Enteroids and organoids derived in vitro from pluripotent stem cells, and uses thereof

Abstract

Disclosed herein are enteroids, obtained by dissociating an epithelial cell population from a non-transplanted human intestinal organoid (HIO), such as an HIO produced in vitro from human pluripotent stem cells (hPSCs). Also disclosed herein are methods of producing and using the same, including methods of transplantation involving said enteroids, particularly for treating a gastrointestinal disease or condition. Also disclosed herein are methods of transplantation involving HIOs and / or HIO components, particularly for treating a gastrointestinal disease or condition.

Description

Attorney Docket No.: CHMC.P0086WO PCTENTEROIDS AND ORGANOIDS DERIVED / VITKO S PLURIPOTENT STEM CELLS, AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 683,103, ENTEROIDS AND ORGANOIDS DERIVED IN VITRO FROM PLURIPOTENT STEM CELLS, AND USES THEREOF, filed on filed August 14, 2024, and U.S. Provisional Application No. 63 / 758,849, ENTEROIDS AND ORGANOIDS DERIVED IN VITRO FROM PLURIPOTENT STEM CELLS, AND USES THEREOF, filed on filed February 14, 2025, which are currently co-pending herewith and which are incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. U01 DK103117 and P30 DK078292 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] Aspects of the present disclosure generally relate to gastrointestinal organoids, methods of preparation, and compositions including the same, as well as uses thereof.BACKGROUND

[0004] Ulcers can occur throughout the gastrointestinal tract due to many conditions, including peptic ulcers, and ischemic injuries associated with surgical conditions and radiation therapies which result in significant morbidity. First line treatments of the underlying condition typically restore the damaged epithelium; however, transmural bowel injury can unfortunately persist in chronic conditions where the underlying mesenchyme is damaged. For this subset of patients, there is a paucity of therapeutic options to regenerate chronically damaged bowel. Even when a treatment successfully controls the underlying damaging condition, it does not always result in bowel regeneration, leaving patients experiencing symptoms impacting quality of life. Apotential cause may be chronic damage within the supporting mesenchymal and vascular compartments, which arc important for epithelial regeneration.

[0005] Currently, few clinical treatment options exist when this happens. While some patients may benefit from surgical intervention when their diseased region is focal, it is also associated with additional morbidity, and it does not address all patients with complex or multifocal disease. Thus, there is a need for novel therapeutic strategies in order to broaden the treatment modalities available for these patients.

[0006] There is also a need for a deeper understanding of human gastrointestinal (GI) disease in general. For example, as many as 11 percent of all Americans have acute inflammatory conditions that present throughout the GI tract due to numerous conditions that are commonly treated with current medical management. Unfortunately, a small yet significant percentage of these patients will develop persistent GI inflammation despite therapy and are left with limited medical options, often leading to non-healing ulcers or fibrotic intestine refractory to repeat dilations and subsequent surgical resections or transplantation. These outcomes are associated with significant morbidity. Improved GI disease models, such as through development and testing of models relevant to one or more of the various GI diseases, are necessary in order to study disease mechanisms and translational use based on such research, e.g. identifying and evaluating potential treatment options.SUMMARY

[0007] Various embodiments of the disclosure relate to in vitro methods of producing an enteroid, the method including: a) dissociating an epithelial cell population from a nontransplanted human intestinal organoid (HIO); and b) substantially removing one or more mesenchymal cell population from the dissociated epithelial cell population; thereby providing an enteroid. In some embodiments, the method further includes expanding the dissociated epithelial cell population in vitro, to provide the enteroid.

[0008] Particular' embodiments of the disclosure relate to in vitro methods of producing an enteroid derived from human pluripotent stem cells (hPSCs), the method including: a) obtaining or isolating an epithelial cell population from a non-transplanted human intestinal organoid (HIO), wherein the HIO is obtained by in vitro expansion of one or more precursorcells (hPSCs, iPSCs); and b) substantially removing one or more mesenchymal cell population from the dissociated epithelial cell population; thereby providing an cntcroid.

[0009] In some embodiments of the methods, the enteroid substantially includes the dissociated epithelial cell population. In some embodiments, the dissociated epithelial cell population self-organizes, or self-assembles, into the enteroid. In some embodiments, the enteroid does not include a mesenchymal, neuronal, endothelial, and / or immune cell population. In some embodiments, all or substantially all of the mesenchymal cell population is removed. In some embodiments, the enteroids are free, or is substantially free, of mesenchymal cells. In some embodiments, the enteroid can further include progenitor cells, intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and / or a proliferative compartment.

[0010] In some embodiments, the method is performed in vitro. In some embodiments, the HIO has been differentiated and proliferated entirely in vitro.

[0011] In some embodiments, the methods further include culturing the dissociated epithelial cell population for at least about one day; optionally at least about 1 to 10 days, 7 to 10 days, 7 to 15 days, 7 to 20 days, or longer. In some embodiments, the enteroids are cultured for at least about one day; optionally at least about 1 to 10 days, 15 days, 20 days, or longer. In some embodiments, the dissociated epithelial cell population is passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times; optionally at least about 3-7 times; optionally at least about 3-5 times; optionally 5 times. In some embodiments, the dissociated epithelial cell population is cultured for at least about 1 to 10 days prior to each passaging; optionally at least about 7 days.

[0012] In some embodiments, the epithelial cells and / or mesenchymal cells can be dissociated from the HIO by enzymatic dissociation and / or mechanical dissociation. In some embodiments, enzymatic dissociation can include dissociating the HIO with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof. In some embodiments, mechanical dissociation can include manual dissociation and / or passing the HIO through a mesh and / or successively narrower bore channels. In some embodiments, epithelial cells and / or mesenchymal cells can be dissociated from the HIO by using an extracellular matrix depolymerization solution and / or mechanical dissociation. In some embodiments, during dissociation, a majority of the epithelial cell population is free from surrounding cell populations. In some embodiments, following dissociation, the epithelial cellpopulation is intact. In some embodiments, the dissociated epithelial cell population can be separated from surrounding cell populations; optionally using a cell strainer. In some embodiments, the enteroid is obtained by in vitro expansion of the dissociated epithelial cell population. In some embodiments, the dissociation step can be performed in vitro. In some embodiments, the enteroids can be cryoprcscrvcd; optionally wherein the enteroids are cryopreserved for at least about 30 days, 60 days, 90 days, 120 days, 1 year, 2 years, or longer.

[0013] In various embodiments, the methods can further including producing the HIO from human pluripotent stem cells (hPSCs). In some embodiments, the hPSCs comprise embryonic stem cells or induced pluripotent stem cells.

[0014] Further embodiments of the disclosure relate to in vitro enteroids derived from human pluripotent stem cells (hPSCs), including: an epithelial cell population derived from a non-transplanted human intestinal organoid (HIO), wherein the HIO is obtained via in vitro expansion of one or more hPSCs, wherein the enteroids are substantially free of a mesenchymal, neuronal, endothelial, and / or immune cell population. Further embodiments of the disclosure relate to in vitro enteroids derived from human pluripotent stem cells (hPSCs), including: an epithelial cell population derived from a non-transplanted human intestinal organoid (HIO), wherein the HIO is obtained via in vitro expansion of one or more hPSCs, wherein the enteroids are substantially free of a mesenchymal, neuronal, endothelial, and / or immune cell population; wherein the enteroids are produced by any of the aforementioned methods.

[0015] In some embodiments, the enteroids are free, or is substantially free, of mesenchymal cells. In some embodiments, the enteroids further include progenitor cells, intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and / or a proliferative compartment. In some embodiments, the enteroids can include MKI67+ cells. In some embodiments, the enteroids express one or more intestinal epithelial cell marker, proliferation marker, surrogate marker of stem cell activity and intestinal crypt, stem cell marker, Paneth cell marker, and / or pan-mesenchymal marker. In some embodiments, the one or more intestinal epithelial cell marker can include CDX2; the one or more proliferation marker can include MKI67; the one or more surrogate marker of stem cell activity and intestinal crypt can include OLFM4; the one or more stem cell marker can include LGR5; the one or more Panethcell marker can include LYZ; and / or the one or more pan-mesenchymal marker can include VIM.

[0016] In some embodiments, the HIO is derived from pluripotent stem cells (PSCs). In some embodiments, the PSCs include embryonic stem cells or induced pluripotent stem cells. In some embodiments, the HIO is an artificial or engineered HIO and is derived from human PSCs in vitro. In some embodiments, the enteroids can include an artificial or engineered enteroid and / or can be generated in vitro. In some embodiments, the enteroids arc three-dimensional and / or generally spherical.

[0017] Further embodiments of the disclosure include in vitro compositions including any of the aforementioned enteroids.

[0018] Further embodiments of the disclosure include any of the aforementioned enteroids or compositions, for use in methods of treating a gastrointestinal-related disease or disorder, methods of screening for therapeutic efficacy in treating a gastrointestinal-related disease or disorder, methods of modeling human gastrointestinal development and / or disease, or the manufacture of a medicament for treating a gastrointestinal-related disease or disorder. Additional embodiments relate to the use of any of the aforementioned enteroids or compositions, in methods of treating a gastrointestinal-related disease or disorder, methods of screening for therapeutic efficacy in treating a gastrointestinal-related disease or disorder, methods of modeling human gastrointestinal development and / or disease, or the manufacture of a medicament for treating a gastrointestinal-related disease or disorder.

[0019] Further embodiments of the disclosure include methods of modeling human gastrointestinal (GI) development and / or disease, the methods including: culturing any of the aforementioned enteroids in the presence of one or more exogenous condition for a period of time; and assessing one or more effects of the exogenous condition on the enteroids. In some embodiments, the one or more exogenous condition includes a physical, mechanical, or chemical stimulus.

[0020] In some embodiments, the enteroids are derived from an HIO including cells associated with, or having effects of, a GI disease or disorder. In some embodiments, the gastrointestinal-related disease or disorder can involve exposed mesenchyme. In some embodiments, the gastrointestinal-related disease or disorder includes one or more refractivetransmural ulcerative disease, gastrointestinal (GI) damage, bowel damage, inflammatory bowel disease (IBD), IBD with refractory ulcer, colonic ulcer, peptic ulcer, gastrointestinal cancer, cancer associated with an APC mutation, and / or ischemic injuries associated with surgical conditions and / or radiation therapies.

[0021] Further embodiments of the disclosure include methods including administering at least one components or fragments of a human pluripotent stem cell (hPSC)-derived human intestinal organoid (HIO), enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids, and / or compositions, to a subject in need thereof. Further embodiments of the disclosure include methods for treating a gastrointestinal-related disease or disorder in a subject in need thereof, including administering at least one components or fragments of a human pluripotent stem cell (hPSC)-derived human intestinal organoid (HIO), and / or an enteroids produced from a transplanted HIO. Further embodiments of the disclosure include methods for treating a gastrointestinal-related disease or disorder in a subject in need thereof, including administering any of the aforementioned enteroids, and / or compositions, to the subject.

[0022] In some embodiments, the HIO components or fragments, and / or the enteroid produced from a transplanted HIO, is administered in combination with any of the aforementioned enteroids and / or compositions. In some embodiments, administering includes transplanting the HIO components or fragments, the enteroids produced from a transplanted HIO, or any of the aforementioned enteroids or compositions into a tissue and / or organ of the subject. In some embodiments, transplanting includes luminal delivery; optionally luminal delivery to a region including ulcerated tissue. In some embodiments, administering includes transplanting the HIO components or fragments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids or compositions into a small bowel region of the subject. In some embodiments, the subject has damaged and / or injured small bowel prior to administration. In some embodiments, administering the HIO components or fragments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids or compositions into the subject includes enteroid engraftment, tissue growth, and / or regeneration of tissue and / or organ structure and / or function. In some embodiments, regeneration of tissue and / or organ structure can include restitution of one or more mucosal and / or muscularis layerand / or mesenchymal-epithelial niche regeneration. In some embodiments, the HIO component, the cntcroids produced from a transplanted HIO, and / or any of the aforementioned cntcroids or compositions engraft onto one or more region of the gastrointestinal system of the subject transmurally.

[0023] In some embodiments, the subject has damaged and / or injured small bowel, and the HIO components or fragments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids or compositions engrafts onto and regenerates the small bowel of the subject. In some embodiments, small bowel regeneration includes regenerating small bowel epithelium.

[0024] In some embodiments, the HIO components or fragments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids, following transplantation, matures, proliferates, and / or persists in vivo. In some embodiments, the HIO components or fragments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids, following transplantation, is incorporated into one or more muscularis and / or vascular endothelium region. In some embodiments, the HIO components or fragments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids, following transplantation, expresses one or more proximal intestinal marker and does not express one or more distal marker; optionally wherein said proximal intestinal marker includes GATA4. In some embodiments, following administration, the tissue and / or organ has sustained cell type presence within the transplant region, has human cellular incorporation within the transplant region, has proliferative zonation of the intestine within the transplant region, retains proximal regionalization and / or neo-epithelia function, cyclic adenosine monophosphate regulation, and / or includes one or more epithelial secretory cell types and / or brush border enzyme. In some embodiments, said brush border enzyme includes one or more of human specific DPPIV, ALP, and / or SI. In some embodiments, the HIO components or fragments, enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids, is vascularized and / or innervated in vivo following administration.

[0025] In some embodiments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids includes a dissociated epithelial cell population passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times; optionally at least about 3-7 times; optionally at leastabout 3-5 times, after dissociation and prior to transplanting. Tn some embodiments, the enteroids produced from a transplanted HIO, and / or any of the aforementioned enteroids, has been produced from cells derived from the subject. In some embodiments, the cells derived from the subject comprise induced pluripotent stem cells from the subject. In some embodiments, the subject is a mammal; optionally a human or a mouse.

[0026] In some embodiments, the gastrointestinal-related disease or disorder involves exposed mesenchyme. In some embodiments, the gastrointestinal-related disease or disorder includes one or more refractive transmural ulcerative disease, gastrointestinal (GI) damage, bowel damage, inflammatory bowel disease (IBD), IBD with refractory ulcer, colonic ulcer, peptic ulcer, gastrointestinal cancer, cancer associated with an APC mutation, and ischemic injuries associated with surgical conditions and / or radiation therapies.

[0027] Further embodiments of the disclosure include methods for determining the transplant and / or therapeutic efficacy of any of the aforementioned enteroids, the methods including transplanting the enteroids into an animal model and / or a subject, and assessing one or more effects of the enteroids on the animal model and / or subject. Further embodiments of the disclosure include methods for screening, including contacting any of the aforementioned enteroids with a candidate compound or composition, and assessing the effects of the candidate compound or composition on the enteroids.

[0028] In some embodiments, the enteroids can be a model for a gastrointestinal-related disease or disorder, and assessing one or more effects of the candidate compound or composition on the recombined organoid can include assessing the effects of the candidate compound or composition on the gastrointestinal-related disease or disorder. In some embodiments, the enteroids have been produced from cells derived from a subject, optionally wherein the cells derived from the subject are induced pluripotent stem cells. In some embodiments, the subject can have a gastrointestinal-related disease or disorder.

[0029] In some embodiments, the methods include assessing the intestinal metabolism of the candidate compound or composition. In some embodiments, the methods include measuring oral bioavailability of the candidate compound or composition.

[0030] Further embodiments of the disclosure include kits including means for conducting any of the aforementioned methods. Further embodiments of the disclosure includekits including the compositions or means for generating any of the aforementioned enteroids. Further embodiments of the disclosure include uses of any of the aforementioned methods, any of the aforementioned enteroids or compositions, or any of the aforementioned kits, as a medicament, means for treatment and / or prevention of a disease, means of diagnosis, and / or tool for medical research.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0032] Figure 1. Experimental design and mucosectomy surgical injury model.

[0033] FIG. 1A) Experimental design schematic.

[0034] FIG. IB) Surgical imagery of loop creation, damage, and application of cell therapy product. The arrowhead indicates anastomosis. The dashed line outlines the loop.

[0035] FIG. 1C) Histology of healthy and injured rat small bowel.

[0036] FIG. ID) Topography of healthy and injured rat small bowel.

[0037] FIG. IE) Kaplan-Meier curve associated with the surgeries.

[0038] FIG. IF) Harvest images of damaged loops reseeded with txp-enteroids and HIOs after 10 weeks.

[0039] All scale bars = 100 pm.

[0040] Figure 2. Organoid fragmentation was essential for expansion during regeneration.

[0041] FIG. 2A) Representative histology of a Loop + Whole HIOs and serial section stained for human cells (KU80, brown).

[0042] FIG. 2B) Brightfield and live-GFP fluorescence imagery of HIOs and txp- enteroids before and after fragmentation.

[0043] FIG. 2C) Histograms of HIO and txp-enteroid fragment sizes.

[0044] FIG. 2D) Graph of cell number in HIOs and txp-enteroids represented as mean ± SD.

[0045] FIG. 2E) Brightfield and live-GFP fluorescence image of a harvested Loop + Fragmented HIOs after 10 weeks; dashed line indicates tissue edge.

[0046] FIG. 2F) Quantification of live-GFP surface area in Loop + Fragmented HTOs samples.

[0047] FIG. 2G) Representative section stained for human cells (KU80, brown) in a live- GFP+ region in a Loop + Fragmented HIO. Boxed inset depicts margin / boundary of human (KU80, brown) and rat tissue.

[0048] FIG. 2H) Quantification of KU80+ cells as a percentage of cells in each 20x field of view for areas with positive human cellular incorporation in Loop + Fragmented HIOs samples.

[0049] FIG. 21) Brightfield and live-GFP fluorescence image of a harvested Loop + Fragmented txp-enteroids after 10 weeks; dashed line indicates tissue edge.

[0050] FIG. 2J) Quantification of live-GFP surface area in Loop + Fragmented txp- enteroids samples.

[0051] FIG. 2K) Representative section stained for human cells (KU80, brown) in a live- GFP+ region in a Loop + Fragmented txp-enteroids.

[0052] FIG. 2L) Quantification of KU80+ cells as a percentage of cells in each 20x field of view for areas with positive human cellular incorporation in Loop + Fragmented txp-enteroid samples.

[0053] All scale bars = 100 pm. For F vs. J, p=0.0023 and for H vs. L, p<0.0001 from Mann- Whitney Tests.

[0054] Figure 3. Enteroid Characterization from Three Sources: human tissue, in vitro HIOs and ex vivo HIOs.

[0055] FIG. 3A) Representative brightfield images of enteroids. Scale bars = 100 pm.

[0056] FIG. 3B) Maximum intensity projections of confocal stacks of enteroids stained for an intestinal epithelial marker (CDX2, red, left) and a mesenchymal marker (VIM, green, left) and a proliferation marker (MKI67, white, right). Scale bars = 100 pm.

[0057] FIG. 3C) Transcriptomic gene expression data for intestinal related genes (CDX2, MKI67, OLFM4, LGR5, LYZ, and VIM) in Enteroids, iv-Enteroids, and txp-enteroids. No significant differences were observed.

[0058] FIG. 3D) Flow plots of Enteroids stained for epithelium (EPCAM) and proliferation (MKI67).

[0059] FIG. 3E) Flow plots of iv-Enteroids stained for epithelium (EPCAM) and proliferation (MKI67).

[0060] FIG. 3F) Flow plots of txp-enteroids stained for epithelium (EPCAM) and proliferation (MKI67).

[0061] FIG. 3G) Summary table of flow data from FIGs. 3D-F.

[0062] Figure 4. Fragmented HIOs contribute to tissue regeneration of damaged bowel in vivo after 10 weeks.

[0063] FIG. 4A) Tile scan of a Loop + Fragmented HIOs stained for human cells (KU80, brown); scale bar = 0.5 cm. Higher magnification images of KU80+ cells within the mucosa and muscularis from tile scan; scale bar’ = 50 pm.

[0064] FIG. 4B) Representative images of Sham Loop and Loop + Fragmented HIOs stained for a proliferation marker (MKI67, red), epithelium (CDH1, white) and human cells (GFP, green).

[0065] FIG. 4C) Representative images of Sham Loop and Loop + Fragmented HIOs stained for a surrogate marker of stem cell activity and the intestinal crypt (OLFM4, red) and epithelium (CDH1, green).

[0066] FIG. 4D) Representative images of Sham Loop and Loop + Fragmented HIOs stained for human cells (KU80, brown) and goblet cells (alcian blue for mucin, blue).

[0067] FIG. 4E) Representative images of Sham Loop and Loop + Fragmented HIOs stained for human cells (GFP, green) and Paneth cells (DEFA5, pink).

[0068] FIG. 4F) Representative images of Sham Loop and Loop + Fragmented HIOs stained for human cells (GFP, green), and enteroendocrine cells (CHGA, white).

[0069] FIG. 4G) Representative images of Sham Loop and Loop + Fragmented HIOs stained for human cells (GFP, green), and smooth muscle (ACTA2, red).

[0070] FIG. 4H) Representative images of Sham Loop and Loop + Fragmented HIOs stained for a pan-neuronal marker (TUBB3, brown). Arrowheads indicate neuronal bundles.

[0071] FIG. 41) Representative images of Sham Loop and Loop + Fragmented HIOs stained for human cells (GFP, green), blood vessels (VWF, red), and human blood vessels (hCD31, white). Single channel insets are within the white dashed box.

[0072] Scale bars for FIGS. 4A-I) = 50 pm.

[0073] Figure 5. HIO seeding material retains its proximal specification after engraftment.

[0074] FIG. 5A) Representative immunofluorescence staining for a proximal transcription factor (GATA4, red) and epithelium (CDH1, green) in human small bowel and Loop + Fragmented HIOs.

[0075] FIG. 5B) Representative staining for brush border enzymes (DPPIV, Active ALP, and SI, all in red) in human small bowel and Loop + Fragmented HIOs.

[0076] FIG. 5C) Representative staining for a distal transcription factor (SATB2, red), a colonic mucin (MUC5B, brown) and colonocytes (MS4A12, brown) in human colon and Loop + Fragmented HIOs.

[0077] All scale bars = 50 pm.

[0078] Figure 6. Neo-epithelia of reseeded loops are responsive to chemical stimuli after 10 weeks.

[0079] FIG. 6A) Representative merged brightfield and live-GFP images of healthy rat small bowel (proximal to the loop), Sham Loop and Loop + Fragmented HIOs mounted in an Ussing assay slider; dashed circle outlines slider opening.

[0080] FIG. 6B) Representative time course of healthy rat small bowel Isc measured in Ussing Chamber experiments.

[0081] FIG. 6C) Representative time course of Sham Loop Isc measured in Ussing Chamber experiments.

[0082] FIG. 6D) Representative time course of Loop + Fragmented HIOs Isc measured in Ussing Chamber experiments.

[0083] FIG. 6E) Graphs of calculated changes in Isc in response to 10 pM Forskolin, 100 pM IB MX, and 100 pM Bumetanide across sample groups.

[0084] FIG. 6F) Graph of baseline TEER across sample groups.

[0085] FIG. 6G) Graph of FITC-dextran permeability across sample groups.

[0086] FIG. 6H) Calculated FITC flux from graph in FIG. 4G.

[0087] Bar graphs are represented as mean ± SD. Kruskal-Wallis test p-values were not significant between groups in FIGs. 6E-H.

[0088] Figure 7. Characterization of the biodistribution of fragmented HIO in vivo.

[0089] FIG. 7A) Representative images of tissues 10 weeks post-operatively stained for a human specific marker (KU80, brown) and nuclei (hematoxylin, blue). In a transplanted HIO, there are primarily KU80+ cells, however in various organs of rats undergoing a seeded mucosectomy procedure, KU80+ cells were not observed. Scale bar = 100 pm.

[0090] FIG. 7B) Dot plot of Ct values from Alu PCR on organs collected from rats in a. Dotted line indicates Ct value threshold for a positive signal (presence of human cells). An open circle indicates an undetermined value, or Ct of greater than 40. No data points fell within the range of human cellular presence.

[0091] FIG. 7C) Dot plot of Ct values from Alu PCR on rat small bowel, GFP+ loops supplemented with HIOs, and human small bowel taken from slides. GFP+ loops supplemented with HIOs and human samples fell within the range demonstrating human cellular presence.DETAILED DESCRIPTION

[0092] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0093] The following description of various embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.

[0094] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0095] It should be understood that any use of subheadings herein are for organizational purposes, and should not be read to limit the application of those subheaded features to the various embodiments herein. Each and every feature described herein is applicable and usable in all the various embodiments discussed herein and that all features described herein can be used in any contemplated combination, regardless of the specific example embodiments that are described herein. It should further be noted that exemplary description of specific features are used, largely for informational purposes, and not in any way to limit the design, subfeature, and functionality of the specifically described feature.Overview

[0096] Therapeutic use of organoids to repair damaged or diseased intestine holds great promise to improve care of patients with GI disorders, such as intestinal failure, inflammatory bowel disease, and autoimmune enteritis. The present inventors have pioneered efforts to develop and utilize human pluripotent stem cell (hPSC)-derived organoids with therapeutic use as the ultimate goal and have shown that PSC-derived small human intestinal organoids (HIOs) can efficiently engraft with similar development in murine and rat models in both the renal subcapsular space (RSS) and mesentery of the host bowel and mature to form well-vascularized grafts with functional mucosal, submucosal, and muscular- layers, including well-formed crypts and villi.

[0097] As described herein, hPSC-derived organoid products can be used to develop gastrointestinal (GI) disease models. Further, hPSC-derived organoid products can be used as a cell therapy to repair damaged GI tissue and organs.

[0098] For example, luminal delivery of hPSC organoid products to damaged regions of the GI system, such as ulcerated regions, damaged bowel, and the like, can prove beneficial in sustained transmural restoration beyond the mucosa. Such organoid products can include whole, partial, and / or fragmented human intestinal organoids (HIOs) and / or enteroids, and / or combinations thereof.

[0099] HIOs are three-dimensional structures that recapitulate the tissue architecture, cellular diversity, and functionality of the developing human intestine. HIOs can be generated via stepwise differentiation of hPSCs, which can be patient specific (see, e.g., Spence et al., Nature, 2011, 470, 105-109; Singh et al., Am J Physiol Gastrointest Liver Physiol, 2020, 319,G375-G381), and contain both epithelial and mesenchymal cell types in vitro, which can differentiate into laminated structures upon transplantation. HIOs have been shown to produce intestinal crypts and villi analogous to those found in humans. To further their development, HIOs can be transplanted into rodent kidney capsules, allowing them to vascularize and mature.

[0100] In contrast, enteroids are self-proliferative organoids comprised of intestinal epithelial cells, that can be produced from human intestinal organoids (HIOs) or patient derived tissues, effectively providing an intestinal epithelium in culture. The intestinal crypts from HIOs and human tissues can be isolated and grown independently as enteroids. Enteroids are epithelial-only structures that were first derived from patient intestinal crypts and can be made from biopsies and full-thickness surgical samples (e.g. mature human tissues) throughout the intestine (referred to herein as “b-enteroids”; also referred to as somatic enteroids). Other types of enteroids can be made from the isolated crypts of full-thickness HIOs which have been previously transplanted into an animal (referred to herein as “txp-enteroids” or “it-enteroids”; also referred to as transplanted enteroids), such as the kidney capsule of a mouse, or derived from the epithelium of in vitro HIOs and grown solely in culture (referred to herein as “iv- enteroids”; also referred to as in vitro enteroids), as described herein. While enteroids contain the capacity to generate differentiated intestinal epithelial cell subtypes, such as enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, they can lack the mesenchymal, neuronal, endothelial, and immune compartments present in the human intestine. Enteroids can be easily made from healthy patients or from diseased patients.

[0101] Enteroids have been used to replace damaged intestinal epithelium in animal models. Furthermore, a b-enteroid-based therapeutic is currently being clinically evaluated wherein biopsies from healthy gut regions are obtained from patients with inflammatory bowel disease (IBD) with refractory ulcers and used to generate enteroids. Through a second procedure, the patients’ own healthy b-enteroids, biopsied and then expanded in vitro, can be delivered as a reinforcing material / cell therapy product to heal ulcers.

[0102] The use of txp-enteroids and iv-enteroids have the advantage of representing the patient from which the cells are derived, using hPSCs, such as induced pluripotent stem cells (iPSCs) as stalling material, and fully recapitulating a patient genome and / or disease state. In particular, iv-enteroids provide an unlimited amount of material from which cellular productscan be derived, since the production of iv-enteroids does not involve the invasiveness of taking a biopsy and / or the time involved with transplantation. The ability to fully recapitulate a patient disease state is particularly important as well, since different patients with a specific disease do not all respond equally to a given treatment. For example, a patient with cystic fibrosis may respond to one treatment, while a different patient having cystic fibrosis may not respond, or may not respond as effectively, to the same treatment. The presently described methods therefore provide ability to grow an enteroid, and generate a cellular product, which is patient-specific.

[0103] The fundamental goal of tissue engineering is to functionally restore or improve damaged tissues or organs. As described herein, tissue engineering has been successfully used to functionally restore or improve damaged tissues and / or whole organs, using an in vivo xenograft preclinical acute damage model in the small bowel. Specifically, the therapeutic capacity of human intestinal organoids (HIOs), which are generated de novo from human pluripotent stem cells (hPSCs), to repair damaged small bowel was investigated.

[0104] Utilizing an immunocompromised host as a bioreactor for engraftment and maturation, transplanted HIOs develop into structures reminiscent of human intestine, including a crypt / villus axis, vasculature, and smooth muscle layers. In the present disclosure, HIOs are demonstrated as a cell therapy source for damaged small bowel, and HIOs contribution to repair beyond the mucosa. In particular, regeneration of the mesenchymal-epithelial niche is important for robust and sustained engraftment of damaged bowel for functional transmural restoration. These preclinical data allow for the planning of human trials using HlO-based cellular products.

[0105] The cellular complexity found in HIOs can allow for sustained transmural engraftment. Through luminal delivery, in a rodent injury model, fragmented HIOs including both endoderm and mesoderm were found to engraft, proliferate, and persist throughout the bowel following repair. Not only was restitution of the mucosal layer observed, but significant incorporation was observed in the muscularis and vascular endothelium. Further analysis characterized sustained cell type presence within the regenerated regions, along with retention of proximal regionalization and the neo-epithelia’ s function.

[0106] As described herein, HlO-derived enteroids (txp-enteroids and iv-enteroids) were found to grow similarly to somatic enteroids in growth medium and differentiate similarly to somatic enteroids in differentiation medium, thus showing comparable proliferation andexpansion. HTO-derived enteroids had similar appearance and growth (regeneration) characteristics in several media, had similar response to differentiation cues, formed electrically tight barriers and responded to physiologic stimuli, and had generally similar bulk transcriptome.

[0107] These findings demonstrate the therapeutic use of HIOs and enteroids, for treating damaged gastrointestinal tissue and / or organs, and the importance of mesenchyme for intestinal injury repair. In addition to the therapeutic use of HIOs, enteroids derived from HIOs are capable of being used in modeling GI disease, to understand disease mechanisms and translational use based on such research, such as developing treatments therefor.Definitions of Terms

[0108] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant ail. For purposes of the present disclosure, the following terms are explained below.

[0109] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0110] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment. As used herein “another” may mean at least a second or more.

[0111] The term “ones” means more than one.

[0112] As used herein, the term “plurality” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0113] As used herein, the term “set of’ means one or more. For example, a set of items includes one or more items.

[0114] As used herein, the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, step, operation, process, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, without limitation, “at least one of item A, item B, or item C” means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or item A and C. In some cases, “at least one of item A, item B, or item C” means, but is not limited to, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0115] As used herein, “substantially” means sufficient to work for the intended purpose. The term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent.

[0116] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0117] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “anadditional embodiment,” or “a further embodiment” or combinations thereof means that a particular' feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in various embodiments.

[0118] As used herein, the terms “treatment,” “treating,” “treat,” and the like, with respect to a disease or condition, can refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. For example, a treatment can include executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appealing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0119] ‘ ‘Treatment,” as used herein, thus can cover any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition.

[0120] The term “therapeutically effective” or “therapeutically effective amount” as used throughout this application can refer to an amount effective to achieve a desired and / or beneficial effect, and / or anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reductionin the frequency or severity of one or more signs or symptoms of a disease. An effective amount can be administered in one or more administrations. In the methods, a therapeutically effective amount is an amount appropriate to treat an indication. By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease progression, increase the quality of life, or to prolong life. Such achievement can be measured by any suitable method, such as measurement of tumor size or blood cell count, or any other suitable measurement.

[0121] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to that amount of a recited composition or compound that, results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that, is effective to achieve the desired response for a particular’ subject and / or application. The selected dosage level wall depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drags or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0122] The term “disease state” as used herein, can generally refer to a condition that affects the structure or function of an organism. Disease states can include, for example, stages of a disease progression.

[0123] As used herein, the term “assessing” can include any form of measurement, and includes determining if an element is present or not. The terms “determining,” “measuring,” “evaluating,” “assessing” and “assaying” can be used interchangeably and can include quantitative and / or qualitative determinations.

[0124] As used herein, the terms “modulated” or “modulation,” or “regulated” or “regulation” and “differentially regulated” can refer to both up regulation (z.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition orsuppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

[0125] As used herein, the term “subject” can refer to any member of the animal kingdom. In some embodiments, a subject is a human patient.

[0126] As used herein, the term “marker” or “biomarker” can refer to any measurable substance taken as a sample from a subject whose presence is indicative of some phenomenon. Non-limiting examples of such phenomenon can include a disease state, a condition, or exposure to a compound or environmental condition. In various embodiments described herein, biomarkers may be used for diagnostic purposes (e.g., to diagnose a disease state, a health state, an asymptomatic state, a symptomatic state, etc.). The term “biomarker” may be used interchangeably with the term “marker”. The term “marker” or “biomarker” can include a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expression pattern can be utilized as pail of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, or which, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.

[0127] As used herein, the term “cellular phenotype” can refer to any determinable, observable, and / or measurable characteristic associated with a cell population.

[0128] As used herein, a “model” can include one or more in vitro or in vivo disease models; a model can also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof. A model can be used in a process and / or applied to an assay, in accordance with various embodiments as disclosed herein.

[0129] As used herein, a “process” can include one or more steps involving one or more features of one or more model as disclosed herein.

[0130] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to a biological, enzymatic, or therapeutic function.

[0131] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and can refer to the reduction or prevention of abiological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay, A partial inhibition or delay may be realized.

[0132] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and can refer to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” can refer to a cell not contained in a multi - cellular organism or tissue.

[0133] As used herein, “m vivo” is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0134] As used herein, “ex vivo” is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method outside a living organism with little alteration of natural conditions.

[0135] As used herein, “zn vitro” is given its plain and ordinary' meaning as understood in light of the specification and can refer to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0136] The terms “nucleic acid” or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with statically and electronically similar structures, such as aza- sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoramlidate, or phosphoramidate. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g. plasmid, vims, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificialchromosome (BAG), yeast artificial chromosome (YAC), or human artificial chromosome (HAG)) that can be used for amplification and / or expression of the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0137] A nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “upstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the 5’- end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “grouped” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.

[0138] The nucleic acids described herein comprise nucleobases. Primary, canonical, natural, or unmodified bases arc adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5- methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0139] The terms “peptide”, “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N- terminus of a subsequent sequence.

[0140] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity' can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0141] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including ail decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0142] The term “% w / w” or “% wt / wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0143] The term “basement membrane matrix” or “extracellular matrix” as used herein has its plain and ordinary meaning m light of the specification and refers to any biological or synthetic compound, substance, or composition that enhances cell attachment and / or growth. Any extracellular matrix, as well as any mimetic or derivative thereof, known in the art can be used for the methods disclosed herein. Some examples of extracellular matrices, or numerics or derivative thereof, include but are not limited to cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, osteopontin, basement membrane, Matrigel, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof.

[0144] The terms “passage” and “passaging” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to the conventional approaches performed in biological cell culture methods to maintain a viable population of cells for prolonged periods of time. As cells are generally proliferative in cell culture, they undergo multiple cycles of mitosis until occupying the available space, which is typically a surface of a cell culture container (e.g., a plate, dish, or flask) submerged under culture medium. For example, the cells may grow out as a monolayer on a cell culture container surface. If the growing cells occupy the entire available space of surface, they cannot proliferate further and may exhibit senescent behavior. In order to continue growth of the cells, which may be performed to maintain the viability and proliferative nature of the cells and / or to expand the number of cells for downstream purposes, the cells may be passaged by taking a fraction of the cells and seeding this fraction onto a fresh surface (e.g., of a cell culture container) in culture medium. This fraction of the cells will continue to proliferate and multiply until they occupy the available space of the new surface, upon which this passaging can be repeated successively.

[0145] The term “organoid” as used herein refers to a population of cells differentiated in vitro to form self-organizing structures, which generally are three-dimensional (3D), and include one or more functional cell types. As such, organoids are man-made, artificial constructs which undergo controlled differentiation in order to provide a specific desired cell population or combination of cell populations. Organoids can have one or more cell types and / or structural and / or functional characteristics which recapitulate one or more such feature (e.g. celltype, structural characteristic, and / or functional characteristic) of a specific type of tissue or organ. Many types of organoids have been developed to date and arc known in the art and can recapitulate features of, for example, intestinal, colonic, gastric, esophageal, liver, pancreatic, kidney, and / or other types of tissues and / or organs. As organoids are differentiated in vitro, by human direction (e.g. through carefully selected promotion and / or inhibition of one or more signaling pathways, addition / removal of growth factors, culture conditions, etc.), they are generally differ from naturally occurring tissue in a number of ways, such as in overall maturity, cell types present, ratios of cells types, structural features, functional characteristics, shape, size, and / or other such qualities.

[0146] The term “enteroid” as used herein refers to an in vitro cellular composition including epithelial cells and lacking mesenchymal cells. As described herein, enteroids can be derived from crypts isolated from biopsied patient intestinal tissue (termed herein “b-enteroids”). Alternatively, enteroids can be derived from crypts isolated from transplanted human intestinal organoids (HIOs) (termed herein “txp-enteroids”) which have been transplanted into an organism and grown in vivo for a period of time. Alternatively, enteroids can be derived from the epithelium of in vitro hPSC-derived HIOs (“iv-enteroids”), as described herein; iv-enteroids thus are prepared entirely in vitro. Enteroids also have the ability to proliferate and can be passaged multiple times in vitro.

[0147] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In certain embodiments, a construct and / or vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.Enteroids

[0148] Enteroids, as described above, are in vitro cellular compositions which include epithelial cells and lack mesenchymal cells. While enteroids contain the capacity to generatedifferentiated intestinal epithelial cell subtypes from proliferating multipotent progenitors, such as cntcrocytcs, goblet cells, Paneth cells, and enteroendocrine cells, they arc generally thought to lack the mesenchymal muscularis, neuronal, endothelial, and immune compartments present in the human intestine.

[0149] Different types of enteroids can be produced, based on differing source / starting material. There can be various advantages and disadvantages associated with the source / starting materials as well as the use of the type of enteroid produced, as described below.

[0150] b-Enteroids are derived from biopsied patient intestinal tissue. Methods and protocols for isolating b-enteroids have been published previously and are known to those skilled in the art (see, e.g., Mahe et al., Curr Protoc Mouse Bio, 2013, 3:217-240). Some variations in the methods have been implemented over the years, including, for example, the use of improved culture media (e.g. Intesticult from Stem Cell Technologies). Culture media requirements can differ between intestinal regions and source (e.g. mouse vs human). In addition, high quality media that support many of these conditions are now commercially available (e.g. Intesticult), which has resulted in process improvements. Various growth factors, additives, and culture media can be used in the process of isolating and / or culturing b-enteroids, as would be appreciated by those skilled in the art.

[0151] While the methods for producing b-enteroids are arguably the simplest of the enteroid types, and the most well-known and documented, this procedure requires tissue from a live patient donor. In many cases, live patient donor tissue often is not available or is not readily obtained, rendering this option unsuitable or less than ideal. This is a major disadvantage of b- enteroids, as it can be challenging to access b-enteroid starting material outside of an academic hospital setting. Although it is reported that b-enteroids can be propagated “indefinitely”, it has been observed that they adapt to culture over time, e.g. after -15-20 passages, and are less readily differentiated after that. Cryopreservation is simple.

[0152] b-Enteroids also retain the characteristics of the tissue from which they are derived (e.g., duodenal enteroids are distinct from ileal enteroids). This means that multiple, separately cultured biopsies are needed in order to represent the full GI tract.

[0153] It is also challenging to do genomic editing on b-enteroids. In addition, genomic engineering is only is done in the regionalized enteroid. Thus, the b-enteroid approachdoes not permit for studying genetic modifications or using fluorescent tags used to identify cellular products.

[0154] In addition, while each line is a match for the donor, there are no universal donors, and patient variability is known to be relatively large, thus limiting widespread applicability of b-enteroids.

[0155] txp-enteroids can be derived from crypts isolated from transplanted human intestinal organoids (HIOs) which have been transplanted into an organism and grown in vivo for a period of time. Methods and protocols for isolating txp-enteroids from in vivo transplanted HIOs are similar to those used for isolating b-enteroids from biopsied patient intestinal tissue. Many types of transplantation are possible and contemplated within the context of the present disclosure. For example, HIOs can be xenografted under the capsule of the kidney of NSG recipient mice, xenografted into the intestinal mesentary of RRG recipient rats, xenografted into the RRG rat mesentary and then connected via anastomosis to the rat intestine, engrafted to the debrided intestinal mucosa of an RRG rat, etc. One skilled in the art will appreciate various types of transplantation relevant to the disclosure. The HIOs can optionally be manipulated in various ways prior to xenografting. Various growth factors, additives, and culture media can be used in the process of isolating and / or culturing txp-enteroids, as would be appreciated by those skilled in the art.

[0156] The methods for producing txp-enteroids are the most complex, for various reasons. In particular, producing txp-enteroids is much more time and resource intensive, given the transplantation process prior to biopsy, and involving both iPSCs and immunocompromised animal recipients (e.g., NSG mice or RRG rats). In addition, txp-enteroids are likely to be stably patterned.

[0157] iv-Enteroids can be derived from the epithelium of in vitro cultured human pluripotent stem cell (hPSC)-derived HIOs and are prepared entirely in vitro. Methods and protocols for preparing iv-enteroids include passaging the HIO multiple times to remove a mesenchymal cell population. In some embodiments, the HIO is passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, all or substantially all of the mesenchymal cell population is removed such that the iv-enteroid is free, or substantially free, of mesenchymalcells. Various growth factors, additives, and culture media can be used in the process of isolating and / or culturing iv-cntcroids, as would be appreciated by those skilled in the ail.

[0158] The methods for producing iv-enteroids have some complexity given that HIOs are first generated, which takes much longer than culturing b-enteroids from human biopsies. However, producing iv-enteroids is significantly less time and resource intensive than producing txp-enteroids. It has also been observed that iv-enteroids retain properties that can regenerate bowel epithelium, unlike b-enteroids or txp-enteroids.

[0159] In addition, iv-enteroids have the tremendous advantage of being able to be made from a donor for whom the equivalent tissue (e.g. small intestine) is absent or inaccessible, as iPSCs can be generated from any donor tissue (e.g., blood, oral scraping, etc.). Further, iPSCs are widely generated, and starting material is readily available, as buccal swab, skin biopsy, blood draw, etc., are all very easy to obtain. It is also relatively straightforward to genetically modify / correct mutations etc. in the iPSCs and resulting HIOs. Also, iv-enteroids can be derived from a “universal donor” via HLA knockout iPSCs. These features allow iv-enteroids to have broad clinical applicability.

[0160] In contrast to b-enteroids, genomic editing can be performed on enteroids derived from iPSC-derived HIOs (including txp-enteroids and iv-enteroids), which allow for genomic editing in all cell lines. This includes the use of labels such as fluorescent tags used to identify cellular products. Accordingly, enteroids derived from iPSC-derived HIOs (including txp-enteroids and iv-enteroids) have tremendous advantages over b-enteroids in various aspects of developing GI disease models, studying GI diseases, and identifying and evaluating potential treatment modalities for the same.

[0161] Exemplary embodiments of methods for producing in vitro enteroids are provided herein. In some embodiments, the methods include dissociating an epithelial cell population from a human intestinal organoid (HIO) derived in vitro from induced pluripotent stem cells (iPSCs); and substantially removing one or more mesenchymal cell population from the dissociated epithelial cell population. The in vitro enteroid thus produced can substantially include an epithelial cell population, which can self-organize into the in vitro enteroid, and can be absent of a mesenchymal, neuronal, endothelial, and / or immune cell population. In various embodiments, all or substantially all of the mesenchymal cell population is removed, such thatthe in vitro enteroid is free, or is substantially free, of mesenchymal cells. Other cell types may optionally be present in the in vitro enteroid, such as, for example, comprises cntcrocytcs, goblet cells, Paneth cells, enteroendocrine cells, and the like.

[0162] In general, methods of preparing in vitro enteroids as described herein are performed entirely in vitro, as the HIO from which the in vitro enteroid has been derived has not been transplanted in vivo. As mentioned previously, this provides a significant advantage over other types of enteroids, which are derived from in vivo cell populations, tissues, or organs.

[0163] In some embodiments, the dissociation and removal of the mesenchymal cell population from the epithelial cell population involves enzymatic dissociation and / or mechanical dissociation, and / or passaging the HIO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Enzymatic dissociation can include dissociating the HIO with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof. Mechanical dissociation can include manual dissociation and / or passing the HIO through a mesh (e.g. cell strainer) and / or successively narrower bore channels. In some embodiments, epithelial cells and / or mesenchymal cells can be dissociated from the HIO by using an extracellular matrix depolymerization solution and / or mechanical dissociation. In some embodiments, during dissociation, a majority of the epithelial cell population is free from surrounding cell populations. In some embodiments, following dissociation, the epithelial cell population is intact or can selforganize, e.g. in culture.Transplantation of hPSC-Derived Organoids, and Components / Fragments Thereof

[0164] Various embodiments of the disclosure pertain to the establishment of a preclinical model to assess intestinal repair using hPSC-generated HIOs. As described in Examples 1-6, HIOs derived from hPSCs have been demonstrated to engraft and contribute to sustained regeneration within an acutely damaged loop of host bowel in vivo and reconstitution of both the mucosa and muscularis. functionally amalgamating with existing rat tissues.

[0165] From a basic science standpoint, this model system is advantageous because of the efficient engraftment and related ability to study the regenerated tissue ex vivo. Clinically, these data support the strategy of new treatment paradigms for refractive transmural ulcerative diseases.

[0166] As described herein, HIO fragment therapy results in improved engraftment. For example, an exemplary study herein showed an average engraftment of 17% by surface area after ten weeks, as compared to 2% when using txp-enteroids, which is an improvement of an order of magnitude. Further, only iv-enteroids have the capability to engraft to generate entire bowel with complex epithelium. Likewise, a limited engraftment / expansion rate of donor cells in colitic mice as 0.02% of cells or about 100 cells per mouse four weeks post-transplantation when using b-enteroids has been reported. Although several similar reports have also demonstrated the ability of b-enteroids to contribute to intestinal and colonic epithelial healing, efficiencies were rarely reported. Consistent with the present data data on retained regional identity following engraftment, sites of ileal enteroid engraftment, using HIO fragments that contain endoderm, have been shown to retain their small bowel identity within the mouse colon. In separate data, this has been demonstrated using iv-enteroids recombined with mesenchyme). In contrast, recombining txp-enteroids and b-enteroids with the same mesenchyme, does not have the capability to form full bowel.

[0167] To present knowledge, the results described herein represent the first report of using multi-lineage seeding material, namely having epithelial and mesenchymal components, in whole or in part, to achieve in vivo transmural intestinal repair as a cell therapy without loop / segment removal from the host. This is of high clinical relevance because it allows for an epithelial stem cell niche to be generated exclusively by the cell therapy source in areas where the native mesenchyme has failed to support natural restitution and healing or in which the mesenchyme is itself contributing to the disease etiology. Upon incorporation, the epithelial stem cell compartment and proliferative zonation had remerged by ten weeks, thus indicating that the tissue was homeostatic in the physiologic context of luminal content exposure. Epithelial secretory cell types and brush border enzymes important for digestion were also present after regeneration. Furthermore, human regions of loop neo-epithelia were functional and responsive to chemical stimuli with appropriate barrier integrity, as observed through ex vivo electrophysiology studies. With more recent efforts in generating hPSC-derived organoids that represent more diverse and specific regions of the gastrointestinal tract, this platform can be extended to additional areas, such as the colon, stomach and esophagus.

[0168] To enhance therapeutic potential, a refined delivery method can he developed, c.g. using a biological adhesive or hydrogel, to target specific sites of ulcerative damage. Furthermore, next generation organoid models that incorporate neuronal and immune components can provide enhanced clinical impact.Methods of Producing Gastrointestinal Organoids

[0169] Generating various types of organoids, including gastrointestinal organoids (e.g. human intestinal organoids), from pluripotent stem cells, and precursors thereof, such as definitive endoderm, gut endoderm, foregut endoderm, mid / hindgut endoderm, lateral plate mesoderm, splanchnic mesoderm, splanchnic mesenchyme, vascularized mesoderm, vascularized mesenchyme, and the like, are generally known in the art. Exemplary methods may be found in PCT publications WO 2011 / 140441, WO 2015 / 183920, WO 2016 / 061464, WO2017 / 192997, WO 2018 / 085622, WO 2018 / 085623, WO 2018 / 106628, WO 2018 / 200481, WO2018 / 226267, WO 2019 / 074793, WO 2020 / 023245, WO 2020 / 160371, WO 2020 / 243633, WO2021 / 030373, WO 2021 / 041443, WO 2022 / 072553, WO 2023 / 102133, WO 2023 / 023180, WO2023 / 137467, and WO 2023 / 278676. Any methods for producing definitive endoderm, gut endoderm, foregut endoderm, mid / hindgut endoderm, lateral plate mesoderm, splanchnic mesoderm, splanchnic mesenchyme, vascularized mesoderm, vascularized mesenchyme, and the like, from pluripotent stem cells disclosed herein or otherwise known in the art are applicable to the methods described herein.

[0170] In some embodiments, intestinal and colonic organoids are differentiated through the culture of definitive endoderm cells. These definitive endoderm cells can be differentiated from pluripotent cells by activating and / or inhibiting one or more pathway in the definitive endoderm, such as the TGFP pathway. In some embodiments, activating and / or inhibiting includes contacting the cells with with the Nodal, Activin, and / or BMP subgroups of the TGFP superfamily of growth factors. In some embodiments, the pluripotent stem cells are contacted with Nodal, Activin A, Activin B, BMP4, or any combination thereof, to differentiate the pluripotent stem cells to definitive endoderm. In some embodiments, the pluripotent stem cells can be contacted with Activin A to differentiate the pluripotent stem cells to definitive endoderm.

[0171] Definitive endoderm can further be subjected to FGF / Wnt-induced posterior endoderm patterning to direct hindgut specification.

[0172] In some embodiments, to produce intestinal and colonic organoids, a Wnt signaling pathway and an FGF signaling pathway, and optionally an EGF signaling pathway, are activated in definitive endoderm to posteriorize the definitive endoderm to hindgut endoderm. In some embodiments, activating a Wnt signaling pathway and an FGF signaling pathway, and optionally an EGF signaling pathway, includes contacting with a Wnt signaling pathway activator and an FGF signaling pathway activator, and optionally an EGF signaling pathway activator. During this culture process, hindgut endoderm grows as monolayer but also spontaneously buds off as clumps of cells called hindgut spheroids in suspension. In some embodiments, the Wnt signaling pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, or Wntl6, or any combination thereof. In some embodiments, the Wnt signaling pathway activator is Wnt3a. In some embodiments, the Wnt signaling pathway activator comprises a glycogen synthase kinase-3 (GSK3) inhibitor, which acts as a Wnt signaling pathway activator. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the FGF signaling pathway activator comprises FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15 / FGF19), FGF16, FGF17, FGF18, FGF20, FGF21, FGF22, FGF23, or any combination thereof. In some embodiments, the FGF signaling pathway activator is FGF4. In some embodiments, the EGF signaling pathway activator is EGF. The hindgut endoderm and hindgut spheroids produced comprise CDX2+ polarized epithelium surrounded by CDX2+ mesenchyme, and lack Alb and Pdxl, which denote foregut endoderm.

[0173] Following formation of hindgut endoderm, or hindgut spheroids, which can be manipulated in suspension and embedded in a basement membrane matrix (e.g. Matrigel) for three-dimension culture, the BMP signaling pathway regulates formation of distinct regional types of intestine. Inhibition of BMP signaling after the hindgut stage promotes a proximal intestinal fate (duodenum / jejunum). Activation of BMP signaling after the hindgut stage promotes a more distal intestinal cell fate (cecum / colon). In some embodiments, a BMP signaling pathway can be activated to differentiate the hindgut endoderm into an intestinalorganoid. Tn some embodiments, BMP signaling pathway activation in the hindgut endoderm is achieved by contacting with a BMP signaling pathway activator to differentiate the hindgut endoderm into an intestinal organoid. In some embodiments, a BMP signaling pathway can be activated to differentiate the hindgut endoderm into a colonic organoid. In some embodiments, the BMP signaling pathway activation is achieved by contacting the hindgut endoderm with a BMP signaling pathway inhibitor to differentiate the hindgut endoderm into a colonic organoid. In some embodiments, the BMP signaling pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP signaling pathway activator comprises BMP2. In some embodiments, the BMP signaling pathway inhibitor comprises Noggin, RepSox, LY364947, LDN193189, or SB431542, or any combination thereof. In some embodiments, the BMP signaling pathway inhibitor comprises Noggin.Gastrointestinal-Related Diseases and Disorders

[0174] The in vitro enteroids and / or HIOs (in whole or in part) of the disclosure can be used in treatment and / or studying or modeling gastrointestinal (Gl)-related diseases and disorders, for which their ability to regenerate the GI region, including small bowel, is particularly advantageous and renders them applicable to a wide range of conditions. In particular, the organoid products described herein are relevant for treating and / or studying or modeling Gl-related diseases which involve exposed mesenchyme, such as any disease or condition which involves an ulcer.

[0175] In some embodiments, various methods according to the disclosure include using enteroids, including b-enteroids, txp-enteroids, and / or iv-enteroids, to model Gl-related diseases and disorders in vitro. In some embodiments, the methods include using enteroids derived from HIOs differentiated from progenitors to model Gl-related diseases and disorders in vitro. In some embodiments, the methods include using txp-enteroids and / or iv-enteroids derived from HIOs differentiated from progenitors to model Gl-related diseases and disorders in vitro. In some embodiments, the methods include using iv-enteroids derived from HIOs differentiated from progenitors to model Gl-related diseases and disorders in vitro, e.g. using such models to effectively reproduce human disease in a dish. In some embodiments, modeling Gl-related diseases and disorders can include studying disease mechanisms and translational use based onsuch research, and / or identifying and / or evaluating potential therapeutic options, e.g. screening potential treatment conditions, compounds, etc..

[0176] In some embodiments, various methods according to the disclosure include administering any of the in vitro enteroids and / or HIOs, or cell compositions including the same, disclosed herein. Also disclosed herein are the in vitro enteroids and / or HIOs, or cell compositions including the same, disclosed herein for use in the manufacture of a medicament for the treatment of a Gl-related disease or disorder. Also disclosed herein are the in vitro enteroids and / or HIOs, or cell compositions including the same, disclosed herein for use in the treatment of a Gl-related disease or disorder in a subject in need thereof.

[0177] Gl-related diseases and disorders relevant to the in vitro enteroids and / or HIOs of the disclosure can include conditions such as refractive transmural ulcerative disease, gastrointestinal (GI) damage, bowel damage, inflammatory bowel disease (IBD), IBD with refractory ulcer, colonic ulcer, peptic ulcer, ischemic injuries associated with surgical conditions and radiation therapies, and the like. One skilled in the art will appreciate other Gl-related diseases and conditions for which the in vitro enteroids and / or HIOs, or cell compositions including the same, as disclosed herein could have relevance.

[0178] For example, the in vitro enteroids and / or HIOs can be transplanted into a subject having GI dysfunction, injury, and / or failure, where the transplanted in vitro enteroids and / or HIOs engraft onto the GI tissue and / or organ of the subject. Following transplantation, the subject can have increased survival rate and or improved symptoms, and / or the tissue and / or organ can have sustained cell type presence within the transplant region, human cellular incorporation within the transplant region, and / or proliferative zonation of the intestine within the transplant region. The tissue and / or organ can also retain proximal regionalization and / or neo-epithelia function, cyclic adenosine monophosphate regulation, and / or include one or more epithelial secretory cell types and / or brush border enzyme, such as human specific DPPIV, ALP, SI, and the like.

[0179] For example, these in vitro enteroids and / or HIOs organoids can be used an in vitro human model system for studying GI cell function and developmental divergence, studying Gl-related disease, identifying and / or screening for therapeutic targets, and / or identifying therapeutic compounds and / or compositions effective in treating a Gl-related disease or disorder.Accordingly, the in vitro enteroids and / or HIOs of the disclosure can allow for new developments in GI disease treatment and study.Stem Cells

[0180] The term “totipotent stem cells” (also known as omnipotent stem cells) as used herein has its plain and ordinary meaning as understood in light of the specification and are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.

[0181] The term “embryonic stem cells (ESCs),” also commonly abbreviated as ES cells, as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that are pluripotent and derived from the inner cell mass of the blastocyst, an early- stage embryo. For purpose of the present disclosure, the term "ESCs" is used broadly sometimes to encompass the embryonic germ cells as well.

[0182] The term “pluripotent stem cells (PSCs)” as used herein has its plain and ordinary' meaning as understood in light of the specification and encompasses any cells that can differentiate into nearly all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system), PSCs can be the descendants of inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0183] The term “induced pluripotent stem cells (iPSCs),” also commonly abbreviated as iPS cells, as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a type of pluripotent stem cells artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing a “forced” expression of certain genes. hiPSC refers to human iPSCs. In some methods known in the art, iPSCs may be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral transduction using viruses such asretroviruses or lentiviruses. Transfected genes may include the master transcriptional regulators Oct-3 / 4 (PUU5F1) and Sox2, although other genes may enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. As used herein, iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, a lentiviral system is used to transform somatic cells with GCT4, SOX2, NANOG, and LIN28. Genes whose expression are induced in iPSCs include but are not limited to Oct-3 / 4 (POU5F1); certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Soxl5); certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5), certain members of the Mye family (e.g., C-myc, L-myc, and N- myc), Nanog, LIN28, Tert, Fbxl5, ERas, EC ATI 5- 1, ECAT15-2, Tell, b-Catenm, EC ATI, Esgi, Dnmt3L, EC ATS, Gdf3, FthlU, Sall4, Rexl, UTF1, Stella, Stat3, Grb2, Prdml4, Nr5al, Nr5a2, or E-cadherin, or any combination thereof.

[0184] The term “precursor cell” as used herein has its plain and ordinary meaning as understood in light of the specification and encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types. In some embodiments, a precursor cell is pluripotent or has the capacity to becoming pluripotent. In some embodiments, the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell. In some embodiments, a precursor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein / peptide treatment. Precursor cells include embryonic stem cells (ESC), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSC).

[0185] In some embodiments, one step can include obtaining stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, pluripotent stemcells are derived from embryonic stem cells, which are in turn derived from totipotent cells of the early mammalian embryo and arc 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 blastocytes are well known in the art. It would be understood by one of skill in the ait that the methods and systems described herein are applicable to any stem cells.

[0186] Additional stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to those provided by or described in the database hosted by the National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); WISC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (IIW- SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Techmon at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to SA01 (SA001); SA02 (SA002); ESDI (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 (13); TE04 (14); TE06 (16); UCO1 (HSF1); UC06 (HSF6); WA01 (HI); WA07 (H7); WA09 (H9); WA13 (HI 3); WA14 (HI 4). Exemplary human pluripotent cell lines include but are not limited to TkDA3-4, 1231 A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34- 1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, 1.20012. C213, 1383D6, FF, or 317-12 cells.

[0187] In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. As used herein, the term “directed differentiation” describes a process through which a less specialized cell becomes a particular specialized target cell type. The particularity of the specialized target cell type can be determined by any applicable methods that can be used to define or alter the destiny of the initial cell Exemplary methods include but are not limited to genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0188] In some embodiments, an adenovirus can be used to transport the requisite four genes, resulting in iPSCs substantially identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated, in some embodiments, non-viral based technologies are employed to generate iPSCs. In some embodiments, reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies. In other embodiments, direct deliver}' of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiment, generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.

[0189] The term “feeder cell” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying on the cell surface. Feeder cells are generally adherent cells and may be growth arrested. For example, feeder cells are growth-arrested by irradiation (e.g. gamma rays), mitomycin-C treatment, electric pulses, or mild chemical fixation (e.g. with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily have to be growth arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, the feeder cells are allogeneic or xenogeneic to the supported target stem cell, which may have implications m downstream applications. In some embodiments, the feeder cells arc mouse cells. In some embodiments, the feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human foreskin fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used in lieu of feeder cell co-culture or incombination with feeder cell co-culture. In some embodiments, feeder cells are not used during the proliferation of the target stem cells.Differentiation of PSCs

[0190] Known methods for producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some embodiments, pluripotent cells are derived from a morula. In some embodiments, pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells can be derived from the embryonic inner cell mass or from the embryonic gonadal ridges. Embryonic stem cells or germ cells can originate from a variety of animal species including, but not limited to, various mammalian species including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSCs are used to produce definitive endoderm, in some embodiments, human iPSCs (hiPSCs) are used to produce definitive endoderm.

[0191] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic geim layer cells, organ tissue progenitor cells, and then into tissue such as GI tissue or any other biological tissue. In some embodiments, the directed differentiation is done in a stepwise manner to obtain each of the differentiated cell types where molecules (e.g. growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses. In some embodiments, the directed differentiation is done in a nonstepwise manner where molecules (e.g. growth factors, ligands, agonists, antagonists) are added at the same time. In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in the PSCs or any downstream cells.

[0192] In some embodiments, the embryonic stem cells or genu cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 6 hours to 300 hours, 24 hours to 120 hours, 48hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours, in some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors arc added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0193] In some embodiments, the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / niL, 10000 ng / mL, or 15000 ng / mL, or any concentration that is within a range defined by any two of the aforementioned concentrations, for example, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is varied during the course of the treatment. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can differ in concentrations.

[0194] In some embodiments, the ESCs or iPSCs, or the ESCs, germ cells, or iPSCs are cultured in growth media that supports the growth of stem cells. In some embodiments, the ESCs or iPSCs, or the ESCs, germ cells, or iPSCs, are cultured in stem cell growth media. In some embodiments, the stem cell growth media is RPMI 1640, DMEM, DMEM / F12, or Advanced DMEM / F12. In some embodiments, the stem cell growth media comprises fetal bovine serum (FBS). In some embodiments, the stem cell growth media comprises FBS at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0,6%, 0.7%, 0.8%, 0,9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%. In some embodiments, the stemcell growth media does not contain xenogeneic components. In some embodiments, the growth media comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0195] In some embodiments, populations of cells enriched in definitive endoderm cells are used. In some embodiments, the definitive endoderm cells are isolated or substantially purified. In some embodiments, the isolated or substantially purified definitive endoderm cells express one or more (e.g. at least 1, 3) of SOX17, FOXA2, or CXRC4 markers to a greater extent than one or more (e.g. at least 1, 3, 5) of GCT4, AFP, I'M, SPARC, or SGX7 markers.

[0196] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are cryopreserved. In some embodiments, the somatic cells are cryopreserved. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate.

[0197] In some embodiments, stem cells are treated with one or more growth factors to differentiate to definitive endoderm cells. Such growth factors can include growth factors from the TGF-beta superfamily. In some embodiments, the one or more growth factors comprise the Nodal / Activin and / or the 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 combinations of any of these growth factors. In some embodiments, the stem cells are contacted with Activin A. In some embodiments, the stem cells are contacted with Activin A and BMP4.

[0198] In some embodiments, activin-induced definitive endoderm (DE) can further undergo anterior endoderm pattering, foregut specification and morphogenesis, dependent on FGF, Wnt, EGF, or retinoic acid, or any combination thereof, or on FGF, Wnt, EGF, BMP, or retinoic acid, or any combination thereof, and a GI culture system that promotes GI growth, morphogenesis and cytodifferentiation. In some embodiments, human PSCs arc efficientlydirected to differentiate in vitro into GI epithelium and mesenchyme, it will be understood that molecules such as growth factors can be added to any stage of the development to promote a particular type of hepatic tissue formation.

[0199] It will be understood by one of skill in the art that altering the concentration, expression or function of one or more Wnt signaling proteins in combination with altering the concentration, expression, or function of one or more FGF proteins can give rise to directed differentiation in accordance with the present disclosure. In some embodiments, cellular constituents associated with the FGF, Wnt, EGF, or retinoic acid (RA) signaling pathways, or with the FGF, Wnt, EGF, BMP, or retinoic acid (RA) signaling pathways, for example, natural inhibitors, antagonists, activators, or agonists of the pathways can be used to result in inhibition or activation of the FGF, Wnt, or retinoic acid signaling pathways, or of the FGF, Wnt, EGF, BMP, or retinoic acid signaling pathways. In some embodiments, siRNA and / or shRNA targeting cellular constituents associated with the FGF, Wnt, or retinoic acid signaling pathways, or the FGF, Wnt, EGF, BMP, or retinoic acid signaling pathways, are used to inhibit or activate these pathways.

[0200] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream GI cell types are contacted with a Wnt signaling pathway activator or Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway activator comprises a Wnt protein, in some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt signaling pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, WntSa, WntSb, Wnt6, Wnt7a, Wnt7b, Wnt8a, WntSb, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l Wntl6, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt signaling pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway activator comprises CHIR99Q21, CfflR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpauilone, lithium chloride, TDZD 8, or TWS119, or any combination thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises C59, PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, or XAV939, or any combination thereof. In some embodiments, the cells are not treated with a Wnt signaling pathway activator or Wnt signaling pathway inhibitor. The Wnt signaling pathway activator or Wnt signaling pathwayinhibitor provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0201] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream GI cell types are contacted with an FGF signaling pathway activator. In some embodiments, the FGF signaling pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF signaling pathway activator comprises one or more of FGF1 , FGF2, FGF3, FGF4, FGF4, FGF 5, FGF6, FGF7, FGF 8, FGF8, FGF9, FGF 10, FGF11, FGF 12, FGF 13, FGF 14, FGF 15 (FGF 19, FGF15 / FGF19), FGF 16, FGF 17, FGF 18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with an FGF signaling pathway activator. The FGF signaling pathway activator provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0202] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream GI cell types are contacted with an EGF signaling pathway activator. In some embodiments, the EGF pathway activator is EGF. In some embodiments, the cells are not treated with an EGF pathway activator or inhibitor. EGF pathway activation and / or inhibition as used with embodiments provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein. The EGF pathway activator provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0203] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream GI cell types are contacted with a retinoic acid signaling pathway activator or retinoic acid signaling pathway inhibitor. In some embodiments, the retinoic acid signaling pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof, in some embodiments, the retinoic acid signaling pathway inhibitor comprises guggulsterone. In some embodiments, the cells are not treated with a retinoic acid signaling pathway activator or retinoic acid signaling pathway inhibitor. The retinoic acid signaling pathway activator or retinoic acidsignaling pathway inhibitor provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0204] In some embodiments, pluripotent stem cells are converted into GI cell types via a “one step” process. For example, one or more molecules that can differentiate pluripotent stem cells into DE culture (e.g., Activin A) are combined with additional molecules that can promote directed differentiation of DE culture (e.g., FGF4, CHIR99021, RA; or e.g., FGF4, Wnt, Noggin, RA) to directly treat pluripotent stem cells.

[0205] In some embodiments, iPSCs are expanded in cell culture. In some embodiments, pluripotent stem cells are expanded in a basement membrane matrix. In some embodiments, iPSCs are expanded in Matrigel, In some embodiments, the iPSCs are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632). In some embodiments, the iPSCs are differentiated into definitive endoderm cells. In the iPSCs are differentiated into definitive endoderm cells by- contacting the iPSCs with Activin A, BMP4, or both. In some embodiments, the iPSCs are contacted with a concentration of Activin A that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or any concentration of Activin A within a range defined by any two of the aforementioned concentrations, for example, 10 to 200 ng / mL, 10 to 100 ng / mL, 100 to 200 ng / mL, or 50 to 150 ng / mL. In some embodiments, the pluripotent stem cells are contacted with Activin A at a concentration of 100 ng / mL or about 100 ng / mL. In some embodiments, the iPSCs are contacted with a concentration of BMP4 that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or any concentration of BMP4 within a range defined by any two of the aforementioned concentrations, for example, 1 to 200 ng / mL, 1 to 100 ng / mL, 25 to 200 ng / mL, 1 to 80 ng / mL, or 25 to 100 ng / mL, In some embodiments, the pluripotent stem cells are contacted with BMP4 at a concentration of 50 ng / mL or about 50 ng / mL.

[0206] In some embodiments, the PSCs are differentiated into definitive endoderm cells. In some embodiments, the PSCs are differentiated into posterior foregut cells, in some embodiments, the PSCs are differentiated into a GI organoid.

[0207] In some embodiments, any of the cells disclosed herein may be cryopreserved for later use. The cells can be cryoprcscrvcd according to methods generally known in the art, optionally including one or more cryoprotectants.

[0208] Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals. Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3 -phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxy ethyl starch. Cryoprotectants can be used as part of a cryopreservation medium, which include other components such as nutrients (e.g. albumin, serum, bovine serum, fetal calf serum (FCS)) to enhance post-thawing survivability of the cells, in these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.Gene Editing

[0209] Embodiments of the disclosure can include PSCs, iPSCs, definitive endoderm cells, posterior foregut spheroids, or organoids which have been or which can be genetically modified or edited according to methods known in the ait. For example, gene editing using CRISPR nucleases such as Cas9 are explored in PCT Publications WO 2013 / 176772, WO 2014 / 093595, WO 2014 / 093622, WO 2014 / 093655, WO 2014 / 093712, WO 2014 / 093661, WO 2014 / 204728, WO 2014 / 204729, WO 2015 / 071474, WO 2016 / 115326, WO 2016 / 141224, WO 2017 / 023803, and WO 2017 / 070633.Pharmaceutical Compositions

[0210] Embodiments of the disclosure can include pharmaceutical compositions. Such pharmaceutical compositions can include one or more additional pharmaceutically acceptable components, which can include carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable”“diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and arc intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or earner approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or earners can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular' weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, saltforming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0211] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes,ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, b-propiolactone, gelatin, cell debris, nucleic acids, peptides, ammo acids, or growth medium components or any combination thereof. The amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0212] Pharmaceutical compositions can include one or more “pharmaceutically acceptable salts”, which can include relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; ammo acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl ammoethane.

[0213] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein arc known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular’, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0214] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and can refer to a compound, particle, solid, semi- solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs.

[0215] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and can refer to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the ait is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.Dosage and Administration Routes

[0216] Embodiments of the disclosure can include methods of administering or treating an animal, which can involve administering an amount of at least one treatment, that is effective to treat the disease, condition, or disorder that the organism has, or is suspected of having, or is susceptible to, or to bring about a desired physiological effect. In some embodiments, the disease, condition, or disorder can be a Gl-related disease or disorder.

[0217] In some embodiments, at least one treatment can include a composition or pharmaceutical composition, which can be administered to an animal (e.g., mammals, primates, monkeys, or humans) in an amount of about 0.005 to about 50 mg / kg body weight, about 0.01 toabout 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12 mg / kg, or about 15 mg / kg. In regal'd to some conditions, the dosage can be about 0.5 mg / kg human body weight or about 6.5 mg / kg human body weight. In some instances, some subjects (e.g., mammals, mice, rabbits, feline, porcine, or canine) can be administered a dosage of about 0.005 to about 50 mg / kg body weight, about 0.01 to about 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 80 mg / kg, about 100 mg / kg, or about 150 mg / kg. Of course, those skilled in the art will appreciate that it is possible to employ many concentrations in the methods of the present disclosure, and using, in part, the guidance provided herein, will be able to adjust and test any number of concentrations in order to find one that achieves the desired result in a given circumstance. In some embodiments, a dose or a therapeutically effective dose of a compound disclosed herein will be that which is sufficient to achieve a plasma concentration of the compound or its active metabolite(s) within a range set forth herein, e.g., about 1-10 nM, 10-100 nM, 0.1-1 pM, 1-10 pM, 10-100 pM, 100-200 pM, 200-500 pM, or even 500-1000 pM, preferably about 1-10 nM, 10-100 nM, or 0.1-1 pM.

[0218] In other embodiments, a treatment can be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

[0219] The compounds and pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disease or disorder, age and body weight of the subject, different daily doses can be used.

[0220] Under certain circumstances, however, higher or lower daily doses can be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.

[0221] A treatment can be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease or disorder and the weight and general state of the subject. Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for treatment of particular disorders.

[0222] Dosages for parenteral administration of active pharmaceutical agents can be converted into corresponding dosages for oral administration by multiplying parenteral dosages by appropriate conversion factors. As to general applications, the parenteral dosage in mg / mL times 1.8 = the corresponding oral dosage in milligrams (“mg”). As to oncology applications, the parenteral dosage in mg / mL times 1.6 = the corresponding oral dosage in mg. An average adult weighs about 70 kg.

[0223] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0224] In some embodiments, the administration can include a unit dose of one or more treatments in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. In certain embodiments, the earner, vehicle or excipient can facilitate administration, delivery and / or improve preservation of the composition. In other embodiments, the one or more carriers, include but are not limited to, saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. Carriers can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. In other embodiments, the one or more excipients can include, but are not limited to water, saline, dextrose, glycerol, ethanol, or the like,and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

[0275] The quantity of active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.

[0225] A treatment can be administered to subjects by any number of suitable administration routes or formulations. The treatment, such as an immunotherapy, can also be used to treat subjects for a variety of diseases. Subjects include but are not limited to mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats. In particular’ embodiments described herein, the subject is a human.

[0226] The route of administration of the compounds of the treatments described herein can be of any suitable route. Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route. In other embodiments, administration routes can be parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. The choice of administration route can depend on the compound identity (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the particular disease (e.g., type of cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of administration routes can be administered, as desired.

[0227] Some embodiments of the disclosure include a method for providing a subject with a treatment which comprises one or more administrations of one or more compositions; the compositions may be the same or different if there is more than one administration.Toxicity

[0228] The ratio between toxicity and therapeutic effect for a particular treatment is its therapeutic index and can be expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and ED50 (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from in vitro assays, cell culture assays and / or animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.l, p.l, 1975. The exact formulation, route of administration, and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used. For in vitro formulations, the exact formulation and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used.

[0229] Having described the disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0230] The following non-limiting examples are provided to further illustrate embodiments of the disclosure herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.EXAMPLE 1Materials and methodsHuman Tissue

[0231] Human tissue collection was performed with the prior approval of Cincinnati Children’s Hospital Medical Center’s (CCHMC) Institutional Review Board (Tissue Characterization, Study No. 2014-0427). Surgical samples of pathologically normal adult human small bowel and colon were obtained from patients undergoing bariatric or revision / resection procedures between the ages of 14 and 25 years old. Informed consent or assent was obtained from all patients and / or parent / legal guardians as appropriate. Additional de-identified samples of pathologically normal colon were obtained through CCHMC ’s Discover Together Biobank. All human tissue was utilized in accordance with institutional ethics guidelines.Immunocompromised Rats

[0232] All animal procedures and experiments were performed with the prior approval of CCHMC’s Institutional Animal Care and Use Committee (Protocol No. 2021-0060). Both males and females were utilized for experiments. Adult immunodeficient rats with Ragl and I12rg gene deletions (RRG) with ages between three and six months of age were used for mucosectomy experiments (founders were purchased from Transgenesis Rat ImmunoPhenomic Platform, Nantes, France, then bred in-house). Rats were housed in CCHMC’s banner animal vivarium and handled humanely in accordance with the NIH Guide for the Care and Use of Laboratory Animals. RRG rats were primarily fed standard autoclaved chow and provided water bottles supplemented with fluconazole (0.1 mg / ml, NorthStar Rx, LLC.). Both food and water were provided ad libitum before and after surgeries. A single dose of carprofen (5 mg / kg) was administered for pain management at the end of the mucosectomy procedure. Rats were monitored daily for three days postoperatively for signs of pain and distress and additional analgesics administered as needed.Hl-GFP Cell Line Generation

[0233] CRISPR-Cas9 was used for introduction of the green fluorescent protein (GFP) sequence to the AAVS1 safe-harbor site in commercially available Hl human embryonic stem cells (hESCs, WiCell Research Institute, Inc.) using modified standard reagents known to those skilled in the ail and used in this assay. Briefly, single-stranded donor oligonucleotides (ssODNs) encoding the validated guide RNA sequence (5’-GGGGCCACTAGGGACAGGAT-3’) for targeting the AAVS1 locus were annealed and subcloned into PX458M-HF, a modified version of pSpCas9(BB)-2A-GFP (PX458) (Addgcnc #48138) generated by the CCHMC Transgenic Core, which carries an optimized single guide RNA using TransIT-LTl, according to the manufacturer’s recommendations (Mirus Bio). Transfection was performed in mTeSRl media containing lOpM Y27632 on hESC-qualified Matrigel-coated plates (Corning). Four hours post-transfection, media was removed and replaced with mouse embryonic fibroblast (MEF) -conditioned hESC media (DMEM / F12, 20% KOSR, O.lmM nonessential amino acids, 2 mM L-glutamine, 0.1 mM P-Mercaptoethanol and 4 ng / mL bFGF) containing lOpM Y-27632 with daily media changes with MEF-conditioned hESC media. At 2 days post-transfection, a single-cell suspension of cells was generated with Accutase (STEMCELL Technologies) and replated at an approximate density of 10,000 cells / cnr for Geneticin (G418) selection. Beginning three days post-transfection, G418 selection (100 pg / mL) was performed for eight days, after which daily feeds were performed using mTeSRl. After two weeks, the remaining G418- resistant colonies were harvested using Accutase and plated at cloning density in mTeSR with CloneR supplement (STEMCELL Technologies). Recovered clones were manually excised, expanded in mTeSRl media, and subjected to genotyping. Correctly targeted insertion of the 2A- NeoR-CAG-GFP cassette in the AAVS1 locus was confirmed by PCR, sanger sequencing, copynumber analysis and GFP expression. This cell line was used in all experiments. Monthly mycoplasma testing was performed using the MycoAlert Plus Detection Kit and Control Set (Lonza #LT07-705 & LT07-518) on all cell cultures and results were consistently negative.Generation of Human Intestinal Organoids

[0234] HIOs were generated and maintained, using previously described methods (see, e.g. Spence et al., Nature, 2011, 470, 105-109; Watson et al., Nat Med, 2014, 20:1310- 1314; McCracken et al., Nat Protoc, 2011, 6:1920-1928). Briefly, Hl-GFP cells (CCHMC Pluripotent Stem Cell Core Facility) were grown in feeder-free conditions in Matrigel (BD Biosciences) coated six-well Nunclon surface plates (Nunc) and maintained in mTESRl media (STEMCELL Technologies). For definitive endoderm (DE) induction, cells were passaged as single cell suspensions generated with Accutase (STEMCELL Technologies) and plated in 24- well Nunc plates at a density of approximately 100,000 cells / well. Cells grew in mTESRl media for two days before treatment with 100 ng / ml of Activin A for three days. DE was then treatedwith hindgut induction medium (RPMT 1640, lOOx NEAA, 2% dFCS,) for four days with 100 ng / ml FGF4 (R&D) and 3 pM Chiron 99021 (Tocris) to induce mid-hindgut spheroids. Spheroids were then plated in Growth Factor Reduced (GFR) Matrigel® and maintained in intestinal growth medium (Advanced DMEM / F-12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng / ml EGF (R&D) to generate HIOs. Media was changed twice weekly, and HIOs were replated in fresh Matrigel® on day 14. HIOs were utilized for surgical transplantation between days 28 and 34.Generation of b-Enteroids and txp-enteroids

[0235] b-Enteroids were generated from human surgical samples, txp-enteroids were generated from transplanted Hl GFP HIOs for ease of use and downstream analysis. Crypts were isolated from transplanted HIOs, using processes known to those skilled in the art (Mahe et al., J Vis Exp, 2015, 97:52483). Briefly, segments of HIO tissue were pinned down in a SYLGARD 184 (Dow) coated petri dish, gently scraped to remove villi, washed with 2 mM chelation buffer before a 30 minute incubation in 2 mM chelation buffer. Then, to release the crypts the tissue was again gently scraped. The chelation buffer containing the crypts was removed from the petri dish, filtered through a 150 pm nylon mesh, and spun down at 50 g for 5 min at 4°C to pellet the crypts for use in cell culture. Crypts were plated in Matrigel® (Coming) and IntestiCult media (STEMCELL Technologies) was used to generate b-enteroids and txp-enteroids. Media was changed twice weekly and passages occurred every 7 to 10 days, txp-enteroids were used after a minimum of 3 passages to minimize mesenchymal contamination.Generation of iv-Enteroids

[0236] Matrigel bubbles containing singular HIOs were removed from plates and pooled within a 15 mL tube. 500 pL of Cell Recovery Solution (Coming) per HIO was added. Then, incubations took place for 30 min at 4°C with agitation. Tubes were then centrifuged at 300g for 5 min. Supernatant was removed and the HIOs were resuspended in 5 mL of fresh Cell Recovery Solution. Then, using a serological pipette, dissociation was performed by pipetting up and down. Dissociation was achieved when epithelial structures were free from mesenchyme. As a final step, the mixture was filtered through a 40 pm mesh, discarding the flow through, and thoroughly washing with PBS. iv-Enteroids were plated in Matrigel® (Corning), and IntestiCult media (STEMCELL Technologies) was used. Media was changed twice weekly and passagesoccurred every 7 to 10 days. iv-Enteroids were used after a minimum of 3 passages to minimize mesenchymal contamination.Mucosectomy Surgical Procedure

[0237] The mucosectomy procedure was optimized for present purposes from standard procedures known in the art (see, e.g., Avansino et al, Surgery, 2006, 140:423-434.).Loop Creation

[0238] One day prior to the procedure, chow diet was removed, and rats were placed on GelDiet 76A (ClearH2O), to be continued seven days postoperatively before returning to chow. Rats were anesthetized with 2% inhaled isoflurane (Butler Schein), and their abdomen shaved and prepped in sterile fashion using swabs coated with isopropyl alcohol and povidine- iodine. A midline laparotomy of approximately 3 cm was made. A single dose of piperacillin and tazobactam (100 mg / kg) was administered within the abdominal cavity using an 18G blunt tip fill needle affixed to a 5 mL syringe. Then, the cecum was identified, and the intestines eviscerated, using saline warmed to 37C to maintain tissue moisture throughout the procedure. A suitable stretch of bowel (~2-3 cm) was first identified for blind loop creation. To create the loop, the bowel and mesentery at the proximal end of what will become the loop was transected using scissors and a Bovie pen, as necessary. Then, the distal end of the blind loop was partially transected using scissors. An anastomosis was performed using 7-0 silk suture (PERMA-HAND; Ethicon) between the open end of the fully transected bowel and the partially transected distal end of the blind loop in a simple interrupted fashion. This establishes full continuity for the rat and maintains the blind loops’ distal connection.Injury Creation

[0239] A bulldog clamp was applied to the distal end of the blind loop, blocking flow into the continuous bowel. First, the chemical damage was induced. Using a 20 mL syringe equipped with a cannula, the loop was flushed with saline warmed to 37 C for 2 min. Then, the loop was flushed with 1 mM dithiothreitol (DTT) in saline warmed to 37 C for 2 min using a 20 mL syringe equipped with a cannula. The loop was subsequently flushed with warm saline again before flushing with 5 mM isotonic ethylenediamine tetra-acetic acid (EDTA) buffer warmed to 37 C for 10 min using a 20 mL syringe equipped with a cannula. Approximately 50 mL ofEDTA solution was used for flushing over the 10 min period. This series of flushes was repeated and then followed by a final flush with saline warmed to 37 C. To induce the mechanical damage, a dental go-between style brush flosser of appropriate diameter was inserted and removed from the loop, slightly twisting during entry and exit three times. After injury creation, the bulldog clamp was removed, and the distal end of the blind loop was tied off using 4-0 absorbable ChromicGut suture (ETHILON; Ethicon).Reseeding the Loop

[0240] Loops were reseeded with fragmented HIOs, including endoderm and mesoderm, or media void of cellular content. To fragment structures, they were collected with their media and pooled. Then, they were drawn into a syringe affixed with an 18G blunt tip fill needle, then the needle was exchanged for a 20G needle and the contents evacuated from the syringe into the well of a 24-well plate. This process was repeated using sequentially smaller needles ending at 25G, doing three syringe evacuations per needle size. Then, the fragments were drawn into a 1 mL syringe equipped with a canula and deposited within the loop. Approximately 100,000 cells per 4 mm of bowel length was used for reseeding with fragmented HIOs. After reseeding, the proximal end of the loop was closed using 5-0 silk suture (PERMA- HAND; Ethicon) as the canula was removed. The bowel was then carefully replaced within the abdominal cavity. To close the incision, the muscle was sutured in a running fashion using 4-0 coated absorbable suture (VICRYL RAPIDE; Ethicon). Then, the skin was closed in a buried inteiTupted fashion again using 4-0 coated absorbable suture (VICRYL RAPIDE; Ethicon). A single dose of carprofen (5 mg / kg) was administered for pain management at the end of the procedure. Tissues were harvested seven days postoperatively as an early time point during the healing process, and ten weeks postoperatively as a long term, recovered time point.In vitro HIO Dissociation and Cell Quantification

[0241] Nuclei were isolated from HIOs and txp-enteroids using the Minute Detergent Free Nuclei Isolation Kit (Invent Biotechnologies, Inc.) following manufacturer guidelines. To have sufficient cell quantities, structures were pooled for isolation. Four d28 HIOs were pooled for single nuclear isolations. Immediately following completion, nuclei were automatically quantified using a TC20 (Bio-Rad Laboratories, Inc.). The total count was then divided by four to determine the number of cells per HIO.In vitro Enteroid Dissociation and Flow Cytometry

[0242] All three enteroid types (b-cntcroids, txp-cntcroids, and iv-cntcroids) were dissociated into single-cell suspensions using Accutase (Stemcell Technologies Cat # 07920) according to the manufacturer’s instructions. The single cells were first stained with fixable viability dye eFluor 780 (eBioscience, Cat # 65-0865-14) to assess viability. Following this, cells were fixed and permeabilized using fixation and permeabilization reagents (Invitrogen, Cat # 00- 5521-00). Subsequently, the cells were stained with anti-human EpCAM AF647 (Biolegend, Cat # 324212) and anti-MKI67 PeCF594 (BD Biosciences, Cat # 567120) antibodies. After staining, the cells were washed and resuspended in flow buffer. The prepared cell suspensions were analyzed using a BD FACS Canto™ III flow cytometer (BD Biosciences). Data acquisition and analysis were performed using Facs DIVA (BD Biosciences).Scanning Electron Microscopy

[0243] Segments of healthy rat distal small bowel and freshly damaged rat distal small bowel were fixed overnight in 3% glutaraldehyde in 0.175 M sodium cacodylate buffer of pH 7.4. Samples were then buffer rinsed and post fixed in 1% osmium tetroxide in 0.175 M cacodylate buffer for 1 hour at 4°C. After another buffer rinse, samples were put through a graded ethanol series (25%, 50%, 75%, 95%, 3x 100%) for dehydration. Specimens were then critical point dried in an EM CPD300 (Leica), stub mounted and sputter coated 10 nm thick with 60 / 40 gold palladium using an EM ACE600 (Leica). An SU8010 transmission electron microscope (Hitachi) was used to image samples.Tissue Processing and Immunostaining

[0244] Samples were harvested and fixed overnight in 4% paraformaldehyde (PFA), processed and embedded in paraffin blocks. Sections were deparaffinized and either stained immediately with hematoxylin and eosin or subjected to antigen retrieval, and antibody stained. Antibody incubations took place at 4°C overnight in 1% bovine serum albumin in phosphate buffered saline (PBS). Antibodies used are listed in the Key Resources section. The Vectastain ABC system was used for amplification and the diaminobenzidine substrate kit was used for signal detection (Vector Laboratories). Mucins were demonstrated using 1% Alcian Blue pH 1 (Newcomer Supply, Inc.). Lillie-Mayer’s Hematoxylin (Agilent Technologies) or 1% NuclearFast Red (Polysciences, Inc.) was used as a counterstain. For biodistribution, serial sections were made and every tenth slide was stained over 2 mm of tissue thickness.Image Acquisition

[0245] Surgical imagery was acquired using an M80 microscope outfitted with a MC 170HD camera (Leica Microsystems). Gross images of harvested structures were acquired using a V60 ThinQ (LG Electronics, Inc.). Harvests were performed using a M165 FC microscope outfitted with a DCF7000 T camera (Leica Microsystems). Slides were imaged using an Eclipse Ti microscope (Nikon Corporation) an Eclipse Ti2 microscope (Nikon Corporation). Whole mount staining was imaged using an Eclipse Til AIR confocal microscope (Nikon Corporation). Subsequent analysis performed using Nikon Elements Imaging Software, including the Object Count function for cell quantification, (Nikon Corporation) and ImageJ software (NIH) for surface area quantification and measurements..Ex vivo Epithelial Characteristics and Permeability

[0246] The epitheliums of freshly harvested healthy distal small bowel and reseeded loops were carefully dissected through a technique similar’ to seromuscular’ stripping. Samples were opened, and dissection was done in ice cold Kreb’s buffer (NaCl, 117 mM; KC1, 4.7 mM; MgC12, 1.2mM; NaH2PO4, 1.2 mM; NaHCO3, 25 mM; CaC12, 2.5 mM and glucose, 11 mM). Full thickness tissue segments were then pinned in a dish containing cured Sylgard (Electron Microscopy Sciences). Reseeded loops were verified to be GFP positive before further dissection and use in the Ussing chamber assay. The seromusculature was then micro-dissected from the epithelium using Dumont #5 and #7 forceps along with Vannas scissors (Fine Science Tools, Inc.). Gross tissue integrity was assessed using the stereoscope’s bottom lighting for uniformity in appearance and any damaged areas were removed. Some remnant subepithelial mucosa remained after dissection. The central portion of the epithelium, with the least amount of handling, was then positioned for mounting between the hemi-chambers of an Ussing apparatus (Physiologic Instruments). 0.031 cm2of tissue was exposed to 5 mL of oxygenated Krebs buffer at 37 °C throughout the assay. The transepithelial potential difference was detected with two paired electrodes affixed to a salt bridge containing 3.75% agar in 3 M KC1. The electrodes were connected to a VVC MC8 voltage clamp amplifier (Physiologic Instruments, San Diego). Electrode potential difference and fluid resistance values were offset to zero immediately beforesliders were mounted between the chambers. A 30 min period was allowed for the establishment of equilibrium. Then, tissues were voltage-clamped at 0 mV while continuously measuring the short circuit current (Isc) and chemical stimuli applied (10 pM Forskolin, 100 pM IBMX, and 100 pM Bumetanide). For FITC-dextran permeability, 2.2 mg / ml FITC-dextran was added into apical side, and a sample was taken from the basolateral side every 30 minutes for 3 hours, replacing the same amount of fresh modified Kreb’s buffer in the basolateral side to maintain pressure across the sample. Once all aqueous samples were collected, they were quantified with a plate-reader (Synergy 2, BioTek).Genomic DNA Isolation

[0247] Samples were harvested 10 weeks post-operatively from reseeded mucosectomy rats (brain, colon, heart, kidney, liver, lung, and small intestine) and flash frozen in liquid nitrogen. As a positive control, Hl GFP cells were also used. Samples were homogenized, filtered, and digested using an ISOLATE II Genomic DNA kit (Meridian Biosciences) according to manufacturer guidelines. After the gDNA was isolated, quality and quantity was analyzed using a NanoDrop Microvolume Spectrophotometer.Human Specific Alu PCR

[0248] Primers and Probes: Detection of human DNA was done using previously described primers and probes. Briefly, Alu PCR was performed on gDNA extracted from various organs from reseeded mucosectomy rats and the Hl GFP cell line. The forward primer was designed to anneal upstream of the human specific Alu sequence (5'-TGGTGG CTCTCT CCT GTA AT-3') and the reverse primer was designed to primarily anneal within the human-specific Alu sequence (5'-GAT CTC GGC TCA CTG CAA C-3'), resulting in a 96 base pair amplicon. The probe was designed to bind between the two primers (5'- TGA GGC AGG AGA ATC GCT TGA ACC-3’) quencher-MGB-6FAM upstream of the hAlu-Fspecific sequence. The primers and probes were custom ordered from Integrated DNA Technologies.

[0249] Alu PCR: Quantitative real time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) on 200 ng of target template gDNA. Each sample was sequenced in triplicate using a OneStep thermocycler (Applied Biosystems). Standard curves were generated by adding 10-fold serial dilutions (200 ng- 0 ng) of hDNA (Millipore Sigma) and Hl GFP cells on each PCR plate. QunatoStudio software (Applied Biosystems) was used tocalculate crossing threshold (Ct) values for presence of human cells based on the standard values.Data Representation, Statistics and Reproducibility

[0250] For bar graphs, data are represented as a mean ± standard deviation, with all individual data points represented. For violin plots, a dashed line indicates the mean and interquartile ranges, with all individual data points represented. For statistics comparing three groups of data, Kruskal-Wallistests were performed. For statistics comparing unpaired data a student t-test was performed. The statistical significance cutoff was p < 0.05 and confidence 95%.EXAMPLE 2An acute injury model was used to test the therapeutic capacity of HIOs

[0251] This experimentation utilized the Hl embryonic stem cell line, which was modified in house to constitutively express green fluorescent protein (GFP) for ease of downstream xenograft analysis. The modified cell line was characterized and determined to pass quality control metrics (G-banded karyotype analysis demonstrated normal (46, XY) karyotype of Hl embryonic stem cell line after GFP insertion, and electropherogram of short tandem repeat analysis of Hl embryonic stem cell line after GFP insertion displayed a pass result). HIOs and txp-enteroids, both made from the Hl-GFP line to reduce genetic variability between groups, were used as cell therapy sources in damaged small bowel limbs of rodents, as shown in Figure 1A. Immunocompromised Ragl and I12rg knockout rats were utilized as host subjects to surgically create an isolated segment of small bowel that underwent chemical and mechanical injury before reseeding with HIOs, txp-enteroids or media alone as sham (Figure 1 A).

[0252] The surgical procedure resulted in a blind end-to-side “Y” segment, or loop, which was temporarily tied off with a fast absorbing chromic suture, such that after a few days the segment could drain distally and be exposed to host enteric content (Figure IB). A significant advantage of this model is the ability to provide a brief window for HlO / txp-enteroid retention, epithelial restitution, and engraftment to occur following acute transmural damage in an immunocompromised host. Bowel continuity was retained with this approach, so as not to impair the host’s overall digestive function.

[0253] Immediately after injury, the isolated segment of bowel was predominantly denuded of epithelium with exposed and damaged mesenchyme, as observed histologically and via topographical micrographs, when compared to healthy bowel (Figure 1 C-D). Mortality and survival rates were similar between the three experimental groups (Figure IE).EXAMPLE 3Fragmented HIOs and txp-enteroids incorporate during intestinal repair

[0254] At the ten-week post-operative time point, loops were harvested and evaluated (Figure IF). In loops supplemented with GFP HIOs, discrete engraftment as organoid units was first observed (Figure 2A). Therefore, a fragmentation strategy was devised to promote widespread incorporation of the cellular products as opposed to isolated engraftment (Figure 2B). By doing so, fragments of varying sizes were created, with the highest proportion being around 30 pm in size for both fragmented HIOs and fragmented txp-enteroids (Figure 2C). The average number of cells per structure was also quantified after dissociation, so that consistent amounts of cellular product could be administered (Figure 2D).

[0255] When administering fragmented GFP HIOs, regions expressing live-GFP were observed and comprised an average of 17% of the loop’s surface area (Figure 2E-F). Immunohistochemistry for a human-specific DNA binding protein (KU80), revealed robust human cellular incorporation within the dissected GFP+ regions of HIO fragment reseeded loops (Figure 2G). In addition, KU80+ cells were observed throughout the thickness of the intestine. This was further highlighted by the quantification of KU80+ cells within full-thickness fields of view that contained human cellular incorporation. The average proportion of KU80+ human cells per field was quantified to be 52% (Figure 2H). In loops supplemented with fragmented GFP txp-enteroids, regions expressing live-GFP were also observed; however they were significantly reduced and comprised 2% of the loop’s surface area on average (Figure 2I-J). KU80+ cell incorporation was observed within the dissected GFP+ regions of txp-enteroid fragment supplemented loops and presented exclusively within the epithelium (Figure 2K). The proportion of KU80+ cells in full-thickness samples supplemented with fragmented txp-enteroids was 3% per field of view, a significant reduction compared to loops supplemented with fragmented HIOs (Figure 2L).

[0256] txp-Enteroids from the H1 -GFP line were chosen for ease of analysis, taking advantage of the livc-GFP expression. Because cntcroids can be made from three different sources (c-enteroids, txp-enteroids, and iv-enteroids), baseline analysis of each type was performed in case a phenotypic difference specific to the enteroid source accounted for the low engraftment. Through brightfield and confocal imaging, c-enteroids, iv-enteroids and txp- enteroids all demonstrated a similar spherical phenotype in WNT-rich media with a high proportion of cells MKI67+ throughout (Figure 3A-B). Quantitative real-time PCR was also performed and there were no differences between groups for the transcription of the intestinal epithelial cell marker CDX2, the proliferation marker MKI67, the surrogate marker of stem cell activity and the intestinal crypt 0LFM4, the stem cell marker EGR5, the Paneth cell marker LYZ, and the pan-mesenchymal marker VIM (Figure 3C). The goblet cell marker MUC2, and the enteroendocrine cell marker CHGA did not cycle above background in any of the groups. Flow cytometry was performed to quantify the proliferation across groups, which was over 95% for each enteroid type, indicating that they were all similarly enriched for sternness (Figure 3D- G). After validation of enteroid engraftment in the above-described damage model, more in- depth analysis was continued using only fragmented HIOs as the cell source, because it allowed for testing of the overarching hypothesis.EXAMPLE 4HIOs contributed to a diverse assortment of sustained cell types after regeneration

[0257] To further characterize the cellular components of the regenerated loops a series of immuno staining s was performed. First, to demonstrate the extent of human cellular contributions within loops supplemented with fragmented GFP HIOs, a tile scan was performed on a region of GFP+ loop stained for KU80 (Figure 4A, left). Human cells were observed within the mucosa and the muscularis, which displayed proper alignment (Figure 4A, right). Immuno staining for MKI67 demonstrated that the stereotypical proliferative zonation of the intestine had been reestablished in both sham and HIO supplemented loops (Figure 4B). To further support crypt formation and stem cell activity, an antiapoptotic factor (OLFM4) was stained for and observed to localize correctly in both groups (Figure 4C). Mucin producing goblet cells, antimicrobial Paneth cells, and hormone secreting enteroendocrine cells were also observed in both groups indicating that epithelial cell specification was occurring in both groups(Figure 4D-F). The humanization of the muscularis was further highlighted by co- immuno staining for smooth muscle (ACTA2) and GFP (Figure 4G).

[0258] Next, it was explored whether the host innervation, part of the enteric nervous system (ENS), responsible for a variety of functions including motility, maintained its localization within regenerated tissue. Immuno staining for a pan-neuronal marker (TUBB3) confirmed that the host innervation was not displaced after human cellular reconstitution indicating that its function can also be preserved (Figure 4H). Vascularization was also investigated through co-staining for a cross-species endothelial marker (VWF) and a human specific marker (hCD31) using GFP expression as a secondary confirmation for human cells (Figure 41). Human vasculature was found to be exclusively present within loops supplemented with HIOs, indicating that HIOs also possess competent endothelial cell precursors that contributed to vascular regeneration.

[0259] Clinically relevant regional differences exist throughout the gastrointestinal tract, so regionality was further investigated through protein expression compared to native human bowel. As HIOs are patterned as proximal small bowel and were engrafted into the distal small bowel, it was determined whether regionalization was maintained.

[0260] Immuno staining for a regional marker expressed in the small intestine but not the colon or rectum (GATA4), revealed positive expression in HIO supplemented loops similar to that in human small bowel samples (Figure 5A). Next, brush border enzymes important for digestion and specific to the small bowel were interrogated. Three such enzymes, human specific DPPIV, ALP and SI, were detected in HIO supplemented loops with localization comparable to that of human small bowel (Figure 5B). Finally, distal and colonic markers were also interrogated as a form of further validation of regional specification. A distal transcription factor (SATB2), colonic mucin (MUC5B) and colonocyte marker (MS4A12) were not detected in HIO supplemented loops but present in human colon samples (Figure 5C). Taken together, positive protein expression of proximal intestinal markers along with the absence of distal markers demonstrate that the regionality of the loop’s HIO derived neo-epithelium was maintained during the engraftment and expansion of the HIO fragments.EXAMPLE 5Electrophysiology ofHIO neo-epithelia demonstrates functionality

[0261] To gain functional insight, Ussing chamber assays were performed ten weeks postoperatively on the epithelia of healthy rat small bowel, sham loops, and live-GFP expressing loop regions (Figure 6A). Epithelia were exposed to a series of chemical stimuli while electrophysical properties were continuously recorded. Epithelia from all three groups were responsive to challenges with forskolin, 3-isobutyl-l-methylxanthine (IBMX) and bumetanide, as observed in real time recordings (Figure 6B-D). Changes in short circuit current (Isc), a reflection of active ion transport, were consistent with the known method of action of each compound applied for all groups (Figure 6E). This demonstrates that appropriate cyclic adenosine monophosphate regulation and function of ion channels in the neo-epithelia was occurring.

[0262] Baseline readings of transepithelial electrical resistance (TEER) were also measured (Figure 6F). Resistance values without the muscularis and serosal layers have been previously documented between 20 and 45 Q*cm2for rat small intestine. In the case of adult human small intestine, TEER values have been previously documented between 50 and 100 □*cm2. While some of the HIO supplemented loop TEER values fall within the established TEER range for adult human small intestine, others were observed to be lower, likely reflective of the immature nature of HIOs and the contribution from regenerated rat bowel.

[0263] To further assess the integrity of the loop neo-epithelium, paraccllular permeability was evaluated. Fluorescein isothiocyanate (FITC)-dextran, 4kDa in size, was added to the apical chamber of each specimen and samples were collected from the basolateral chamber for evaluation of fluorescence intensity over time (Figure 6G). From this dataset, FITC-dextran flux values were calculated and no difference between groups was observed (Figure 6H), demonstrating that HIO- supplemented loop neo-epithelium established a functional and sufficient barrier during the regeneration and healing process.EXAMPLE 6Fragmented HIOs were retained within the site of administration

[0264] As proliferation of HIO cellular products was demonstrated, the biodistribution and potential for undesirable expansion was then investigated. As a first lineassessment of safety, samples were collected from various off target organs from ten-week postoperative rats supplemented with HIOs. Portions of the brain, colon, heart, kidney, liver, lung, and small bowel were collected for histology and PCR.

[0265] No gross abnormalities or tumor formation was observed, which was consistent with previous experiences of performing whole organoid transplants where hosts have been void of tumors. When immunostaining for KU80 across serial sections through 2 mm of tissue thickness, KU80+ cells were only observed in the transplanted HIO positive control (Figure 7A).

[0266] To further investigate the presence of human cells, Alu-based real-time PCR was performed to discern human from rodent cells. To determine the threshold for significance, a titration was performed on a known human gDNA sample and related back to cellular equivalence (Table 1). Experimental samples were then run, including the Hl GFP cell line and the various off target organs. No Ct data points were found to be below the Ct value threshold for human cell presence (Figure 7B raw values in Table 2), supporting the lack of human cell migration within the hosts.

[0267] As an additional measure, tissue samples from slides were rehydrated and their gDNA isolated for Alu-based real-time PCR. Rat small bowel, serial sections from loops with KU80+ cells, and human small bowel samples were all. Only the samples with known human cellular content were observed below the Ct value threshold for human cell presence (Figure 7C). This served as a third measure to validate human cell incorporation into the regenerated loops receiving HIO fragment therapy.Table 1. Control Human DNA and determination of Ct Value cutoff for human cellular presence. Titration of human DNA content in relation to Ct values and cell presence equivalency used for tumorigenicity.Table 2. Human DNA Ct Values in rat tissues post mucosectomy. Titration of human DNA content in the Hl GFP cell line used to generate HIOs and their associated Ct values. Quantification of Human DNA content by PCR throughout the major organs of rats having undergone mucosectomy procedures with fragmented HIOs used as a reseeding material. Undetermined Ct values are above 40 as per the sensitivity of the thermocycler. No values were found to be below the determined Ct threshold of 29, thus human cellular presence was not found within the samples.EXAMPLE 7 b-Enteroids, txp-enteroids, and iv-enteroids regenerate and respond to injury in a similar manner

[0268] Crypts predominantly contain stem cells which differentiate into other intestinal epithelial cell types depending on culture conditions. Growth medium contains factors such as Wnt-3a and R-Spondin-1 that maintain the stem- like state of the enteroids. This allows enteroids, including txp-enteroids and iv-enteroids, to remain maximally proliferative. Differentiation medium contains fewer growth factors, encouraging the specialization of stemlike cells into functional cell types, such as enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. Enteroids were grown in growth medium and differentiation medium and were compared to each other, as described below.

[0269] Enteroids can form a confluent monolayer whose barrier integrity is measurable via electrical resistance across the monolayer. Electrodes were placed on the apical and basolateral sides of the membrane, and a small current (10 pA) was run through the cells. From there, the resistance was detected in ohms. This transepithelial electrical resistance (TEER) increases when the cells of the monolayer increase in density, differentiate, and produce tight junctions, thereby decreasing barrier permeability. TEER decreases as the monolayer accrues damages brought on by cell starvation, toxin exposure, inflammation, or mechanical disruption. Therefore, TEER measurements serve as a versatile in vitro tool for investigating the effects of different treatments on epithelial tissue integrity.

[0270] The TEER measurements illustrate the differences in peak epithelial barrier resistance in b-enteroids, txp-enteroids, and iv-enteroids when grown in growth medium and differentiation medium. In both culture environments, txp-enteroids and iv-enteroids were found to perform similarly to b-enteroids, showing that b-enteroids, txp-enteroids, and iv-enteroids regenerate and respond to injury in a similar manner, as shown in Table 3.

[0271] Analysis was conducted demonstrating the change in epithelial barrier integrity of intestinal epithelial organoids when exposed to the exemplary chemotherapy medication Doxorubicin. Doxorubicin is a drug used to treat a variety of different cancers, including breast, bone, lung, ovarian, thyroid, and stomach cancer. While its main effects are notintestinal, a common side effect is a severe and sometimes life-threatening inflammation of the GI tract.

[0272] The effects of Doxorubicin on enteroid monolayer TEER values, expressed as the proportion of each monolayer's peak TEER value prior to drug exposure, were determined. In all treatment conditions, txp-enteroids and iv-enteroids were shown to respond in a comparable manner to b-enteroids.

[0273] Further experiments demonstrated similar growth and differentiation effects in txp-enteroids and iv-enteroids as compared to b-enteroids. All showed robust response to forskolin / cAMP-mediated CFTR activation in the swelling assay, although the txp-enteroids and iv-enteroids showed slightly decreased activity as compared to b-enteroids.Table 3. TEER measurements of b-enteroids, txp-enteroids, and iv-enteroids grown in growth and differentiation medium. Experiments were conducted on multiple samples (14-30 separate experiments per enteroid type), with the results averaged.

[0274] Taken together, these results show that HIOs and enteroids can be used in tissue engineering or cell therapy approaches to treating gastrointestinal diseases and disorder, including conditions involving damaged small bowel. These results demonstrate the uses of these platforms in preclinical experimentation and clinical treatment.

[0275] The various methods and techniques described above provide a number of ways to carry out the disclosure. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0276] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0277] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the disclosure extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0278] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0279] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0280] Preferred embodiments of this application are described herein. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art uponreading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0281] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0282] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the disclosure. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

1. CLAIMSWhat is claimed is:

1. An in vitro method of producing an enteroid derived from human pluripotent stem cells (hPSCs), the method comprising: a) obtaining or isolating a dissociated epithelial cell population from a non-transplanted human intestinal organoid (HIO), wherein the HIO is obtained by in vitro expansion of one or more precursor cells (hPSCs, iPSCs); and b) substantially removing one or more mesenchymal cell population from the dissociated epithelial cell population; thereby providing an enteroid.

2. The method of claim 1, wherein the enteroid substantially comprises the dissociated epithelial cell population.

3. The method of any preceding claim, wherein the dissociated epithelial cell population self-organizes into the enteroid.

4. The method of any preceding claim, wherein the enteroid does not comprise a mesenchymal, neuronal, endothelial, and / or immune cell population.

5. The method of any preceding claim, wherein all or substantially all of the mesenchymal cell population is removed.

6. The method of any preceding claim, wherein the enteroid is free, or is substantially free, of mesenchymal cells.

7. The method of any preceding claim, wherein the enteroid further comprises progenitor cells, intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and / or a proliferative compartment.

8. The method of any preceding claim, wherein the method is performed in vitro.

9. The method of any preceding claim, wherein the HIO has been differentiated and proliferated entirely in vitro.

10. The method of any preceding claim, further comprising culturing the dissociated epithelial cell population for at least about one day; optionally at least about 1 to 10 days, 7 to 10 days, 7 to 15 days, 7 to 20 days, or longer.

11. The method of any preceding claim, further comprising culturing the enteroids; optionally wherein the enteroids are cultured for at least about one day; optionally at least about 1 to 10 days, 15 days, 20 days, or longer.

12. The method of any preceding claim, wherein the dissociated epithelial cell population is passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times; optionally at least about 3-7 times; optionally at least about 3-5 times; optionally 5 times.

13. The method of claim 12, wherein the dissociated epithelial cell population is cultured for at least about 1 to 10 days prior to each passaging; optionally at least about 7 days.

14. The method of any preceding claim, wherein epithelial cells and / or mesenchymal cells are dissociated from the HIO by enzymatic dissociation and / or mechanical dissociation.

15. The method of claim 14, wherein enzymatic dissociation comprises dissociating the HIO with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof.

16. The method of claim 14, wherein mechanical dissociation comprises manual dissociation and / or passing the HIO through a mesh and / or successively narrower bore channels.

17. The method of any preceding claim, wherein epithelial cells and / or mesenchymal cells arc dissociated from the HIO by using an extracellular matrix depolymerization solution and / or mechanical dissociation.

18. The method of any preceding claim, wherein, during dissociation, a majority of the epithelial cell population is free from surrounding cell populations.

19. The method of any preceding claim, wherein, following dissociation, the epithelial cell population is intact.

20. The method of any preceding claim, wherein the dissociated epithelial cell population is separated from surrounding cell populations; optionally using a cell strainer.

21. The method of any of preceding claim, wherein the enteroid is obtained by in vitro expansion of the dissociated epithelial cell population, and / or wherein the dissociation step is performed in vitro.

22. The method of any of preceding claim, further comprising cryopreserving the enteroid; optionally wherein the enteroid is cryoprcscrvcd for at least about 30 days, 60 days, 90 days, 120 days, 1 year, 2 years, or longer.

22. The method of any preceding claim, the method further comprising producing the HIO from human pluripotent stem cells (hPSCs).

23. The method of claim 1 or claim 22, wherein the hPSCs comprise embryonic stem cells or induced pluripotent stem cells.

24. An in vitro enteroid derived from human pluripotent stem cells (hPSCs), comprising: an epithelial cell population derived from a non-transplanted human intestinal organoid (HIO), wherein the HIO is obtained via in vitro expansion of one or more hPSCs; andwherein the enteroid is substantially free of a mesenchymal, neuronal, endothelial, and / or immune cell population.

25. An in vitro enteroid derived from human pluripotent stem cells (hPSCs), comprising: an epithelial cell population derived from a non-transplanted human intestinal organoid (HIO), wherein the HIO is obtained via in vitro expansion of one or more hPSCs; and wherein the enteroid is substantially free of a mesenchymal, neuronal, endothelial, and / or immune cell population; wherein the enteroid is produced by the method of any of claims 1-23.

26. The enteroid of any of claims 24-25, wherein the enteroid is free, or is substantially free, of mesenchymal cells.

27. The enteroid of any of claims 24-26, wherein the enteroid further comprises progenitor cells, intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and / or a proliferative compartment.

28. The enteroid of any of claims 24-27, wherein the enteroid comprises MKI67+ cells.

29. The enteroid of any of claims 24-28, wherein the enteroid expresses one or more intestinal epithelial cell marker, proliferation marker, surrogate marker of stem cell activity and intestinal crypt, stem cell marker, Paneth cell marker, and / or pan-mesenchymal marker.

30. The enteroid of claim 29, wherein the one or more intestinal epithelial cell marker comprises CDX2; the one or more proliferation marker comprises MKI67; the one or more surrogate marker of stem cell activity and intestinal crypt comprises OLFM4; the one or more stem cell marker comprises LGR5; the one or more Paneth cell marker comprises LYZ; and / or the one or more pan-mesenchymal marker comprises VIM.

31. The enteroid of any of claims 24-30, wherein the HIO is derived from pluripotent stem cells (PSCs).

32. The enteroid of claim 31, wherein the PSCs comprise embryonic stem cells or induced pluripotent stem cells.

33. The enteroid of any of claims 24-32, wherein the HIO is an artificial or engineered HIO and is derived from human PSCs in vitro.

34. The enteroid of any of claims 24-33, wherein the enteroid comprises an artificial or engineered enteroid and / or is generated in vitro.

35. The enteroid of any of claims 24-34, wherein the enteroid is three-dimensional and / or generally spherical.

36. An in vitro composition comprising the enteroid of any of claims 24-35.

37. The enteroid of any of claims 24-35, or the composition of claim 36, for use in a method of treating a gastrointestinal-related disease or disorder, a method of screening for therapeutic efficacy in treating a gastrointestinal-related disease or disorder, a method of modeling human gastrointestinal development and / or disease, or the manufacture of a medicament for treating a gastrointestinal-related disease or disorder.

38. Use of the enteroid of any of claims 24-35, or the composition of claim 36, in a method of treating a gastrointestinal-related disease or disorder, a method of screening for therapeutic efficacy in treating a gastrointestinal-related disease or disorder, a method of modeling human gastrointestinal development and / or disease, or the manufacture of a medicament for treating a gastrointestinal-related disease or disorder.

39. A method of modeling human gastrointestinal (GI) development and / or disease, the method comprising: culturing the enteroid of any of claims 24-35 in the presence of one or more exogenous condition for a period of time; and assessing one or more effects of the exogenous condition on the enteroid.

40. The method of claim 39, wherein the one or more exogenous condition comprises a physical, mechanical, or chemical stimulus.

41. The method of any one of claims 39-40, whereiisn the enteroid is derived from an HIO comprising cells associated with, or having effects of, a GI disease or disorder.

42. The method of any of claims 39-41, wherein the gastrointestinal-related disease or disorder involves exposed mesenchyme.

43. The method of any of claims 39-42, wherein the gastrointestinal-related disease or disorder comprises one or more refractive transmural ulcerative disease, gastrointestinal (GI) damage, bowel damage, inflammatory bowel disease (IBD), IBD with refractory ulcer, colonic ulcer, peptic ulcer, gastrointestinal cancer, cancer associated with an APC mutation, and / or ischemic injuries associated with surgical conditions and / or radiation therapies.

44. A method comprising administering at least one component or fragment of a human pluripotent stem cell (hPSC)-derived human intestinal organoid (HIO), an enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35, and / or the composition of claim 36, to a subject in need thereof.

45. A method for treating a gastrointestinal-related disease or disorder in a subject in need thereof, comprising administering at least one component or fragment of a human pluripotent stem cell (hPSC) -derived human intestinal organoid (HIO), and / or an enteroid produced from a transplanted HIO.

46. A method for treating a gastrointestinal-related disease or disorder in a subject in need thereof, comprising administering the enteroid of any one of claims 24-35, and / or the composition of claim 36, to the subject.

47. The method of claim 45, wherein the HIO component or fragment, and / or the enteroid produced from a transplanted HIO, is administered in combination with the enteroid of any one of claims 24-35, and / or the composition of claim 36.

48. The method of any one of claims 44-47, wherein administering comprises transplanting the HIO component or fragment, the enteroid produced from a transplanted HIO, the enteroid of any one of claims 24-35, or the composition into a tissue and / or organ of the subject.

49. The method of claim 48, wherein transplanting comprises luminal delivery; optionally luminal delivery to a region comprising ulcerated tissue.

50. The method of any one of claims 44-49, wherein administering comprises transplanting the HIO component or fragment, the enteroid produced from a transplanted HIO, the enteroid of any one of claims 24-35, and / or the composition into a small bowel region of the subject.

51. The method of any one of claims 44-50, wherein, prior to administration, the subject has damaged and / or injured small bowel.

52. The method of any one of claims 44-51, wherein administering the HIO component or fragment, the enteroid produced from a transplanted HIO, the enteroid of any one of claims 24- 35, and / or the composition into the subject comprises enteroid engraftment, tissue growth, and / or regeneration of tissue and / or organ structure and / or function.

53. The method of claim 52, wherein regeneration of tissue and / or organ structure comprises restitution of one or more mucosal and / or muscularis layer and / or mcscnchymal-cpithclial niche regeneration.

54. The method of any one of claims 44-53, wherein the HIO component, the enteroid produced from a transplanted HIO, the enteroid of any one of claims 24-35, and / or the composition engrafts onto one or more region of the gastrointestinal system of the subject transmurally.

55. The method of any one of claims 44-54, wherein the subject has damaged and / or injured small bowel, and wherein the HIO component or fragment, the enteroid produced from a transplanted HIO, the enteroid of any one of claims 24-35, and / or the composition engrafts onto and regenerates the small bowel of the subject.

56. The method of claim 55, wherein small bowel regeneration comprises regenerating small bowel epithelium.

57. The method of any one of claims 44-56, wherein the HIO component or fragment, the enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35, following transplantation, matures, proliferates, and / or persists in vivo.

58. The method of any one of claims 44-57, wherein the HIO component or fragment, the enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35, following transplantation, is incorporated into one or more muscularis and / or vascular endothelium region.

59. The method of any one of claims 44-58, wherein the HIO component or fragment, the enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35, following transplantation, expresses one or more proximal intestinal marker and does not expressone or more distal marker; optionally wherein said proximal intestinal marker comprises GATA4.

60. The method of any one of claims 44-59, wherein, following administration, the tissue and / or organ has sustained cell type presence within the transplant region, has human cellular incorporation within the transplant region, has proliferative zonation of the intestine within the transplant region, retains proximal regionalization and / or neo-epithelia function, cyclic adenosine monophosphate regulation, and / or comprises one or more epithelial secretory cell types and / or brush border enzyme.

61. The method of claim 60, wherein said brash border enzyme comprises one or more of human specific DPPIV, ALP, and / or SI.

62. The method of any of claims 44-61, wherein the HIO component or fragment, enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35, is vascularized and / or innervated in vivo following administration.

63. The method of any one of claims 44-62, wherein the enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35 comprises a dissociated epithelial cell population passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times; optionally at least about 3-7 times; optionally at least about 3-5 times, after dissociation and prior to transplanting.

64. The method of any one of claims 44-63, wherein the HIO component or fragment, the enteroid produced from a transplanted HIO, and / or the enteroid of any one of claims 24-35, has been produced from cells derived from the subject.

65. The method of claim 64, wherein the cells derived from the subject comprise induced pluripotent stem cells from the subject.

66. The method of any one of claims 44-65, wherein the subject is a mammal; optionally a human or a mouse.

67. The method of any of claims 44-66, wherein the gastrointestinal-related disease or disorder involves exposed mesenchyme.

68. The method of any one of claims 44-67, wherein the gastrointestinal-related disease or disorder comprises one or more refractive transmural ulcerative disease, gastrointestinal (GI) damage, bowel damage, inflammatory bowel disease (IBD), IBD with refractory ulcer, colonic ulcer, peptic ulcer, gastrointestinal cancer, cancer associated with an APC mutation, and ischemic injuries associated with surgical conditions and / or radiation therapies.

69. A method for determining the transplant and / or therapeutic efficacy of the enteroid of any one of claims 24-35, the method comprising transplanting the enteroid into an animal model and / or a subject, and assessing the effects of the enteroid on the animal model and / or subject.

70. A method for screening, comprising contacting the enteroid of any one of claims 24-35 with a candidate compound or composition, and assessing one or more effects of the candidate compound or composition on the enteroid.

71. The method of claim 70, wherein the enteroid is a model for a gastrointestinal-related disease or disorder, and assessing the effects of the candidate compound or composition on the recombined organoid comprises assessing the effects of the candidate compound or composition on the gastrointestinal-related disease or disorder.

72. The method of claim 70 or 71, wherein the enteroid has been produced from cells derived from a subject, optionally wherein the cells derived from the subject a e induced pluripotent stem cells.

73. The method of claim 72, wherein the subject has a gastrointestinal-related disease or disorder.

74. The method of any of claims 70-73, wherein the method comprises assessing the intestinal metabolism of the candidate compound or composition.

75. The method of any of claims 70-74, the method further comprising measuring oral bioavailability of the candidate compound or composition.

76. A kit comprising means for conducting the method according to any one of claims 1-23 or 39-75.

77. A kit comprising the compositions or means for generating the enteroid of any one of claims 24-35.

78. Use of the method according to any of claims 1-23 or 39-75, the enteroid of any one of claims 24-35, the composition of claim 36, or the kit according to any one of claims 76-77, as a medicament, means for treatment and / or prevention of a disease, means of diagnosis, and / or tool for medical research.

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