.

[0049] Fibroblast growth factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. It will be understood by those skilled in the art that in some embodiments, any FGF can be used with proteins from the Wnt signaling pathway. In some embodiments, soluble FGFs include, but are not limited to, FGF4, FGF2, and FGF3. In some embodiments, the FGF signaling pathway is activated by contacting progenitor cells with one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. In some embodiments, siRNA and / or shRNA targeting cellular components related to the FGF signaling pathway may be used to activate these pathways. It will be understood by those skilled in the art that the methods and compositions described herein in relation to the Wnt and FGF signaling pathways are provided as examples. Similar methods and compositions are applicable to the other signaling pathways disclosed herein.

[0050] In some embodiments, the DE cultures are treated with one or more modulators of a signaling pathway described herein for 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 120 hours or more, 150 hours or more, 180 hours or more, 200 hours or more, 240 hours or more, 270 hours or more, 300 hours or more, 350 hours or more, 400 hours or more, 500 hours or more, 600 hours or more, 700 hours or more, 800 hours or more, 900 hours or more, 1,000 hours or more, 1,200 hours or more, or 1,500 hours or more.

[0051] In some embodiments, the DE culture is treated with one or more modulators of a signaling pathway described herein at a concentration of 10 ng / ml or more, 20 ng / ml or more, 50 ng / ml or more, 75 ng / ml or more, 100 ng / ml or more, 120 ng / ml or more, 150 ng / ml or more, 200 ng / ml or more, 500 ng / ml or more, 1,000 ng / ml or more, 1,200 ng / ml or more, 1,500 ng / ml or more, 2,000 ng / ml or more, 5000 ng / ml or more, 7,000 ng / ml or more, 10,000 ng / ml or more, or 15,000 ng / ml or more. In some embodiments, the concentration of the signaling molecule is maintained constant throughout the treatment. In other embodiments, the concentration of the signaling pathway modulator is changed during the course of the treatment. In some embodiments, the signaling molecule according to the present invention is suspended in a medium comprising DMEM and fetal bovine serine (FBS). FBS can be at a concentration of 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 50% or more. One of skill in the art will appreciate that the dosing regimens described herein are applicable to any known modulator of the signaling pathways described herein, alone or in combination, including but not limited to any molecule in the Wnt signaling pathway and the FGF signaling pathway.

[0052] In embodiments in which more than one signaling molecule is used to treat a DE culture, the signaling molecules can be added simultaneously or separately. When more than one molecule is used, the concentration of each can be varied independently.

[0053] Expression of CDX2 can be used to reveal propensity for hindgut formation after the DE is incubated with an activator of FGF signaling and an activator of Wnt signaling, such as FGF4 and Wnt3a, for a period of time, such as 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, or 90 hours or more. In some embodiments, longer periods of incubation are required to achieve a stable posterior endoderm phenotype, as measured by prolonged expression of CDX2. In such embodiments, the period of incubation can be 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 108 hours or more, 120 hours or more, 140 hours or more, 160 hours or more, 180 hours or more, 200 hours or more, 240 hours or more, or 300 hours or more.

[0054] Alternatively, in some embodiments, the absence of cellular constituents such as foregut markers Sox2, Pdx1, Cldn18, and albumin can be used to reveal directed hindgut formation. In some embodiments, intestinal transcription factors CDX2, KLF5, and SOX9 can be used to represent intestinal development. In some embodiments, GATA6 protein expression can be used to represent intestinal development. In these embodiments, the incubation period can be 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, or 90 hours or more. Alternatively, the incubation period can be 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 108 hours or more, 120 hours or more, 140 hours or more, 160 hours or more, 180 hours or more, 200 hours or more, 240 hours or more, or 300 hours or more.

[0055] In some embodiments, the abundance data of cellular constituents, such as protein and / or gene expression levels, are determined by immunohistochemistry using primary and / or secondary antibodies targeting molecules in the relevant signaling pathway. In other embodiments, the abundance data of cellular constituents, such as protein and / or gene expression levels, are determined by microarray analysis.

[0056] Alternatively, morphological changes can be used to represent the progression of directed differentiation.In some embodiments, hindgut spheroids are further subjected to three-dimensional culture conditions for further maturation.In other embodiments, highly convoluted epithelium surrounded by mesenchymal cells can be observed after hindgut spheroid formation.In addition, intestinal organoids, polarized columnar epithelium, goblet cells, or smooth muscle cells can be observed for 6 days or more, 7 days or more, 9 days or more, 10 days or more, 12 days or more, 15 days or more, 20 days or more, 25 days or more, 28 days or more, 32 days or more, 36 days or more, 40 days or more, 45 days or more, 50 days or more, or 60 days or more.

[0057] Transition of mid / hindgut spheroids to colonic organoids In addition to FGF and WNT signaling, it has been identified that bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, can promote posterior / hindgut fates and suppress foregut fates. In addition, BMP signaling regulates the formation of different regional types of intestine. Inhibition of BMPs by Noggin after the hindgut stage promotes proximal intestinal fates (duodenum / jejunum). Activation of BMP signaling after the hindgut stage promotes more distal intestinal cell fates (cecum / colon).

[0058] Activation of BMP can be performed by contacting mid / hind gut spheroids with BMP activators and EGF signaling pathway activators for a time sufficient to form the human colon organoids. The boundaries of the incubation period can be defined by the time point at which the human colon organoids express SATB2. Suitable BMP activators and EGF signaling pathway activators will be readily understood by those skilled in the art. Suitable BMP activators can include, for example, BMP2, BMP4, BMP7, BMP9 and proteins or small molecule agonists such as ventromorphin (Genthe et al. 2017) or proteins that serve as agonists. BMP activators and EGF signaling pathway activators can be contacted with mid / hind gut spheroids for about 1 day to about 3 days. BMP signaling can become active within the first 3 days. In one embodiment, the contact step between the BMP activator and the EGF signaling pathway activator is from 24 hours to about 10 days, or from about 48 hours to about 9 days, or from about 3 days to about 8 days, or from about 4 days to about 8 days, or from about 5 days to about 7 days. Suitable EGF activators may include, for example, TGF alpha, HB-EGF, amphiregulin, epigen, betacellulin, and small molecules such as db-cAMP. The EGF activator may be contacted with the mid / hindgut spheroids at a concentration of about 10 ng / mL to 10,000 ng / ML for about 24 hours to about 10 days, or from about 48 hours to about 9 days, or from about 3 days to about 8 days, or from about 4 days to about 8 days, or from about 5 days to about 7 days.

[0059] The mid / hindgut spheroids can be contacted with BMP activators and / or EGF activators, either alone or in combination, at concentrations of 5ng / ml or more, 20ng / ml or more, 50ng / ml or more, 75ng / ml or more, 100ng / ml or more, 120ng / ml or more, 150ng / ml or more, 200ng / ml or more, 500ng / ml or more, 1,000ng / ml or more, 1,200ng / ml or more, 1,500ng / ml or more, 2,000ng / ml or more, 5,000ng / ml or more, 7,000ng / ml or more, 10,000ng / ml or more, or 15,000ng / ml or more. In some embodiments, the concentration of the signaling molecules is kept constant during the treatment. In other embodiments, the concentration of the molecules of the signaling pathway changes during the course of the treatment. In some embodiments, the signaling molecules according to the present invention are suspended in a medium comprising DMEM and fetal bovine serum (FBS). FBS can be at a concentration of 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, or 50% or more. One of skill in the art will appreciate that the dosing regimens described herein are applicable to any known molecule of the signaling pathways described herein, alone or in combination. EXAMPLES

[0060] The following non-limiting examples are provided to further illustrate the aspects of the invention disclosed herein. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent approaches found to work well in the practice of the invention, and therefore can be considered to constitute examples of the manner of its implementation. However, those skilled in the art will recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0061] The epithelium of the digestive tract is derived from the definitive endoderm, one of the primary germ layers established during gastrulation. The process of gut morphogenesis transforms the definitive endoderm into a primitive gut tube with the foregut, midgut, and hindgut. The midgut gives rise to the small intestine and proximal large intestine, while the hindgut gives rise to the distal large intestine and rectum (Zorn and Wells, 2009). The small intestine is further subdivided into three segments: the duodenum, involved in nutrient absorption and iron uptake, the jejunum, involved in nutrient digestion and absorption, and the ileum, involved in the absorption of bile acids and vitamin B12 (Jeejeebhoy, 2002). The large intestine is subdivided into the cecum, colon, and rectum, all of which are involved in water and electrolyte absorption (Jeejeebhoy, 2002). While recent advances have focused on small intestinal development (Finkbeiner et al., 2015; Spence et al., 2011; Watson et al., 2014), little is known about the development of the large intestine / colon in humans. Furthermore, diseases affecting this region of the gastrointestinal (GI) tract, colitis, colon cancer, polyposis syndromes, and irritable bowel syndrome, are widely recognized (Molodecky et al., 2012; Siegel et al., 2014; Zbuk and Eng, 2007). Animal models of polyposis syndromes and intestinal cancer are limited, as polyps and tumors form preferentially in the small intestine and rarely in the colon or rectum (Haramis et al., 2004; He et al., 2004; Moser et al., 1990).

[0062] We have previously described a method by which human pluripotent stem cells can be differentiated into intestinal tissue through stages of directed differentiation that resemble the embryonic development of the small intestine. First, pluripotent stem cells are differentiated into definitive endoderm by treatment with activin A. Exposure of the definitive endoderm to high levels of Wnt and FGF induces morphogenesis into midgut / hindgut spheroids. Once formed, these midgut / hindgut spheroids, when grown in 3D culture under conditions favorable for intestinal growth, transition through stages that resemble the development of the small intestine in vivo, forming human intestinal organoids (HIOs) (Spence et al., 2011). HIOs have the identity of the small intestine and have proven extremely useful for modeling the biology of the small intestine (Bouchi et al., 2014; Finkbeiner et al., 2015; Watson et al., 2014; Xue et al., 2013). However, to date, PSC-derived colonic organoids have not been developed, and given the prevalence of disease in the large intestine, such a system would enable the investigation of developmental and disease mechanisms in this region of the gastrointestinal tract.

[0063] To develop a method to generate colonic organoids, we first identified Satb2 as a distinct marker of putative colonic epithelium in frogs, mice, and humans. Using Satb2 as a marker, we showed that BMP signaling is required for the specification of posterior gut endoderm in frogs and mice, which is consistent with the known role of BMPs in posterior ventral development (Kumar et al., 2003, Roberts et al., 1995, Sherwood et al., 2011, Tiso et al., 2002, Wills et al., 2008). Moreover, stimulation of BMP signaling in PSC-derived intestinal cultures for 3 days is sufficient to induce the formation of posterior HOX-encoding and SATB2-expressing colonic organoids. Human colonic organoids (HCOs) had marker characteristics and cell types consistent with the large intestine. Furthermore, HCOs, but not HIOs, formed colonic enteroendocrine cells (EECs) in response to expression of NEUROG3, demonstrating that HCOs are functionally involved in colonic regions. In addition, HCOs engrafted under the renal capsule of immunocompromised mice and grown in vivo for 8–10 weeks maintained their regional identity, formed tissues with colonic morphological properties, contained colon-specific cell types, had areas of proliferation and differentiation, and formed a well-formed smooth muscle layer. Intestinal enteroids and colonoids derived from in vivo-grown organoids maintained regional identity. Finally, RNA-seq analysis demonstrated that HIOs and HCOs underwent substantial maturation and expressed regional markers consistent with small and large intestinal identity, respectively. In summary, we identified an evolutionarily conserved BMP-HOX pathway in frogs and mice and used it to direct hindgut patterning and the formation of human colonic organoids.

[0064] result SATB2 expression marks the enteric endoderm of the embryonic and adult posterior gut.

[0065] The molecular pathways that establish the midgut and hindgut, the presumptive small intestine and the large intestine are poorly understood, in part due to the lack of well-defined markers. This has limited the ability to direct the differentiation of human PSCs into regionally distinct intestinal organoids, especially colonic organoids. Therefore, we identified markers that distinguish different domains of mouse embryonic gut and used them to investigate the signaling pathways that pattern the early intestine. Consistent with previous reports, we found that Gata4 labeled the gut endoderm from the posterior foregut to the yolk stalk in E9.5 mouse embryos (Figure 8A) (Aronson et al., 2014; Battle et al., 2008; Beuling et al., 2008a; Beuling et al., 2007a; Beuling et al., 2007b; Beuling et al., 2010; Beuling et al., 2008b; Bosse et al., 2007; Kohlnhofer et al., 2016; Patankar et al., 2012a; Patankar et al., 2012b; Sherwood et al., 2009; Walker et al., 2014). Later in development (embryonic day 11.5–embryonic day 16.5), Gata4 continued to clearly label the foregut but not the hindgut (Fig. 8B–D, I–J), and this expression domain remains intact into adulthood in both mice (not shown) and humans (Fig. 8K–L).

[0066] To identify markers of the posterior fetal gut, we searched public expression databases such as GNCPro™, TiGER and the Human Protein Atlas for genes abundant in the colon and found Satb2 as a promising colon marker. Satb2 is a member of the CUT class of homeobox genes (Holland et al., 2007), which binds nuclear matrix attachment regions and is involved in chromatin remodeling (Gyorgy et al., 2008). Immunostaining showed that Satb2 protein was first detected in the posterior endoderm of mouse embryos at embryonic day 9–9.5 and formed a discreet expression boundary with Gata4 at the yolk stalk (Figure 8A), suggesting that the Satb2+ domain marks the posterior gut, a broader expression domain than previously identified (Dobreva et al., 2006). Satb2 expression continued to label the posterior gut endoderm of the postnatal colon in mice (not shown) and humans (Figure 8L) throughout development (embryonic days 11.5-16.5) (Figure 8B,C,E,F,H,J). Using published human proteome and RNA-seq data, we confirmed that GATA4 and SATB2 differentially label proximal and distal regions of the human fetal and adult intestinal tract, respectively (Bernstein et al., 2010, Fagerberg et al., 2014) (Wang et al., 2015) (Figure 9A-C). These data demonstrate that the expression boundaries of Gata4 and Satb2 are established early in mouse development and label the future boundaries of the developing small and large intestine in mice and humans.

[0067] BMP signaling is required for Satb2 expression in the embryonic hindgut endoderm.

[0068] We next used Satb2 as a marker to identify pathways that promote the posterior gut fate in embryos. Given its known role in patterning endoderm at several stages of zebrafish, Xenopus, chick and mouse development, we first determined whether BMP signaling is active in the posterior gut (Kumar et al., 2003; Roberts et al., 1995; Sherwood et al., 2011; Tiso et al., 2002; Wills et al., 2008). We observed that BMP signaling is highly active in the endoderm and mesoderm of the posterior gut of embryonic day 8.5 mouse embryos, as measured by phosphorylated Smad1 / 5 / 8 (pSMAD1 / 5 / 8) (Figure 1A-B). To determine whether BMP signaling is required for posterior gut patterning, we cultured early frontal stage mouse embryos (embryonic day 7.5) in the BMP signaling inhibitor DMH-1 (Figure 1C). After 48 hours of DMH-1 treatment, we observed a significant reduction in pSmad1 / 5 / 8 levels and loss of Satb2 expression in the posterior gut (Figure 2D-K). In addition, Satb2 expression was lost in the first brachial arch of DMH-1-treated embryos, consistent with previous studies in zebrafish (Sheehan-Rooney et al., 2013). DMH-1 had no effect on TGFI3 signaling as measured by pSmad2 / 3 levels (Figure 1F). Given the evolutionary conservation of Satb2 across vertebrate species (Li et al., 2006), we investigated whether BMPs are required for Satb2 expression in the hindgut of frog embryos (Figure 2L). Similar to mice, treatment of Xenopus embryos with DMH-1 ( Fig. 1M–V ) or transgenic expression of the BMP antagonist Noggin (not shown) resulted in loss of Satb2 expression in the hindgut and brachial arch.BMP signaling has been shown to directly regulate Satb2 expression in mouse embryonic mandibles through direct binding of Smad1 / 5 to a conserved enhancer (Bonilla-Claudio et al., 2012), suggesting that Satb2 may also be a direct BMP target in the intestine. Together, these results reveal a conserved pathway in vertebrates whereby BMP signaling is required to define the posteriormost region of the developing intestine that gives rise to the distal ileum and large intestine.

[0069] BMP signaling promotes posterior fates in human intestinal cultures.

[0070] We next investigated whether BMP signaling could be used to promote a posterior intestinal fate in humans using nascent CDX2+ intestinal spheroids derived from human PSCs as previously described (Spence et al., 2011). We used the BMP inhibitors Noggin or BMP2, respectively, to inhibit or activate BMP signaling (Figure 2A) and monitored BMP signaling levels by accumulation of nuclear pSMAD1 / 5 / 8. Control cultures had low levels of pSMAD1 / 5 / 8 protein, and addition of Noggin abolished this staining (Figure 2B-D). In contrast, addition of BMP2 caused a rapid accumulation of pSMAD158 in both epithelial and mesodermal cells, suggesting that both cell types respond to BMP signals similar to what we observed in mouse embryos (Figure 1A-B). The specificity of pSmad1 / 5 / 8 staining was confirmed using adult mouse colon, which showed that pSmad1 / 5 / 8 staining was restricted to the differentiated compartment of the upper crypts, as previously reported (Hardwick et al., 2004; van Dop et al., 2009; Whissell et al., 2014). Further analysis of organoids revealed that 3 days of BMP2 treatment was sufficient to induce high levels of SATB2 protein in the epithelium compared to noggin and control cultures (Figure 2F-I). This suggests that a short pulse of BMP activity is sufficient to pattern spheroid endoderm towards a posterior intestinal fate.

[0071] Although BMP signaling is known to regulate anterior-posterior patterning of the endoderm, less is known about the transcriptional network that ultimately confers positional identity along the anterior-posterior axis in mammals. We used human intestinal spheroids and RNA-seq to identify how BMP signaling establishes a posterior domain in the developing human intestine. Principal component analysis revealed that intestinal spheroids treated with BMP for 3 days clustered separately from organoids treated with Noggin and control (Figure 2J). Examination of gene ontology terms (GO terms) revealed that regulation of BMP signaling affects multiple biological processes including organ morphogenesis, cell-cell signaling, pattern specification and cellular responses to BMP signaling (Figure 2K). As the most crucial regulators of anterior-posterior patterning are HOX genes, we found that BMP activation results in downregulation of anterior HOX genes and upregulation of posterior HOX genes (Figure 2L). In particular, we observed a BMP-mediated increase in multiple paralogs of HOX groups 10, 11, 12 and 13. These results demonstrate that BMP signaling broadly regulates the anterior and posterior hox codes during human intestinal patterning and suggest a mechanism by which the distal gut is initially specified.

[0072] BMP signaling acts downstream of SHH to induce the posterior HOX cord.

[0073] Previous studies have suggested that Sonic Hedgehog (Shh) acts upstream of Bmp4 and Hox13 expression during posterior gut patterning in chick embryos (Figure 10A) (Roberts et al., 1995). However, the relative epistatic association between BMP and Hox13 (Figure 10B) was not examined due to embryonic lethality caused by Bmp4 overexpression in the midgut and hindgut (De Santa Barbara et al., 2005; Roberts et al., 1995). We used human intestinal cultures to better model the epistatic association of SHH-BMP-HOX13 during posterior gut patterning. Activation of Hedgehog signaling with the smoothened agonist SAG resulted in concentration-dependent activation of the BMP signaling target gene MSX2 and the mesenchymal HOX factors HOXA13 and HOXD13 (Figure 10C). However, SAG-mediated activation of these factors was only a small fraction of that mediated by BMP2 (Figure 10C). We further showed that the ability of HH signaling to activate HOXA13 was entirely dependent on BMP (Figure 10D-E), confirming that BMP signaling functions downstream of SHH as previously reported (Shyer et al., 2015; Walton et al., 2012; Walton et al., 2009; Walton et al., 2016). Whether BMP signaling is sufficient to activate the posterior HOX program downstream of HH signaling remains to be determined. Therefore, we examined HOXA13 induction by BMP in the presence of the SHH inhibitor cyclopamine and found that BMP2 was sufficient to induce HOXA13 when SHH signaling was inhibited (Figure 10F-G). Consistent with this, activation of SHH signaling during BMP patterning did not improve SATB2 expression (Fig. S11A). Experiments in Xenopus confirmed this epistatic association between SHH and BMPs, suggesting that this mechanism is evolutionarily conserved (data not shown).Taken together, our data suggest that BMP signaling is sufficient to activate the posterior HOX code and does so downstream of HH signaling.

[0074] BMP-derived organoids cultured in vitro maintain distal identity.

[0075] We next investigated whether 3 days of BMP treatment was sufficient to confer stable regional identity after 25 days of long-term culture of organoids (Figure 3). The levels of ONECUT1 (a marker of the proximal small intestine) were highest in organoids treated with Noggin and controls and absent in organoids treated with BMP2 (Figure 3A-D). Conversely, SATB2 was absent in the epithelium of organoids treated with Noggin and controls, but was widely expressed in nearly all CDX2+ epithelial cells of organoids treated with BMP2 (Figure 3E-H, Figure 11A). Importantly, modulation of BMP signaling had similar proximal-distal patterning effects on multiple human PSC lines, including embryonic stem cell lines H1 and H9 and induced pluripotent stem cell lines (IPSC 54.1 and IPSC 72.3) (see below). We frequently observed non-epithelial SATB2 expression in Noggin and control organoids, likely due to the presence of other cell types known to reside in HIOs in vitro (data not shown). Examination of HOXB13 and HOXD13, expressed in the posterior epithelium and mesenchyme, respectively, further revealed that BMP-treated organoids maintained posterior patterning after long-term culture in vitro (Figure 11B-C).

[0076] Goblet cells are distributed in a low-to-high gradient from the proximal small intestine to the distal large intestine (Rodriguez-Pineiro et al., 2013), and we investigated whether goblet cell numbers were lower in proximal organoids and higher in distal organoids. Analysis of MUC2 staining at day 28 revealed that BMP2-treated organoids had a higher number of goblet cells as visualized by intracellular MUC2 compared to more proximal Noggin-treated and control organoids (Figure 3I-L), which had only rare intracellular MUC2 staining. We further confirmed the regional identity of goblet cells using MUC5B, a marker expressed by a subset of goblet cells in the colon but not in the small intestine (van Klinken et al., 1998). MUC5B staining was absent in 28-day-old organoids treated with Noggin and controls, but was present in BMP2-treated organoids (Figure 4M-P). The morphological properties of goblet cells are more mature in older organoids (Figure 11D-I), and here, in 44-day-old BMP-treated organoids, we observed goblet cells in the process of secreting mucus into the lumen of the organoid (Figure 11J-L). The ability to observe mucus secretion in BMP-treated organoids suggests that this organoid system will be useful for studying mucus secretion and the role of mucus in intestinal pathophysiology.

[0077] Although the regional pattern of organoids is stable after 28 days in culture, we wanted to examine whether early patterning was fully established after the first 3 days of treatment. To do so, we transferred spheroids treated with Noggin for 3 days to BMP2-containing medium for 3 days, and conversely, transferred spheroids treated with BMP for 3 days to Noggin-containing medium for 3 days. Proximal organoids generated with Noggin did not express SATB2 in response to BMP2, demonstrating that the proximal fate was stable after 3 days of patterning (Figure 11A). In the reverse experiment, 3 days of BMP2 treatment was sufficient to induce a stable distal fate, but a subset of organoids lost SATB2 expression in response to Noggin treatment (Figure 11A). Although 3 days of BMP2 treatment is sufficient to induce a colonic fate that is stable in vitro and in vivo (Figure 12), plasticity remains in the early posterior intestinal tract. This is consistent with the observation that the colonic endoderm of the midgestation rat embryo is regionally more plastic than the small intestinal endoderm (Ratineau et al., 2003).

[0078] Organoid mesenchymal patterning by BMP signaling.

[0079] Although stimulation of BMP signaling conferred regional identity to the organoid epithelium, we also observed upregulation of pSMAD1 / 5 / 8 in the non-epithelial compartment of BMP2-treated organoids during patterning, as well as posterior HOX factors known to be expressed in mesenchyme. To determine whether mesenchymal patterning was stable or required sustained patterning input from the epithelium, we isolated and expanded mesenchymal cell cultures for 2-3 weeks and analyzed them for expression of regional HOX genes. Mesenchymal cultures were devoid of E-cadherin expressing cells, suggesting that they were primarily composed of mesenchyme (Figure 3Q). Analysis of HOXD3, enriched in proximal intestinal mesenchyme (Yahagi et al., 2004), revealed that mesenchyme derived from Noggin- and control-treated organoids had stable proximal identity, whereas BMP-treated organoids had reduced expression of HOXD3 (Figure 3R) and high levels of HOXA13 (Figure 3S), the latter of which continues to be expressed in human colonic fibroblasts (Higuchi et al., 2015). Together, these data suggest that early regulation of BMP signaling patterns both epithelium and mesenchyme, and that mesenchymal patterning is stable even in the absence of epithelium.

[0080] Induction of colonic enteroendocrine cells is limited to BMP2-treated organoids.

[0081] The development of some ECC subtypes is regionally restricted to specific segments of the small and large intestine. For example, expression of the protein INSL5 is restricted to colonic EECs (Burnicka-Turek et al., 2012; Thanasupawat et al., 2013). As a functional test of colonic identity, we determined whether experimental induction of the colonic EEC marker INSL5 was restricted to BMP2-treated distal organoids. To do this, we inducibly expressed the pro-endocrine transcription factor NEUROG3 using an iPSC line carrying a doxycycline (DOX)-inducible NEUROG3 expression cassette (Figure 4A) as previously described (McCracken et al., 2017; McCracken et al., 2014). We performed a 6-h DOX pulse in cultures and after an additional 7 days observed a robust induction of EECs as measured by CHGA-positive cells (Figure 4B-I). However, we observed only INSL5-positive cells in BMP2-treated organoids, which was confirmed by QPCR analysis (Figure 4C-H, J). Given that INSL5-expressing cells are present only in the colon, our data strongly suggest that BMP2-treated organoids are functionally committed to a colon fate. The expression of distal markers such as SATB2, MUC5B and HOXA13 as well as the responsive ability to generate colon-specific ECCs support the conclusion that BMP2-treated organoids are colonic and therefore will be referred to as human colon organoids (HCOs).

[0082] The regional identity of patterned organoids is maintained in vivo.

[0083] Previous studies of mouse and human fetal intestine have demonstrated that regional identity and tissue morphology of different regions of the intestine are maintained after orthotopic transplantation and growth in immunocompromised mice (Duluc et al., 1994; Savidge et al., 1995). To determine whether in vitro patterned HIOs and HCOs maintain regional identity and grow into small and large intestinal tissues, we transplanted HIOs and HCOs under the mouse kidney capsule for 6–10 weeks, and we have already demonstrated results in HIO maturation into small intestinal tissues (Watson et al., 2014). We observed that engraftment of Noggin and control HIOs was more efficient than HCOs (Figure S12A). Consistent with their regional identity, transplanted HIOs and HCOs developed into mature tissues morphologically resembling either the small intestine or the large intestine, respectively (Figure S5A–E). The epithelium of Noggin and control organoids formed well-defined crypts and long villi comparable to those in the human small intestine. In contrast, BMP2-treated organoids contained crypts but lacked villi, similar to the colon.

[0084] In addition to morphological similarities to either the small or large intestine, transplanted HIOs and HCOs expressed distinct regional markers and contained regionally abundant cell types. For example, the majority of epithelium in noggin and control HIOs expressed the proximal marker GATA4 and did not express the colonic marker SATB2 (Figures 5F-I,K-N, Figure 12B-E). Conversely, HCO epithelium was uniformly SATB2+ but did not express GATA4 (Figures 5J,O, Figure 12B-E). In addition, DEFA5-expressing Paneth cells were present in the crypts of noggin and control HIOs but absent in HCOs (Figures 5P-T, Figure 12F), similar to human colon (Wehkamp et al., 2006). We further confirmed the colonic identity of HCOs using MUC5B (van Klinken et al., 1998), a colonic goblet cell marker expressed by a subset of goblet cells in HCOs but not detectable in Noggin or control HIOs (Figure 5U-Y, Figure S12G). In addition, the number of MUC2+ goblet cells was much greater in HCOs compared to HIOs (Figure S12H-L), consistent with the abundance of goblet cells seen in the human colon. The presence of patterning markers, MUC5B-expressing goblet cells, and the absence of Paneth cells all support the conclusion that transplanted HCOs have a colonic epithelium.

[0085] Mature HIOs and HCOs in vivo express regional enteroendocrine hormones.

[0086] There are at least 12 major EEC subtypes found in different regions of the digestive tract, and we analyzed HIOs and HCOs for the presence of regional EECs. Ghrelin and motilin were found primarily in the proximal gut, and correspondingly these hormones were primarily expressed in noggin and control HIOs but not in HCOs (Fig. 6A-D). Similarly, GIP, found in K cells of the small intestine but absent in the colon, was found in noggin and control HIOs but not in HCOs (Fig. 6E-H). Next, we investigated the presence of distally enriched EECs in HCOs by analyzing for expression of GLP-1 and PYY, which are more abundant in the colon. We observed a greater number of GLP-1 and PYY cells and greater expression of preproglucagon and PYY in HCOs than in HIOs (Fig. 61-P). In addition, we observed expression of the colon-specific hormone INSL5 ( Burnicka-Turek et al., 2012 ; Thanasupawat et al., 2013 ) only in HCOs ( Figures 6 Q–T ).

[0087] Analysis of stem and progenitor cells in HIOs and HCOs in vitro and in vivo.

[0088] To determine whether in vitro derived HIOs and HCOs express markers of stem and progenitor cells, we used the previously described H9-BAC-LGR5-eGFP transgenic line (McCracken et al., 2014; Watson et al., 2014). Examination of LGR5-eGFP expression in organoids revealed expression in widespread epithelial domains similar to the expression pattern in Lgr5-eGFP mice as early as embryonic day 13.5 (Shyer et al., 2015) (Figure 13A,B,F,G,K,L). GFP expression was also evident outside the epithelium of organoids as determined by histology and FACS analysis that revealed a population of GFP+ EPCAM- cells (data not shown). In addition, we examined the expression of SOX9, a marker of progenitor cells in the fetal and adult intestine, and found it to be expressed in the epithelium of both HIOs and HCOs (Figure 13C-E,H-J,M-0). These data suggest that embryonic / fetal intestinal progenitor cells marked by LGR5-eGFP and SOX9 are present in HIOs and HCOs in vitro.

[0089] During later stages of intestinal development, progenitor cells become localized to the base of the developing villi, where they will ultimately contribute to intestinal stem cells (ISCs) in the crypts of Lieberkuhn. To determine whether the progenitor cells we observed in vitro undergo this developmental transition, we transplanted HIOs and HCOs and monitored LGR5-eGFP, SOX9, and KI67 protein. Following maturation of organoids in vivo, we observed LGR5-eGFP, SOX9, and KI67 localized to the base presumptive crypts (Figure 13P-X). In addition, SOX9 was also observed in EECs in the villi of HIOs and in the cuffs of colonic epithelium-implanted HCOs, consistent with SOX9 expression in these cell types. Given that Sox9 and Lgr5 mark intestinal and colonic stem cells that can form enteroids and colonoids in mice (Gracz et al., 2010; Ramalingam et al., 2012), we investigated whether the epithelium of transplanted organoids could be isolated and used to generate enteroids and colonoids. Both HIOs and HCOs gave rise to cultures of epithelial organoids that could be grown and passaged (Figure S13Y-A'). Moreover, HCO-derived epithelial cultures expressed colonic markers CKB, FXYD3, SATB2, and HOXB13, but not proximal small intestinal markers PDX1 or GATA4, suggesting that regional identity was maintained (Figure S13B'-D'). These data suggest that HIOs and HCOs grown in vivo contain progenitor and stem cells.

[0090] Global transcriptional analysis of HIOs and HCOs To broadly examine the regional identity and maturation of HIOs and HCOs, we performed RNA-seq analysis of in vivo grown HIOs and HCOs and compared them to published datasets of human fetal and adult small and large intestines. Principal component analysis revealed that primary tissues isolated from adult and fetal intestines clustered together along the principal component 1 (PC1) axis, which accounted for 36.5% of the cumulative variation between samples (Figure 14A). GO analysis revealed that this variation was due to cell types present only in primary tissues and absent in PSC-derived grafts. For example, 6 of the top 10 biological processes present in human primary tissues and absent in grafts were associated with immune cells (Figure 14B-C). The second principal component (PC2), accounting for 17.7% of the cumulative variation, separates samples by maturation (Figure 7A). This component revealed that the transplanted organoids were more mature than human fetal intestine and fetal colon, but not as mature as adult colon and intestine. The third principal component (PC3), accounting for 6.7% of the cumulative variance, separated samples by regional identity, showing that HCOs are more similar to colon, whereas HIOs cluster with small intestine (Figure 7A). Interestingly, human fetal samples did not cluster based on regional identity (small intestine vs. colon), suggesting that these samples may not have been clearly separated from their indicated regions of the digestive tract.

[0091] We next used the hypergeometric mean test to determine the probability that HIO and HCO share similar patterns of small intestine and colonic region-specific gene expression (Figure 7B). A total of 341 transcripts were expressed in the small intestine and in Noggin-treated HIO compared to colon or BMP2-treated HCO, a ratio that is highly unlikely by chance alone (P=1.5×10-143). Similarly, the gene set that is upregulated in control HIO shares a highly significant degree of similarity with the gene set that is upregulated in adult small intestine compared to adult colon (P=2.5×10-203). Conversely, the gene set that is upregulated in HCO is highly enriched for genes that are upregulated in colon compared to small intestine (P=4.1×10-53 and P=6.0×1073, respectively). This analysis concluded that HIO patterning is most similar to human small intestine, and HCO patterning is colonic. To further explore the nature of HIO (NOG and control treatment) and HCO, we performed differential expression analysis (adult small intestine vs. adult colon, HIO vs. HCO). We generated 4-way scatter plots and demonstrated that a high proportion of genes upregulated in colon were also upregulated in HCO, and a majority of genes upregulated in small intestine were also upregulated in HIO (Figure 7C, Table 1). Finally, analysis of enriched biological processes revealed that adult colon and transplanted HCO had highly active Wnt signaling and similar HOX codes (Figure 7D). Taken together, these data suggest that we have developed a robust method for differentiating PSCs into human colon tissue.

[0092] Table 1 Genes upregulated in adult small intestine and colon, which are also upregulated in HIO and HCO, respectively. Column 1, generally upregulated in NOG HIO vs. HCO and adult small intestine vs. adult colon; Column 2, generally upregulated in control HIO vs. HCO and adult small intestine vs. adult colon; Column 3, generally upregulated in HCO vs. NOG HIO and adult colon vs. adult small intestine; Column 4, generally upregulated in HCO vs. control HIO and adult colon vs. adult small intestine [Table 1] TIFF2024096732000003.tif207158TIFF2024096732000004.tif205165TIFF2024096 732000005.tif204158TIFF2024096732000006.tif203156TIFF2024096732000007.t if214154TIFF2024096732000008.tif206157TIFF2024096732000009.tif215163TIF F2024096732000010.tif213157TIFF2024096732000011.tif203150TIFF2024096732 000012.tif211162TIFF2024096732000013.tif210161TIFF2024096732000014.tif2 09155TIFF2024096732000015.tif206149TIFF2024096732000016.tif207153TIFF20 24096732000017.tif213149TIFF2024096732000018.tif201148TIFF2024096732000 019.tif199150TIFF2024096732000020.tif199152TIFF2024096732000021.tif57144

[0093] Consideration Historically, the classification of foregut, midgut, and hindgut has been based on the development of anterior and posterior intestinal portal veins and the mesenteric blood supply (Uppal et al., 2011). Alternative definitions of the midgut and hindgut have been proposed, with the midgut being the portion of the intestine derived from the area anterior to the umbilicus, and the hindgut being derived from the area posterior to the umbilicus (Johnston, 1913; Savin et al., 2011). In either case, the historical reliance on anatomical landmarks, as well as the lack of more precise molecular markers to distinguish the anterior, middle, and hindgut, have made it difficult to develop methods to generate these cells / tissues in vitro from PSCs. Therefore, the identification of markers that clearly distinguish the developing midgut and hindgut regions is essential.

[0094] Using a combination of CDX2, GATA4, ONECUT1 and SATB2, we identified that a clear molecular boundary is established early in midgut and hindgut development in Xenopus, mice and humans. Interestingly, GATA4 and SATB2 expression domains form a boundary at the yolk stalk / presumptive umbilicus in mice, and this boundary is maintained in the adult gut throughout development. The fact that GATA4 expression labels the gut anterior to the umbilicus and SATB2 expression labels the domain posterior to the umbilicus suggests that the umbilicus is the boundary between the midgut and hindgut (Johnston, 1913; Savin et al., 2011).

[0095] While ONECUT1 and SATB2 expression in HIOs is consistent with the proximal and distal identities of HCOs, respectively, GATA4 was not as robustly expressed in proximal HIOs in vitro as would be expected given its embryonic expression (data not shown). In contrast, GATA4 was strongly expressed in enteroids generated from patient biopsies after in vivo maturation of HIOs (data not shown). This may suggest that factors involved in GATA4 expression are absent in culture conditions or that in vivo maturation is required for epithelial expression of GATA4. The data also suggest that high levels of GATA4 expression may be dispensable for early regionalization of the intestine, consistent with intestinal Gata4 knockout mice retaining normal ONECUT factor expression (Battle et al., 2008). In addition, a small subset of BMP-treated organoids lost CDX2 expression and activated expression of bladder markers keratin 13 and uroplakin 1a (data not shown). This is consistent with BMP organoids having a hindgut fate, as urothelial tissue is derived from the hindgut / cloaca ( Georgas et al., 2015 ).

[0096] SATB2 is expressed throughout the development of the distal ileum and colon, but it is not known whether SATB2 is required for distal intestinal development. Mouse knockout studies have focused on the development of craniofacial and cortical neurons, as mutations in SATB2 have been linked to 2q32-q33 deletions and the cleft palate associated with Glass syndrome (FitzPatrick et al., 2003). However, there is indirect evidence that SATB2 may play a role in human colon physiology. SATB2 has been identified as an ulcerative colitis susceptibility gene in a genome-wide association study (McGovern et al., 2010). In addition, loss of SATB2 expression has been shown to be associated with poor prognosis in patients with colon cancer (Eberhard et al., 2012). Future studies with HCOs may allow for the identification of SATB2 targets in the developing colon, which may provide insight into the pathology of ulcerative colitis and colon cancer.

[0097] Several studies in model organisms have implicated the BMP signaling pathway in the patterning of endoderm during hindgut development (Kumar et al., 2003, Roberts et al., 1995, Sherwood et al., 2011, Tiso et al., 2002, Wills et al., 2008). Consistent with this, we have demonstrated that posterior patterning of human definitive endoderm depends on BMP signaling, as inhibition of BMPs abolishes the ability of WNT and FGF to promote posterior endoderm fates (McCracken et al., 2014). However, it is not surprising that BMP signaling plays other temporally distinct roles during intestinal development. For example, after the establishment of the proximal-distal regional domain, BMP signaling functions to establish the crypt-villus axis in the intestine and colon (Li, 2005). Thus, temporal requirements for patterning allow embryos to use the same signaling pathway for versatile gut development, as reported in the Drosophila midgut (Driver and Ohlstein, 2014; Guo et al., 2013). In the context of human disease, mutations in BMPR1A are associated with a subset of juvenile polyposis syndrome patients. The HCO system is well suited to identify HOX codes downstream of BMPs during early development, and it may be interesting to determine whether hamartomatous polyps with BMPR1A mutations have altered HOX gene expression.

[0098] We previously reported in vitro directed differentiation and in vivo transplantation of small intestine, HIO (Spence et al., 2011; Watson et al., 2014). Given the unique physiological properties and pathological conditions affecting the colon, it was essential to develop a colon model system to investigate pathophysiological questions specific to the colon. Developmentally, this system provides an opportunity to investigate fundamental questions about how regional identity is established. HIO and HCO develop unique cell types such as Paneth cells in HIO and colon-specific goblet cells in HCO. Moreover, HIO and HCO harbor distinct sets of EECs that are normally abundant in the small intestine and large intestine, respectively. Regionalized organoids should provide a foundation for future studies of how different regions of the intestine give rise to regionalized stem cells. In addition, the generation of HCO will enable the modeling of diseases that affect the colon, such as ulcerative colitis and colon cancer.

[0099] material and method Animals. Immunodeficient NOD-SCID IL-2Rynu" (NSG) mice aged 8-16 weeks were used for transplantation experiments (obtained from the Comprehensive Mouse and Cancer Core Facility, Cincinnati, OH). Wild-type mice were used for studies on the mouse fetal intestine. All mice were housed in the Cincinnati Children's Hospital Medical Center (CCHMC) animal facility. All experiments were performed with approval from the CCHMC Institutional Animal Care and Use Committee.

[0100] BMP inhibition in frog and mouse embryos. Xenopus tropicalis embryo culture and small molecule treatments were performed as previously described (Rankin et al., 2012; Rankin et al., 2015). DMH-1 (Sigma D8946) was dissolved in DMSO and used at a final concentration of 20 pM, and an equal concentration of DMSO vehicle was used in sibling embryos. Inhibitor treatment experiments were repeated twice with similar effects on the analyzed markers. For Xenopus in situ hybridization analysis, DIG-labeled antisense RNA probes were generated using linearized full-length cDNA plasmid templates (X. tropicalis satb2 was purchased from ATCC, clone 7720194, HinDIII, T7 for probe, X. laevis satb2 was a gift for Tyler Square and Daniel Medeiros, University of Colorado, Boulder, and Xbal, Sp6 for probe). Complete details describing probe synthesis and in situ hybridization protocols are available in Xenbase (hftp: / / wiki.xenbase.orq / xenwiki / index.php / Protocols).

[0101] For mouse whole embryo culture, embryonic day 7.5 embryos were cultured in a 1:1 mixture of Ham's F12 medium and whole embryo culture rat serum (Harlan Labs) containing N-2 supplement (Invitrogen). The containers were placed on a roller culture apparatus (BTC Engineering, Cambridge, UK) and maintained at 37°C for 2 days, aerated with 20% O2 and 5% CO2. DMSO was used as a vehicle control, and BMP signaling was inhibited by treatment with 5 pM DMH-1.

[0102] Generation of human midgut / hindgut spheroids Human intestinal organoids were generated and maintained as previously described (Watson et al., 2014). Human embryonic stem cells and induced pluripotent stem cells were grown under feeder-free conditions in 6-well Nunclon surface plates (Nunc) coated with Matrigel (basement membrane matrix (BD Biosciences)) and maintained in mTESR1 medium (Stem Cell Technologies). For definitive endoderm (DE) induction, human ES or iPS cells were passaged with Accutase (Invitrogen) and seeded at a density of 100,000 cells per well in Matrigel-coated Nunclon surface 24-well plates. For Accutase-splitting cells, 10 pM of Y27632 compound (Sigma) was added to the medium on the first day. After the first day, the medium was replaced with mTESR1 and cells were grown for another 24 h. Cells were then treated with 100 ng / mL activin A for 3 days as previously described (Spence et al., 2011).DEs were then treated with hindgut induction medium (RPMI1640, 2 mM L-glutamine, 2% de-supplemented FBS, penicillin-streptomycin and 100 ng / mL activin A) for 4 days along with 500 ng / mL FGF4 (R&D) and 3 pM Chiron99021 (Tocris) to induce the formation of mid-hindgut spheroids.

[0103] Patterning of midgut / hindgut spheroids into HIOs and HCOs. Spheroids were harvested from 24-well plates and seeded in Matrigel (BD). To generate proximal HIOs, spheroids were overlaid with intestinal growth medium (Applied DMEM / F-12, N2, B27, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng / mL EGF (R&D) alone or 100 ng / mL EGF containing 100 ng / mL Noggin (R&D). To generate HCOs, spheroids were overlaid with 100 ng / mL EGF and 100 ng / mL BMP (R&D). For SHH experiments, 1 μM SAG (Tocris), 5 μM SAG or 2.5 μM Cyclopamine (Tocris) were added to the control medium for the first 3 days after RNA samples were collected. For all patterning conditions, medium was changed at day 3 while maintaining only EGF in the medium. Medium was then changed twice weekly thereafter. HIOs and HCOs were reseeded with fresh Matrigel every 14 days.

[0104] Generation of Neurogenin 3-inducible lines. To generate doxycycline-inducible NEUROG3 lines, we transduced IPSC72.3 cells with pINCUDER21-NEUROG3 lentivirus and selected with 250 μg / mL G418. Both IPSC72.3 cell lines and inducible NEUROG 3 have been previously described (McCracken et al., 2014). Stably transduced cells were differentiated into mid / hindgut spheroids and then patterned into HIOs or HCOs. Spheroids were grown for 28 days and stimulated with 0.5 μg / mL doxycycline for 8 hours. After 35 days, organoids were harvested and analyzed by QPCR and IF.

[0105] Outgrowth of organoids and mesenchymal cells from organoids that attach to the bottom of 24-well plates attach and grow in two dimensions. To grow mesenchymal cells from organoids, DMEM 10% FBS + L-glutamine + penicillin-streptomycin was added to the wells, from which organoids were collected after 14 days. Medium was changed twice a week for a total of 2-3 weeks until nearly 100% confluence was reached.

[0106] Transplantation of human intestinal organoids NSG mice were maintained on an antibiotic-containing diet (275 ppm sulfamethoxazole and 1,365 ppm trimethoprim (test diet)). Food and water were provided ad libitum before and after surgery. Single HIOs matured in vitro for 28 days were removed from the Matrigel, washed with cold phosphate-buffered saline (DPBS; Gibco), and embedded in purified type I collagen (rat tail collagen (BD Biosciences)) 12 h before surgery to allow the formation of solidified gel plugs. These plugs were then placed in standard growth medium overnight in intestinal growth medium (Applied DMEM / F-12, B27, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) (R&D) supplemented with 100 ng / mL EGF. HIOs were then transplanted under the kidney capsule as previously reported (Watson et al., 2014). Briefly, mice were anesthetized with 2% inhaled isoflurane (Butler Schein), then the left side of the mouse was prepared with isopropyl alcohol and povidine-iodine in a sterile fashion. A small left posterior subcostal incision was made to expose the kidney. After creating a subcapsular pocket, collagen-embedded HIO was placed into the pocket. The kidney was then returned to the peritoneal cavity and the mouse was given a quick intraperitoneal lavage of Zosyn (100 mg / kg (Pfizer Inc.)). The skin was closed in a double layer, and the mouse was injected subcutaneously with Buprenex (0.05 mg / kg (Midwest Veterinary Supply)). At 8–10 weeks post-engraftment, the mice were then humanely euthanized or subjected to further experiments.

[0107] Tissue processing, immunofluorescence and microscopy. Depending on the size of the tissue, tissues were fixed in 4% paraformaldehyde (PFA) on ice for 1–3 h. Organoids and graft viabilities were frozen in OCT. OCT sections were blocked with donkey serum (5% serum in 1× PBS + 0.5% Triton-X) for 30 min and incubated with primary antibodies overnight at 4°C. Slides were then washed three times with 1× PBS + 0.5% Triton-X and incubated in secondary antibodies containing DAPI in blocking buffer for 2 h at room temperature. See Table 2 for a list of antibodies and their respective dilutions. Slides were then washed twice with 1× PBS + 0.5% Triton-X followed by a final wash in 1× PBS. Coverslips were then mounted using Fluoromount-G® (SouthernBiotech). Images were captured on a Nikon Al confocal microscope and analyzed using Imaris Imaging Software (Bitplane). For whole mount staining, tissues were processed as described above and then cleared with Murray's solution. Imaging was performed using a Nikon Al confocal microscope.

[0108] [Table 2] TIFF2024096732000023.tif168135

[0109] Quantification of immunofluorescence images Image quantification of whole embryos was performed by splitting the image into separate channels and then measuring the pixel area using ImageJ (NIH). The pixel area was determined for each channel and the ratio between channels was determined, with the ratio for control treated embryos expressed as 100. Quantification of in vitro and in vivo grown organoids was performed on sections from which images were captured, as previously described. The number of CDX2, GATA4 and SATB2 positive nuclei was quantified using the spot function in [marls] after calibration with human biopsy samples.

[0110] RNA isolation and QPCR RNA was extracted using the Nucleospin® RNA extraction kit (Macharey-Nagel) according to the manufacturer's protocol and reverse transcribed into cDNA using Superscript VILO (Invitrogen). QPCR primers were designed using the qPrimerDepot web-based tool (primerdepot.nci.nih.gov). Primer sequences are listed in Table 3. QPCR was performed using the Quantitect SYBR® Green PCR kit (Qiagen) and the QuantStudio™ 6 Flex real-time PCR system (Applied Biosystems).

[0111] [Table 3] TIFF2024096732000025.tif148141

[0112] Identification of SATB2 as a colon marker To identify markers for the large intestine, we first used GNCPro http: / / gncpro.sabiosciences.comigncpro / expression_grapherphp to identify transcription factors that were upregulated in the colon (compared to other tissues) based on the University of Tokyo database. Based on this search, SATB2 was the sixth ranked gene in the colon. To verify that SATB2 is indeed upregulated in the colon, we searched for SATB2 expression using the TiGER database (hftp: / / bioinfo.wilmer.ihu.edu / tiger / db gene / SATB2-index.html). To further confirm the expression of SATB2 in the colon and to examine protein expression across multiple tissues, we used the Human Protein Atlas (http: / / www.proteinatlas.org / search / satb2). Similar approaches were used to identify other markers for the large intestine / colon.

[0113] Public RNA-seq Accession Numbers RNA-seq data for adult small and large intestine were downloaded from the public database E-MTAB-1733. These datasets represent whole organ tissues including epithelial and muscle layers. Accession numbers for small intestine samples: ERR315344, ERR315381, ERR315409, ERR315442, ERR315461. Accession numbers for large intestine samples: ERR315348, ERR315357, ERR315484. For Figure 9B, processed FPKM data were downloaded from https: / / qithub.com / hilldr / Finkbeiner StemCellReports2015. These data include adult duodenum (ERS326992, ERS326976) and the previously listed small intestine samples from E-MTAB-1733, and a human fetal intestine sample (also whole organ) from GSE18927. The accession numbers for human fetal small intestine are GSM1059508, GSM1059521, GSM1059486, GSM1059507, GSM1059517, GSM1220519. For Figure 9C, data were obtained from GEO accession GSE66749 platform GLP5175. The following samples were used: GSM1385160, GSM1385161, GSM1385162, GSM1385163, GSM1385164, GSM1385165, GSM1385166, GSM1385167, GSM1385168, GSM1385169, GSM1385170, GSM1385171, GSM1614646, GSM1614646. Sample values ​​were determined using the “Characteristics Graph” function in GEO2R, ​​searching for GATA4 and SATB2 by their ID numbers (3086100 and 2594089, respectively).

[0114] Estimation of sequence population of RNA-seq RNA library construction and RNA sequencing were performed by the Cincinnati Children's Hospital DNA Sequencing Core using an Illumina HiSeq 2500 platform. The quality of the Illumina sequencing run was assessed by analyzing the FASTQ data of each sample using FastQC version 0.10.1 http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc to identify data features that may indicate quality issues (low quality scores, over-represented sequences, inappropriate GC content, etc.). No major issues were identified in the QC analysis. We used the software package Tuxedo Suite for alignment, differential expression analysis, and post-analysis diagnostics. Briefly, we aligned the reads against the reference transcriptome (UCSC hg19) using TopHat version 2.0.13 and Bowtie version 2.2.5 (Langmead et al., 2009). We used default parameter settings for alignment, except for "-b2-very sensitive" to maximize the accuracy of read alignment, as well as "-non-exhaustive search" and "-non-novel junction" to restrict reads mapping to known transcripts. Cufflinks version 2.2.1 (Trapnell et al., 2012) was used for RNA abundance estimation. UCSC hgl9.fa was used as the reference genome sequence, and UCSC hgl9.gtf was used for transcriptome annotation. We applied the following parameters in Cufflinks: "-multiple read correction" to adjust expression calculations for reads mapping at more than one locus, and "-comparable hit-norm" and "-upper quartile-norm" for normalization of expression values. Normalized FPKM tables were generated using the CuffNorm function. RNA-seq assembly and transcription analysis were performed using a 64-bit Debian Linux stable version 7.10 ("Wheezy") platform.

[0115] Differential expression analysis All plots and statistical analyses were performed in R version 3.3.1 (2016-06-21). Plots were created using the R package 'ggplot2' (Ginestet, 2011). Differential expression analysis and statistical testing of Cufflinks output was completed using the R package 'SeqRetriever' version 0.6 https: / / github.com / hilldr / SeqRetrieyer. The hypergeometric mean test was used to assess the relative abundance of shared gene expression signatures between groups using the R package 'GeneOverlap' http: / / shenlab-sinai.cithub.io / shenlab-sinai / . The complete RNA-seq FASTQ processing pipeline and analysis scripts are available at https: / / qithub.com / hilldr / Munera2016.

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[0193] All percentages and percentages are calculated by weight unless otherwise indicated. All percentages and percentages are calculated based on the total composition unless otherwise indicated.

[0194] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0195] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Rather, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "about 20 mm."

[0196] All documents cited herein, including any cross-referenced or related patents or applications, are incorporated herein by reference in their entirety, unless expressly excluded or specifically limited. The citation of any document shall not be deemed to be prior art to any invention disclosed or claimed herein, or to teach, suggest, or disclose any such invention, either alone or in combination with any other reference(s). Furthermore, if any meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control.

[0197] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

[0198]

Claims

1. A human colon organoid (HCO), wherein the human colon organoid comprises epithelium, mesenchyme, colon-specific goblet cells, colon enteroendocrine cells (EECs), progenitor cells, and stem cells, and the HCO expresses SATB2.

2. 2. The HCO of claim 1, wherein the HCO further expresses CKB, MUC2, MUC5B, CDX2, INSL5, SOX9, FXYD3, HOXA13, HOXB13, and HOXD13.

3. 3. The HCO according to claim 1, wherein the expression of HOXD3, PDX1, and GATA4 is reduced.

4. 4. The HCO according to claim 1, wherein the HCO is characterized by the presence of crypts and is substantially free of villi.

5. 5. The HCO of any one of claims 1 to 4, wherein the HCO is characterized by the presence of areas of proliferation and differentiation.

6. 6. The HCO of any one of claims 1 to 5, characterized by the presence of convoluted epithelium surrounded by mesenchymal cells and / or a well-formed smooth muscle layer.

7. 7. The HCO of claim 1, wherein the HCO comprises CD14+ and / or CD16+ cells and / or fibroblasts.

8. 8. The HCO of any one of claims 1 to 7, wherein the HCO does not contain one or more of immune function, innervation, blood vessels, villi, and Paneth cells.

9. 9. The HCO of claim 8, wherein the HCO is substantially free of Paneth cells.

10. 10. The HCO of any one of claims 1 to 9, wherein the HCO secretes one or more distally concentrated and / or colon-specific hormones.

11. 11. The HCO of any one of claims 1 to 10, wherein the distally enriched and / or colon-specific hormone comprises INSL5.

12. 12. The HCO of any one of claims 11, wherein the distally concentrated and / or colon-specific hormones further comprise GLP-1 and / or PYY.

13. 13. The HCO of any one of claims 1 to 12, wherein the HCO is prepared from definitive endoderm (DE) derived from embryonic stem cells or induced pluripotent stem cells.

14. 14. The HCO of any one of claims 1 to 13, wherein the HCO is prepared in vitro.

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