Methods for repairing intestinal injury using organoid compositions

Abstract

Disclosed herein is an organoid composition and its method of use for the treatment of intestinal injury and damage.The method comprises intraluminal administration of the cell composition derived from stem cell-derived organoid, which comprises both epithelial and mesenchymal components.The administered cells show stable engraftment in the appropriate area of ​​recipient intestinal tissue.The incorporation of multiple cell types present from stem cell-derived organoid results in more complete healing of intestinal injury.

Description

[Technical field]

[0001] (Statement regarding federally sponsored research and development) This invention was made with Government support under U01 DK103117 and NIH P30 DK078292 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] (Reference to sequence listing) This application has been submitted with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named CHMC63_045WO.xml, was created and last updated on January 12, 2023, and is 6021 bytes in size. The electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION Aspects of the present disclosure relate generally to organoid compositions and methods of use for the treatment of intestinal injury. [Background technology]

[0004] A wide range of intestinal diseases are associated with ulceration of the intestinal tissue. Common indications leading to and / or associated with intestinal ulcers include Crohn's disease, ulcerative colitis, enteropathy associated with non-steroidal anti-inflammatory drugs (NSAIDs) and other drugs, radiation-induced enteropathy, and those associated with pathogenic infections, including tuberculosis. However, chronic ulcers of the intestinal tract of unknown etiology have also been reported. Although detection of intestinal ulcers has improved with the use of endoscopic approaches such as capsule endoscopy and balloon endoscopy, a simple approach for the treatment of these ulcers is still lacking. In some situations, improvement may be seen after cessation of use of the suspected drug with its associated side effects, but advanced cases may require surgical resection. Chronic intestinal damage resulting in non-healing ulcerated areas of the intestine poses a clinical challenge, as there is currently a lack of complete restorative treatment options. Thus, there is a continuing need for therapies that are broadly applicable to different manifestations of intestinal ulceration and injury and have long-lasting effects.

[0005] Peptic ulcer disease (PUD), commonly referred to as gastric or duodenal ulcer, is a chronic, potentially life-threatening condition characterized by erosion of the small intestinal epithelium. Generally caused by bacteria or overuse of nonsteroidal anti-inflammatory drugs (NSAIDs), PUD affects up to 10% of the general population and has a mortality rate of up to 10%. Recent advances in the treatment of H. Pylori and more careful use of NSAIDs have reduced the incidence of PUD, but not the overall mortality rate. In fact, the incidence of idiopathic bleeding ulcers with high mortality rates is increasing. Treatment plans for PUD are becoming increasingly complex, pointing out opportunities for the advancement of novel therapeutic approaches. Antibiotic resistance of H. Pylori is increasing, and conventional pharmacological treatments may result in numerous adverse side effects without completely restoring the ulcerated area to a healthy tissue state. It is important to develop novel therapeutic strategies to broaden the available treatment modalities for these patients, as well as to develop treatments for patients with idiopathic bleeding ulcers. The development of these strategies may also be extended to the management and treatment of other major conditions including Crohn's disease, ulcerative colitis, and chronic ulcer disorders. Summary of the Invention

[0006] The present disclosure relates to dissociated cell populations or compositions derived from intestinal organoids, colonic organoids, or both, and methods of using them to treat intestinal injury in subjects. These intestinal and colonic organoids are derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, such that the organoids are composed of many cell types that are normally found in the small intestine and colon, including both epithelial and mesenchymal cell types in particular. Dissociated cell populations or compositions are made by dissociation or fragmentation of intestinal and colonic organoids, and in some embodiments, dissociated cell populations or compositions are composed entirely or predominantly of clumps of living cells (also referred to herein as "fragments") that represent fragments of intestinal or colonic organoids. Dissociated cell populations or compositions are administered to the luminal wall of the intestine of a subject, which can be done by various approaches. The presence of mesenchymal cells in dissociated cell populations or compositions results in superior engraftment into the intestine of a subject, and healing of intestinal injury in a subject.

[0007] Disclosed herein is a method for treating intestinal injury in a subject that requires such treatment.In some embodiments, the method comprises administering dissociated cell populations that are dissociated from intestinal and / or colon organoid to the luminal wall of the intestine of the subject.In some embodiments, the dissociated cell population comprises epithelial cell type and mesenchymal cell type.In some embodiments, the intestine of the subject comprises small intestine and / or colon.

[0008] Also disclosed herein is a method for treating intestinal injury in a subject that needs to treat intestinal injury.In some embodiments, the method comprises producing intestinal and / or colon organoids comprising epithelial cell type and mesenchymal cell type, dissociating intestinal and / or colon organoids to produce a cell population comprising epithelial cell type and mesenchymal cell type, and administering the cell population to the lumen of the intestine of the subject.In some embodiments, the intestine of the subject comprises small intestine and / or colon.

[0009] Also disclosed herein is a dissociated cell population that is dissociated from intestinal and / or colonic organoids for use in a method for treating gastrointestinal disease in a subject in need of such treatment.In some embodiments, the method comprises administering the dissociated cell population to the luminal wall of the intestine of the subject.In some embodiments, the dissociated cell population comprises epithelial cell type and mesenchymal cell type.In some embodiments, the intestine of the subject comprises small intestine and / or colon.

[0010] Also disclosed herein are cell suspensions comprising dissociated cell populations comprising epithelial and mesenchymal cell types. Also disclosed herein are pharmaceutical formulations comprising any of the cell suspensions or dissociated cell populations disclosed herein.

[0011] The embodiments of the present disclosure provided herein are illustrated by the following numbered embodiments: 1. A method of treating intestinal injury in a subject in need thereof, comprising administering a dissociated cell population dissociated from intestinal and / or colon organoids to the luminal wall of the intestine of the subject, wherein the cell population dissociated from intestinal and / or colon organoids comprises epithelial and mesenchymal cell types, and wherein the intestine of the subject comprises the small intestine and / or colon. 2. The method of embodiment 1, wherein administering the dissociated cell population to the luminal wall of the intestine of the subject comprises administering the cell population to a location in the lumen of the intestine affected by intestinal injury, optionally a location immediately adjacent or near the intestine affected by intestinal injury, and optionally administered to a surface of the luminal wall. 3. The method of embodiment 1 or 2, wherein the dissociated cell population is administered to the luminal wall of the subject's intestine as a cell suspension. 4. The method of any one of embodiments 1-3, wherein administering the dissociated cell population to the luminal wall of the intestine of the subject comprises administering the cell population via an enteral catheter, a nasal catheter, or an enema. 5. The method according to any one of the preceding claims, wherein the intestinal and / or colonic organoids are derived from progenitor cells selected from embryonic stem cells, induced pluripotent stem cells, and definitive endoderm cells. 6. The method of any one of embodiments 1 to 5, wherein the intestinal and / or colonic organoids are allogeneic to the subject. 7. The method according to any one of embodiments 1 to 5, wherein the intestinal and / or colonic organoids are derived from cells isolated from a subject, and the intestinal and / or colonic organoids are autologous to the subject. 8. The method of embodiment 7, wherein the intestinal and / or colonic organoids are derived from induced pluripotent stem cells derived from cells isolated from a subject. 9. The method of embodiment 8, wherein the cells isolated from the subject comprise skin fibroblasts or peripheral blood mononuclear cells (PBMCs) from the subject. 10. The method according to any one of embodiments 1 to 9, wherein the dissociated cell population is prepared by enzymatic and / or mechanical dissociation of intestinal and / or colonic organoids. 11. The method of embodiment 10, wherein the enzymatic dissociation comprises dissociating the intestinal and / or colonic organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof. 12. The method of embodiment 10 or 11, wherein the mechanical dissociation comprises passing the intestinal and / or colonic organoids through channels of successively narrower diameter. 13. The method of any one of embodiments 1-12, wherein the percentage of cells in the dissociated cell population that are mesenchymal cell types is, or is about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or any percentage within a range defined by any two of the foregoing percentages. 14. The method of any one of embodiments 1-13, wherein the percentage of cells in the dissociated cell population that are epithelial cell type is, or is about, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or any percentage within a range defined by any two of the foregoing percentages. 15. Dissociated cell populations were cultured over 1 mm of affected intestinal surface area. 2 or 10,000 cells per mm, or within a range defined by any two of the foregoing values. 2 The method of any one of embodiments 1 to 14, wherein the amount of cells administered per 100 μg / mL is any amount of cells administered per 100 μg / mL. 16. The method of any one of embodiments 1-15, wherein the dissociated cell population is administered to the subject multiple times until amelioration of intestinal damage is observed. 17. The method of embodiment 16, wherein the dissociated cell population is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. 18. The method of any one of embodiments 1 to 17, wherein the cells of the dissociated cell population integrate into the mucosa and muscularis of the subject's intestine. 19. The method of embodiment 18, wherein the cells of the dissociated cell population integrated into the subject's intestine maintain their intestinal and / or colonic regionality. 20. The method of embodiment 18 or 19, wherein the cells of the dissociated cell population integrated into the subject's intestine, or a subpopulation thereof, differentiate into smooth muscle actin (SMA) positive smooth muscle cell types. 21. The method of any one of embodiments 1 to 20, wherein the dissociated cell population comprises Marker of Proliferation KI67+ (MKI67+) proliferative cells that integrate into the subject's intestine and promote healing of intestinal injury. 22. The method of any one of embodiments 1-21, wherein the dissociated cell population promotes the formation of an intact intestinal barrier following administration. 23. The method of any one of embodiments 1-22, wherein the dissociated cell population incorporates into at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the surface area of ​​the luminal wall of the intestine of a subject affected by intestinal injury, or any percentage within a range defined by any two of the foregoing percentages. 24. The method of any one of embodiments 1-23, wherein the intestinal damage comprises an intestinal ulcer. 25. The method of any one of embodiments 1-24, wherein the intestinal injury is chemical and / or mechanical. 26. The method of any one of embodiments 1-25, wherein the intestinal damage is associated with a gastrointestinal disease. 27. The method of embodiment 26, wherein the gastrointestinal disease is selected from Crohn's disease, ulcerative colitis, enteropathy associated with nonsteroidal anti-inflammatory drugs (NSAIDs) or other medications, radiation-induced enteropathy, and enteropathy associated with pathogenic infections such as tuberculosis. 28. The method according to any one of embodiments 1 to 27, wherein the intestinal and / or colonic organoids are mammalian. 29. The method according to any one of embodiments 1 to 28, wherein the intestinal and / or colonic organoids are human. 30. The method of any one of embodiments 1-29, wherein the subject is a mammal. 31. The method of any one of embodiments 1 to 30, wherein the subject is a human. 32. The method of any one of embodiments 1 to 31, further comprising dissociating the intestinal and / or colonic organoids to produce a dissociated cell population. 33. The method according to any one of the preceding embodiments, wherein the dissociated cell population is in the form of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments, or is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments. 34. The method according to any one of embodiments 1 to 33, further comprising producing intestinal and / or colonic organoids in vitro, optionally from pluripotent stem cells. 35. A dissociated cell population dissociated from intestinal and / or colonic organoids for use in a method according to any one of embodiments 1 to 34. 36. A cell suspension comprising a dissociated cell population comprising epithelial and mesenchymal cell types. 37. The cell suspension of embodiment 36, wherein the mesenchymal cell types express vimentin (VIM) and / or elastin microfibril interfacer 1 (EMILIN1) and the epithelial cell types express E-cadherin (CDH1) and / or caudal type homeobox 2 (CDX2). 38. The cell suspension of embodiment 36 or 37, wherein the dissociated cell population is dissociated from intestinal and / or colonic organoids, the intestinal and / or colonic organoids comprising epithelial and mesenchymal cell types. 39. The cell suspension of embodiment 38, wherein the intestinal and / or colonic organoids are derived from progenitor cells selected from embryonic stem cells, induced pluripotent stem cells, and definitive endoderm cells. 40. The cell suspension according to any one of embodiments 35 to 39, wherein the cell suspension or the intestinal and / or colonic organoids are allogeneic to the subject. 41. The cell suspension according to any one of embodiments 35 to 40, wherein the cell suspension or the intestinal and / or colon organoids are derived from cells from the subject, and the intestinal and / or colon organoids are autologous to the subject. 42. The cell suspension of embodiment 41, wherein the cell suspension or the intestinal and / or colonic organoids are derived from induced pluripotent stem cells derived from cells isolated from a subject. 43. The cell suspension according to any one of embodiments 35 to 42, wherein the dissociated cell population is prepared by enzymatic and / or mechanical dissociation of intestinal and / or colonic organoids. 44. The cell suspension of embodiment 43, wherein the enzymatic dissociation comprises dissociating the intestinal and / or colonic organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof. 45. The cell suspension of embodiment 43 or 44, wherein the mechanical dissociation comprises passing the intestinal and / or colonic organoids through channels of successively narrower diameter. 46. ​​The cell suspension of any one of embodiments 35 to 45, wherein the dissociated cell population comprises MKI67+ proliferative cells. 47. The cell suspension of any one of embodiments 35-46, wherein the percentage of cells in the dissociated cell population that are mesenchymal cell types is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or a percentage within a range defined by any two of the foregoing percentages. 48. The cell suspension of any one of embodiments 35-47, wherein the percentage of cells in the dissociated cell population that are epithelial cell type is less than or equal to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or a percentage within a range defined by any two of the foregoing percentages. 49. The concentration of the dissociated cell population in the cell suspension is approximately 10 5 , 10 6 , 10 7 , 10 8 , 109 , 10 10 , or 10 11 49. The cell suspension according to any one of embodiments 35-48, wherein the cell suspension is at a cell / mL or any cell concentration within a range defined by any two of the aforementioned concentrations. 50. The concentration of cells that are mesenchymal cell types in a dissociated cell population is approximately 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 50. The cell suspension according to any one of embodiments 35-49, wherein the cell suspension is at a cell / mL or any cell concentration within a range defined by any two of the aforementioned concentrations. 51. The cell suspension of any one of embodiments 35-49, wherein the dissociated cell population is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in the form of multicellular fragments, or at least in the form of multicellular fragments, or any percentage within a range defined by any two of the aforementioned percentages. 52. A pharmaceutical composition comprising an effective amount of a cell suspension according to any one of embodiments 35 to 51 and at least one pharma- ceutically acceptable carrier, excipient, or diluent. 53. A cell suspension according to any one of embodiments 35 to 51 or a pharmaceutical composition according to embodiment 52 for use in the treatment of intestinal damage. [Brief description of the drawings]

[0012] In addition to the features described herein, additional features and modifications will be readily apparent from the following drawings and description of exemplary embodiments, it being understood that these drawings illustrate embodiments and are not intended to be limiting in scope. [Figure 1A]

[0023] Figure 1 depicts an embodiment of a dissociated HIO that contributes to tissue regeneration of the injured intestine in vivo. Figure 1A depicts a schematic embodiment of the experimental design. [Figure 1B-G] FIG. 1B depicts an embodiment of dissociated HIOs contributing to tissue regeneration of injured intestine in vivo. FIG. 1B depicts brightfield (left panel) and GFP (right panel) images of harvested reseeded loops. Presence of viable-GFP demonstrates GFP-HIO fragments incorporated within the loops at 10 weeks post-op. Black and white dashed lines indicate the perimeter of the loop. A dashed line within the white dashed line of the GFP image indicates the perimeter of the viable-GFP expressing area within the loop. Scale bar=1 mm. FIG. 1C depicts an embodiment of quantification of the viable-GFP expressing area within the loops depicted in FIG. 1B. Violin plot of loop surface area expressing viable-GFP as a percentage of the total loop, n=7. FIG. 1D depicts an embodiment of immunostaining for human cells within the dissected area where viable-GFP is present depicted in FIG. 1B. FIG. 1E depicts an embodiment of brightfield (left) and GFP (right) images of harvested loops reseeded with fragmented enteroids. The presence of viability-GFP demonstrates GFP-enteroid fragments incorporated within the loops at 10 weeks post-surgery. Black and white dashed lines indicate the circumference of the loop. Scale bar=1 mm. FIG. IF depicts an embodiment of quantification of viability-GFP expressing area within the loops shown in FIG. IE. Violin plot of loop surface area expressing viability-GFP as a percentage of total loop, n=7. FIG. IG depicts an embodiment of immunostaining for human cells within the dissected area where viability-GFP is present in FIG. IE. [Figure 1H] FIG. 1H depicts an embodiment of a dissociated HIO that contributes to in vivo tissue regeneration of the injured intestine. FIG. 1H depicts an embodiment of a representative tile scan of an injured sham loop stained for human specific markers (KU80) and nuclei (hematoxylin), scale bar=0.5 cm, n=3. No cells of human origin were observed throughout the tissue of the sham loop. [Figure 2A-D]FIG. 2A depicts an embodiment of a representative tile scan of injured and reseeded loops stained for human specific markers (KU80) and nuclei (hematoxylin), scale bar=0.5 cm, n=7. Cells of human origin were observed throughout the reseeded loops. FIG. 2B depicts an embodiment of a high magnification image of the mucosa and muscularis region in FIG. 2A, scale bar=100 μm. FIG. 2C depicts an embodiment of a representative image of a sham-operated and reseeded loop stained for actin alpha 2, smooth muscle (Actin Alpha 2, ACTA2), GFP, and nuclei (DAPI), scale bar=50 μm, n=3 for both groups. FIG. 2D depicts an embodiment of a representative image of a sham-operated and reseeded loop stained for Tubulin Beta 3 Class III (TUBB3) and nuclei (hematoxylin), scale bar=50 μm, n=3 for both groups. Arrowheads indicate nerve bundles from the rat host. [Figure 3A] Illustrates an embodiment of the H1-GFP cell line that retains a normal karyotype after gene editing. G-banded karyotype analysis demonstrates the normal (46,XY) karyotype of the H1 embryonic stem cell line after GFP insertion. [Figure 3B] 1 depicts an embodiment of an electropherogram of short tandem repeat analysis of H1 embryonic stem cell line after GFP insertion showing a passing result. [Figure 4] Illustrates photographic embodiments of key steps during mucosal resection surgery. Panel A depicts loop creation and anastomosis, with the arrowhead pointing to the anastomosis site. Panel B depicts chemically traumatizing the loop from the open proximal end while the distal end is closed with a bulldog clamp. Panel C depicts mechanically traumatizing the loop with a dental interstitial flosser. Panel D depicts reseeding the loop from the proximal opening while the distal end is closed with absorbable suture, with the arrowhead pointing to the absorbable suture. Panel E depicts the resulting anatomy of the end-to-side loop, with the dashed white outline indicating the loop structure. [Diagram 5]Illustrates an embodiment of the acute transmural intestinal injury model. Panel A depicts representative hematoxylin and eosin stained sections of healthy rat jejunum (left) and freshly injured rat jejunal loop (right), scale bar = 50 μm, n = 3 in both groups. Panel B depicts representative scanning electron micrographs of healthy jejunum (left) and freshly injured jejunal loop (right). Scale bar = 100 μm. The epithelium is largely detached with visible disruption in the muscularis layer due to the chemical and mechanical injury model. [Figure 6A] FIG. 6A depicts an embodiment of a Kaplan-Meier curve for mucosal resection procedures with an endpoint of 10 weeks post-op. A total of 32 procedures were performed: sham / medium reseeding n=3, fragmented HIO reseeding n=17 (11), and fragmented enteroid reseeding n=12 (8). FIG. 6B depicts an embodiment of a representative at-harvest image of an end-to-side blind loop 10 weeks post-op. [Figure 6B] FIG. 6A depicts an embodiment of a Kaplan-Meier curve for mucosal resection procedures with an endpoint of 10 weeks post-op. A total of 32 procedures were performed: sham / medium reseeding n=3, fragmented HIO reseeding n=17 (11), and fragmented enteroid reseeding n=12 (8). FIG. 6B depicts an embodiment of a representative at-harvest image of an end-to-side blind loop 10 weeks post-op. [Figure 7A]FIG. 7A depicts an embodiment of initial loop engraftment of HIO regenerating stem cell niches over time. FIG. 7A depicts an embodiment of representative images of loops after 7 days reseeded with medium alone or fragmented HIO (upper panel) or with epithelial marker (CDH1), proliferation marker (MKI67), and nuclei (DAPI) (lower panel) stained for human specific marker (KU80) and nuclei (hematoxylin). The only engrafted human contribution was loops reseeded with dissociated HIO. n=4 for both groups. FIG. 7B depicts an embodiment of representative images of control human jejunum and reseeded loops after 10 weeks stained for proliferation marker (MKI67), epithelial marker (CDH1), surrogate marker of stem cell activity (OLFM4), and telocyte marker (F3). n=3 for both groups. All scale bars=100 μm. [Figure 7B] FIG. 7A depicts an embodiment of initial loop engraftment of HIO regenerating stem cell niches over time. FIG. 7A depicts an embodiment of representative images of loops after 7 days reseeded with medium alone or fragmented HIO (upper panel) or with epithelial marker (CDH1), proliferation marker (MKI67), and nuclei (DAPI) (lower panel) stained for human specific marker (KU80) and nuclei (hematoxylin). The only engrafted human contribution was loops reseeded with dissociated HIO. n=4 for both groups. FIG. 7B depicts an embodiment of representative images of control human jejunum and reseeded loops after 10 weeks stained for proliferation marker (MKI67), epithelial marker (CDH1), surrogate marker of stem cell activity (OLFM4), and telocyte marker (F3). n=3 for both groups. All scale bars=100 μm. [Figure 8A]Figure 8A depicts an embodiment of a representative image of human jejunum and reseeded loops stained for proximal intestinal epithelial transcription factor (GATA4) and epithelium (CDH1) (left panel), Paneth cell marker (DEFA5) (middle panel), and enzymes involved in carbohydrate digestion (SI) (right panel). Figure 8B depicts an embodiment of a representative image of human colon and reseeded loops stained for DNA binding protein present in distal ileum and colon (SATB2), colonocyte marker (MS4A12), and colonic mucin (MUC5B). All scale bars = 100 μm, all groups n = 3. [Figure 8B] Figure 8A depicts an embodiment of a representative image of human jejunum and reseeded loops stained for proximal intestinal epithelial transcription factor (GATA4) and epithelium (CDH1) (left panel), Paneth cell marker (DEFA5) (middle panel), and enzymes involved in carbohydrate digestion (SI) (right panel). Figure 8B depicts an embodiment of a representative image of human colon and reseeded loops stained for DNA binding protein present in distal ileum and colon (SATB2), colonocyte marker (MS4A12), and colonic mucin (MUC5B). All scale bars = 100 μm, all groups n = 3. [Figure 9A-D]9A depicts an embodiment of data showing that the neoepithelium of reseeded loops responds to chemical stimuli. FIG. 9A depicts an embodiment of representative brightfield (left panel) and viable GFP (right panel) images of healthy rat jejunum (proximal to the loop) mounted on a slider for Ussing assay, dashed circle = sider opening. FIG. 9B depicts an embodiment of representative brightfield (left panel) and viable GFP (right panel) images of a reseeded loop reaching the slider for Ussing assay, dashed circle = slider opening. Expression of GFP verifies successful reseeding of the segment and the origin of the cells. FIG. 9C-9D depict an embodiment of representative time courses of short circuit current (Isc) measured in Ussing chamber experiments using healthy jejunum (FIG. 9C) and GFP+ reseeded loops (FIG. 9D). [Figure 9E-H] FIG. 9E depicts an embodiment of data showing that the neoepithelium of reseeded loops responds to chemical stimuli. FIG. 9E depicts an embodiment of a graph of the calculated change in Isc in response to 10 μM forskolin, 100 μM IBMX, and 100 μM bumetanide. FIG. 9F depicts an embodiment of a graph of baseline transepithelial electrical resistance of healthy rat jejunum and GFP+ reseeded loops. FIG. 9G depicts an embodiment of a graph of FITC-dextran permeability of healthy rat jejunum and GFP+ reseeded loops over 3 hours (top line is loop). FIG. 9H depicts an embodiment of FITC flux calculated from the graph of FIG. 9G. Statistical significance between groups in FIG. 9E, FIG. 9F, and FIG. 9H was determined using Wilcoxon signed rank test. n=5 for both groups. [Figure 10A-D]10A depicts an embodiment of in vitro HIO and enteroid fragmentation for reseeding and cell number determination. FIG. 10A depicts an embodiment of representative brightfield and GFP images of intact HIO (left panel) and HIO fragments created by shearing through a series of needles ending in 25G (right panel). Scale bar=500 μm. FIG. 10B depicts the same as FIG. 10A but using enteroids instead of HIOs. FIG. 10C depicts an embodiment of a representative brightfield image acquired during automated cell count quantification of dissociated HIOs or enteroids. FIG. 10D depicts an embodiment of a graph of the average cell number per HIO or enteroid calculated from single nucleus preparations. n=3 HIO and n=4 enteroid preparations. [Figure 11A-B] FIG. 11A depicts an embodiment of a representative image of tissue at 10 weeks post-surgery stained for human specific marker (KU80, brown) and nuclei (hematoxylin, blue). In the implanted HIO, predominantly KU80+ cells are present, whereas no KU80+ cells were observed in various organs of rats undergoing a seeded mucosal resection procedure. Scale bar=100 μm. FIG. 11B depicts an embodiment of a dot plot of Ct values ​​from Alu PCR for organs collected from rats as shown in FIG. 11A. The dotted line indicates the Ct value threshold for a positive signal (presence of human cells). Open circles indicate undetermined values ​​or Cts greater than 40. No data points fell within the range of human cell presence. n=3 per group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Disclosed herein are clinically relevant methods and protocols for intestinal reconstruction and injury repair using dissociated cells from intestinal or colonic organoids generated from pluripotent stem cells. Dissociated cells from intestinal or colonic organoids, such as human intestinal organoids (HIO) and human colonic organoids (HCO), derived from human pluripotent stem cells, can engraft in damaged loops of the host intestine in vivo, contribute to regeneration, and reconstitute both the mucosa and muscularis. From a clinical perspective, this data is exciting, since new therapeutic strategies for chronic refractive bowel disease require transmural regenerative capacity. As disclosed herein, substantial engraftment of the organ surface area was achieved within 10 weeks (mean engraftment of 16.93% by surface area after 10 weeks compared to only 1.68% using enteroids), indicating that intestinal or colonic organoid seed material is not eliminated or washed away over time, making it more efficient than epithelium-only seed material.

[0014] In a previous study, Yui et al. reported the engraftment / proliferation rate of donor cells in colitic mice as 0.02% cells or approximately 100 cells per mouse at 4 weeks after transplantation when using enteroids (i.e., organoid-like structures derived from adult intestinal tissue containing only epithelium and no mesenchyme). Several similar reports have also demonstrated the ability of enteroids to contribute to intestinal and colonic healing, although the efficiency has rarely been reported. Most recently, Sugimoto et al. demonstrated the engraftment of ileal enteroids in the mouse colon.

[0015] Disclosed herein is the first report of using a multi-lineage seed material containing both epithelium and mesenchyme to achieve in vivo intestinal repair as a potential cell therapy. The method herein allows epithelial stem cell niches to be generated exclusively by the seed material in areas where native tissue is non-functioning or damaged. Upon incorporation, the epithelial stem cell compartment re-emerged by 10 weeks and the regional identity of the engrafted HIO fragments was retained. The new epithelium of the loops was functional and responded to chemical stimuli with appropriate barrier integrity as observed in ex vivo physiological Ussing chamber assays. Using pluripotent stem cell-derived organoids representing more diverse and specific regions of the gut, the platform can be extended to additional regions of the gastrointestinal tract, such as the colon.

[0016] Pluripotent stem cell-derived organoid technology, especially for humans, has transformed the landscape of tissue engineering over the past decade. A fundamental goal of tissue engineering lies in the ability of generated materials to functionally restore or improve entire damaged tissues or organs. An in vivo intestinal transmural injury model was used to investigate the therapeutic potential of intestinal organoids. HIOs generated de novo from hPSCs contain both epithelial and mesenchymal components. In a xenograft preclinical injury model, dissociated HIOs of luminal origin were demonstrated to engraft and proliferate during the regeneration process. Not only was restoration of the mucosal layer observed, but significant incorporation was also observed throughout the muscular layer. Further analysis revealed the re-emergence of an epithelial stem cell / progenitor system for homeostatic renewal and maintenance of small intestinal regionalization. As observed through ex vivo physiological readouts, the neoepithelium responded to chemical stimuli and its permeability was similar to that of healthy adjacent host intestine. These findings provide an intriguing proof of concept for the therapeutic use of HIOs to treat chronic non-healing ulcerative intestinal injury, which currently lacks complete restorative treatment options.

[0017] Two potential sources of cell therapy for healing intestinal injury are enteroids and human intestinal organoids (HIOs). Enteroids are epithelial-only structures derived from crypts isolated from the patient's intestine or transplanted HIOs in vitro. They may contain all differentiated epithelial cell subtypes, e.g., enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, but they lack the mesenchymal, nervous, and immune compartments present in the human intestine. Some evidence indicates that enteroids can be used to replace damaged intestinal epithelium, but they cannot fully address transmural injury. Unlike enteroids, HIOs are generated through stepwise differentiation of human pluripotent stem cells using the same small molecule growth factors that promote differentiation of fetal intestinal tissue in utero. HIOs contain both epithelial and mesenchymal cell types, which form laminated structures upon transplantation. Utilizing an immunocompromised host as a bioreactor for engraftment and maturation, transplanted HIOs develop into structures reminiscent of the human intestine, including the crypt / villus axis, vasculature, muscularis mucosae, and both inner circular and outer longitudinal smooth muscle layers.The ability of both enteroids and HIOs to regenerate injured intestine has been investigated in preclinical rodent injury models.

[0018] Methods for producing organoids or enteroids are described in U.S. Pat. Nos. 9,719,068 and 10,174,289, and PCT Publication Nos. WO2015 / 183920, WO2016 / 061464, WO2017 / 192997, WO2018 / 085615, WO2018 / 085622, WO2018 / 085623, WO2018 / 226267, WO2018 / 106628, and the like. , WO2018 / 200481, WO2018 / 191673, WO2019 / 074793, WO2019 / 126626, WO2020 / 056158, WO2020 / 023245, WO2020 / 160371, WO2020 / 243613, and WO2021 / 030373, each of which is expressly incorporated by reference in its entirety.

[0019] definition In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like components unless the 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 expressly contemplated herein.

[0020] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs when read in light of this disclosure. For purposes of this disclosure, the following terms are described below.

[0021] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0022] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 10% from the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0023] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of the recited steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and other elements may not be present. "Consisting essentially of" means the inclusion of any elements recited after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action set forth in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are optional and may or may not be present depending on whether they have a substantial effect on the activity or action of the recited elements.

[0024] The terms "individual", "subject" or "patient" as used herein have their common and usual meaning as understood in the context of this specification, and refer to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as any other vertebrate or invertebrate. The term "mammal" is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like.

[0025] The term "effective amount" or "effective dose" as used herein has its common and usual meaning as understood in the context of this specification and refers to that amount of the described composition or compound that produces an observable effect. The actual dosage level of the active ingredients in the active composition of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound that is effective to achieve a desired response for a particular subject and / or application. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the composition, the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Contemplated herein are the determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments.

[0026] The terms "function" and "functional" as used herein have their common and ordinary meaning as understood in the context of this specification and refer to biological, enzymatic, or therapeutic functions.

[0027] The term "inhibit" as used herein has its common and ordinary meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be, about, at least, at least about, less than, or a percentage that is about or less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values. The term "delay" as used herein has its common and ordinary meaning as understood in light of the present specification and refers to a delay, postponement, or postponement of a biological event to a later time than would otherwise be expected. The delay may be 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, about, at least about, less than, or a percentage less than about, or an amount within a range defined by any two of the preceding values. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0028] As used herein, the term "isolated" has its common and ordinary meaning as understood in light of the present specification and refers to a substance and / or entity that is (1) separated from at least some of the components with which it is associated when originally produced (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 can be separated from equal to, about, at least about, less than, or about less than 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 substantially 100% (or ranges including and / or spanning the foregoing values) of other components with which they are originally associated. In some embodiments, an isolated agent is, about, 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, about, at least about, at least about, less than, or less than (or ranges including and / or spanning the aforementioned values). As used herein, an "isolated" material can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0029] As used herein, "in vivo" is given its ordinary and ordinary meaning as understood in light of the present specification and refers to the performance of methods within living organisms, usually animals, mammals, including humans, and plants, as opposed to tissue extracts or dead organisms.

[0030] As used herein, "ex vivo" is given its ordinary and usual meaning as understood in light of the present specification and refers to the performance of the method outside the body with little change in natural conditions.

[0031] As used herein, "in vitro" is given its common and ordinary meaning as understood in the context of this specification and refers to the performance of methods outside biological conditions, for example in a petri dish or test tube.

[0032] The terms "nucleic acid" or "nucleic acid molecule" as used herein have their common and ordinary meaning as understood in the context of this specification and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those naturally occurring in cells, fragments generated by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, 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 combinations of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Furthermore, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications of the 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 bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotides" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The nucleic acid may be contained in a nucleic acid vector or construct (e.g., a plasmid, a virus, a retrovirus, a lentivirus, a bacteriophage, a cosmid, a fosmid, a phagemid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a human artificial chromosome (HAC)) that can be used for amplification and / or expression of the nucleic acid in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a targeting sequence, a peptide purification tag, or an accessory gene, or any combination thereof.

[0033] A nucleic acid or nucleic acid molecule can include one or more sequences encoding different peptides, polypeptides, or proteins, which can be adjacent in the same nucleic acid or nucleic acid molecule, or can be joined with extra nucleic acid, for example, between linkers, repeats, or restriction enzyme sites, or with any other sequence that is, about, at least, at least about, less than, or about less than 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 in length, or any length within the range defined by any two of the aforementioned lengths. The term "downstream" as used herein with respect to a nucleic acid has its common and usual meaning as understood in the context of this specification and refers to the sequence behind the 3' end of the previous sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded. The term "upstream" as used herein with respect to a nucleic acid has its common and usual meaning as understood in the context of this specification and refers to the sequence ahead of the 5' end of the subsequent sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded.The term "grouping" as used herein with respect to nucleic acids has its ordinary and usual meaning as understood in the context of the present specification and refers to two or more sequences that occur either directly or in close proximity with, for example, extra nucleic acid between linkers, repeats, or restriction enzyme sites, or with any other sequence that is, about, at least, at least about, less than, or about less than 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 in length, but generally not with sequences that are in between that encode a functional or catalytic polypeptide, protein, or protein domain.

[0034] The nucleic acid described herein comprises nucleobases. The primary, normal, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil. Other nucleobases include, but are not limited to, purine, pyrimidine, 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.

[0035] The terms "peptide", "polypeptide" and "protein" as used herein have their common and usual meaning as understood in the context of this specification and refer to a polymer composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides and proteins are known in the art, including, but not limited to, enzymatic, structural, transport, defensive, hormonal or signal transduction. Peptides, polypeptides and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating the nucleic acid template, peptide, polypeptide and protein mutations such as substitution, deletion, truncation, addition, duplication or fusion of two or more peptides, polypeptides or proteins can be performed. These fusions of two or more peptides, polypeptides, or proteins can be adjacent in the same molecule or can be joined with extra amino acids between, for example, linkers, repeats, epitopes, or tags, or any other sequence that is 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 in length, about, at least, at least about, less than, or less than, or any length within the range defined by any two of the aforementioned lengths. As used herein, the term "downstream" in reference to a polypeptide has its ordinary and usual meaning as understood in the context of this specification and refers to a sequence following the C-terminus of the preceding sequence. The term "upstream" as used herein with respect to a polypeptide has its ordinary and usual meaning as understood in the context of this specification and refers to a sequence preceding the N-terminus of a subsequent sequence.

[0036] The term "purity" of any given substance, compound, or material as used herein has its common and ordinary meaning as understood in light of the present specification, and refers to the actual abundance of the substance, compound, or material compared 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 decimal points therebetween. Purity may be affected by undesirable impurities, including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, 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 infectious agents. Purity can be measured using techniques 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 spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0037] The term "yield" of any given substance, compound, or material as used herein has its ordinary and usual meaning as understood in light of the present specification, and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be, about, at least, at least about, less than, or about less than 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, including all decimal points therebetween. Yield may be affected by the efficiency of a reaction or process, undesired side reactions, decomposition, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of production.

[0038] The term "intestinal organoid" as used herein has its general and usual meaning as understood in the context of the present specification, and refers to a three-dimensional cellular structure that presents many characteristics of the small intestine of an organism. In some embodiments, the intestinal organoid is derived from human cells and exhibits the characteristics of the human small intestine. However, intestinal organoids from other mammals are also included. Intestinal organoids as used herein are derived from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) or their intermediates (e.g., definitive endoderm), where the process of differentiating pluripotent stem cells into definitive endoderm, then into hindgut endoderm (which may be in the form of spheroids), and finally into intestinal organoids results in a cellular structure with a composition, structure, and function similar to that of naturally occurring intestine. The significant difference between intestinal organoids as used herein and enteroids, which are cellular structures derived from adult intestinal epithelium, and other so-called organoids that are made from non-pluripotent adult intestinal stem cells, is that intestinal organoids as used herein contain both epithelium and mesenchyme. Mesenchyme plays an important supporting role for epithelium, greatly enhancing the viability and robust function of intestinal organoids. The intestinal organoids used herein can show a lumen with epithelial villi-like involutions, and peristaltic behavior, which are very similar to normal intestine. As a result of the differentiation process from pluripotent stem cells, the intestinal organoids used herein also contain specialized intestinal cell types, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. References that disclose embodiments of intestinal organoids suitable for use herein include WO 2011 / 140441, WO 2016 / 061464, WO 2018 / 200481, WO 2020 / 160371, and WO 2021 / 030373, each of which is incorporated herein by reference in its entirety.

[0039] The term "colon organoid" as used herein has its general and usual meaning as understood in the context of the present specification, and refers to a three-dimensional cellular structure that presents many characteristics of the colon of an organism. In some embodiments, colon organoids are derived from human cells and exhibit the characteristics of the human colon. However, colon organoids from other mammals are also encompassed. Colon organoids as used herein are derived from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) or their intermediates (e.g., definitive endoderm), where the process of differentiating pluripotent stem cells into definitive endoderm, then into hindgut endoderm (which may be in the form of spheroids), and finally into colon organoids results in a structure with composition, structure, and function similar to that of naturally occurring colon. A significant difference between colon organoids as used herein and colonoids, which are cellular structures derived from adult colonic epithelium, and other so-called organoids that are made from non-pluripotent adult colonic stem cells, is that colon organoids as used herein contain both epithelium and mesenchyme. Mesenchyme plays an important supporting role for epithelium, greatly enhancing the viability and robust function of colonic organoids.Colonic organoids used herein may show a lumen with crypts but substantially without villus-like structures.As a result of the differentiation process from pluripotent stem cells, colonic organoids used herein also contain specialized colonic cell types, including a large number of goblet cells (compared to intestinal organoids) and colonic enteroendocrine cells, but substantially without Paneth cells.References that disclose embodiments of colonic organoids suitable for use herein include WO2018 / 106628, which is incorporated herein by reference in its entirety.

[0040] The terms "fragmentation", "fragmented", "dissociation" and "dissociated" as used herein have their common and usual meanings as understood in light of the present specification, and refer to the partial or complete fragmentation or dissociation of organoids or other three-dimensional multicellular structures to generate a population of single cells and viable multicellular structures, fragments, or aggregates without excessive shearing or damage to cells, such that all or most of the dissociated organoids contain intact and healthy cells. Thus, "fragmented" and the like generally do not refer to non-viable subcellular components or fragments of single cells, such as, for example, free intracellular contents or non-viable vesicles, although these components may be present in embodiments of organoid compositions that are fragmented due to natural apoptosis of cells or unintentional damage during dissociation of organoids. Fragmentation or dissociation of organoids can be performed in various ways that are commonly known in the art. The fragmentation or dissociation process may be such that some of the resulting cells are found as small multicellular aggregates / fragments rather than as single cells. Populations of dissociated cells that contain multicellular aggregates / fragments among single cells are contemplated for use herein. In some embodiments, the dissociated cell population or composition exists exclusively as multicellular aggregates / fragments. In some embodiments, the dissociated cell population or composition exists exclusively as single cells without multicellular aggregates / fragments. In some embodiments, the dissociated cell population or composition is primarily (e.g., greater than 70%, 80%, or 90% of the cells) multicellular aggregates / fragments and contains relatively few single cells. In some embodiments, the dissociated cell population or composition exists as a mixture of single cells and multicellular aggregates / fragments.

[0041] The term "enzymatic dissociation" as used herein has its general and ordinary meaning as understood in the context of the present specification, and refers to the fragmentation or dissociation of organoid or other three-dimensional multicellular structures using the catalytic activity of one or more enzymes.Enzymatic dissociation, which is a process generally known in the art, usually involves the use of proteolytic enzymes (e.g., trypsin) or enzymes (e.g., hyaluronidase) that are specific for other molecules involved in attachment to surfaces or intercellular junctions.

[0042] The term "mechanical dissociation" as used herein has its general and ordinary meaning as understood in the context of this specification, and refers to the fragmentation or dissociation of organoid or other three-dimensional multicellular structures using mechanical force.Mechanical dissociation, which is a process generally known in the art, can be achieved by, for example, trituration through narrow-diameter channels, which can be in the form of pipettes, needles, microfluidic channels, etc.

[0043] As used herein, the terms "multicellular aggregates", "cell aggregates", "multicellular fragments", "multicellular organoid fragments", etc., have their common and usual meanings as understood in light of the present specification, and refer to cells collected by adhesion forces such as naturally produced extracellular matrix, and generally, these collections of cells move as a single entity (e.g., in aqueous suspension). These multicellular aggregates or organoid fragments are generated by dissociation of organoids and / or enteroids by classical enzymatic and / or mechanical dissociation means, as described herein. Thus, the skilled artisan can determine the approximate parameters (e.g., number of cells per aggregate / fragment, size, diameter, volume, maximum dimension, etc.) of the multicellular aggregates / fragments generated by these dissociation means. For example, the diameter size of the narrow diameter channel used for mechanical dissociation can affect the resulting size of the aggregates / fragments. These parameters can be quantified by conventional methods such as microscopy or flow cytometry. Also as discussed herein, these "multicellular fragments" refer to groups of living cells derived from the fragmentation or differentiation of a larger three-dimensional cellular structure, such as an organoid, and do not refer to subcellular components, although these subcellular components may be present in the composition due to the method of fragmentation or dissociation of the larger three-dimensional cellular structure.

[0044] As applied to the present disclosure, multicellular aggregates / fragments generated from dissociation of organoids and / or enteroids can be quantified in terms of the number of cells per aggregate / fragment. In some embodiments, multicellular aggregates / fragments can be quantified in terms of the number of cells per aggregate / fragment. 2 , 10 3 , 10 4 , 10 5 , or 10 6 cells, about, at least, at least about, less than, or about, or any number of cells within a range defined by any two of the foregoing cell numbers, e.g., 10 2 ~10 6 cells, 10 2 ~104 cells, 10 4 ~10 6 cells, or 10 3 ~10 5 The number of cells may include cells that are cells. As applied to some embodiments disclosed herein, the multicellular aggregates / fragments produced from dissociation of organoids and / or enteroids can be quantified in terms of the approximate diameter of the multicellular aggregates / fragments (or more generally, the length of the largest dimension of the irregularly cellularized aggregates / fragments). In some embodiments, the multicellular aggregates / fragments have a diameter (or length of the largest dimension) of or are about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 μm. , may have an approximate diameter and / or maximum dimension that is at least those, at least about those, not more than those, or not more than about those, or any diameter (or length of maximum dimension) between the ranges defined by any two of the aforementioned lengths, e.g., 100-400 μm, 100-250 μm, 150-300 μm, 200-400 μm, or 200-250 μm.

[0045] The term "mucosa" as used herein has its common and usual meaning as understood in light of this specification and refers to the innermost layer of the gastrointestinal tract. The epithelium is the innermost layer of the mucosa and is where epithelial cells and other specialized cells such as goblet cells are found. The epithelium also forms the villus structure of the intestine. The epithelium is surrounded by connective tissue called the lamina propria and a thin layer of smooth muscle.

[0046] The term "muscularis layer" as used herein has its common and ordinary meaning as understood in light of the present specification and refers to the muscularis propria of the gastrointestinal tract. The muscularis layer regulates the peristaltic behavior of the intestine and colon and is derived from the mesenchymal layer of the developing new intestine.

[0047] The term "regionality" as used herein has its general and usual meaning as understood in the context of this specification, and refers to the quality and characteristics that distinguish one cell type from another. In the context of intestine and colon (and other gastrointestinal organs), both organs originate from the same definitive endoderm, but early specification leads to the proper development and differentiation of the constituent cells corresponding to the two organs and their functions. As a result, intestinal tissue shows a different regionality from colonic tissue. As shown herein, intestinal and colonic organoids used for engraftment in intestinal injury models retain their respective qualities even after incorporation into cell layers of different organs (e.g., intestinal organoids into host colonic tissue or colonic organoids into host intestinal tissue).

[0048] The term "intestinal barrier" as used herein has its common and usual meaning as understood in the context of this specification and refers to the cellular and mucosal barrier that separates the luminal contents of the gastrointestinal tract from the surrounding tissues and circulatory system while still allowing the exchange of nutrients. This barrier is mediated by the intracellular junctions between the cells of the epithelium. During intestinal injury, this barrier can be disrupted, resulting in abnormal function of the intestine, the passage of potentially pathogenic microorganisms or antigens into the body, and leakage of blood and molecules into the lumen.

[0049] As used herein, "pharmacologically acceptable" has its ordinary and usual meaning as understood in the context of the present specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed at the dosages and concentrations used. As used herein, "pharmacologically acceptable", "diluent", "excipient", and / or "carrier" have their ordinary and usual meaning as understood in the context of the present specification and are intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are those diluents, excipients, and / or carriers approved by federal, state, or other regulatory agencies or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, including humans and non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous solutions of dextrose and glycerol can be used 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.Physiologically acceptable carriers may also include 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, carbohydrates such as amino acids, glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation is typically suited to the mode of administration.

[0050] Cryoprotectants are cell composition additives to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cryopreservation medium that includes other components such as nutrients (e.g., albumin, serum, bovine serum, fetal calf serum, FCS) to increase the survival rate of cells after thawing. In these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, about, at least about, less than, or equal to 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 foregoing numbers.

[0051] Additional excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, 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, sugar, 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 residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of excipient may be found in the composition at, about, at least, at least about, less than, or equal to 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 within a range defined by any two of the foregoing numbers.

[0052] The term "pharmaceutically acceptable salts" has its common and ordinary meaning as understood in the context of this specification and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including, but not limited to, analgesics, 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 inorganic bases suitable for forming salts include 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, classes 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; amino acids, including glycine, arginine, and lysine; guanidine, N-methylglucosamine, N-methylglucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, trihydroxymethylaminoethane.

[0053] The appropriate formulation will vary depending on the route of administration selected. Techniques for the formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques for administering compounds exist in the art, including, but are not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intraarterial, intravenous), intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0054] As used herein, "carrier" has its common and ordinary meaning as understood in light of this specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or body organs.

[0055] As used herein, "diluent" has its common and usual meaning as understood in light of the present specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharma- ceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It can also be a liquid for dissolving a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate buffered saline, which mimics the composition of human blood.

[0056] The disclosure herein uses affirmative language to describe many embodiments, and the disclosure also includes embodiments in which subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is completely or partially excluded.

[0057] The term "w / w%" or "weight / weight %" as used herein has its ordinary and usual meaning as understood in the context of this specification and refers to a percentage expressed in terms of the weight of a component or agent relative to the total weight of the composition, multiplied by 100. The term "v / v%" or "volume / volume %" as used herein has its ordinary and usual meaning as understood in the context of this specification and refers to a percentage expressed in terms of the liquid volume of a compound, substance, component or agent relative to the total liquid volume of the composition, multiplied by 100.

[0058] stem cells The term "totipotent stem cells" (also known as omnipotent stem cells) as used herein has its common and ordinary meaning as understood in the context of this specification, and is a stem cell that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of building complete, viable organisms. These cells are produced from the fusion of egg and sperm cells. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0059] As used herein, the term "embryonic stem cell (ESC)", commonly abbreviated as ES cell, has its common and ordinary meaning as understood in the context of this specification and refers to a cell that is pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of this disclosure, the term "ESC" may be used broadly to encompass embryonic germ cells.

[0060] The term "pluripotent stem cell (PSC)" as used herein has its common and ordinary meaning as understood in the context of this specification and includes any cell that can differentiate into almost any cell type of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs may be the descendants of inner cell mass cells of a preimplantation blastocyst, or may be obtained by induction of non-pluripotent cells, e.g., adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0061] The term "induced pluripotent stem cell (iPSC)" as used herein, commonly abbreviated as iPS cell, has its common and ordinary meaning as understood in the context of this specification, and refers to a type of pluripotent stem cell artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing "forced" expression of certain genes. In theory, induced pluripotent stem cells can be reprogrammed from any type of adult somatic cell. Typically, somatic cells that are relatively easy to isolate are used to reprogram into iPSCs. For example, skin fibroblasts can be isolated from a subject by skin biopsy, and peripheral blood mononuclear cells can be isolated from the subject's peripheral blood. hiPSC refers to human iPSC. In some methods known in the art, iPSCs can be derived by transfection of certain stem cell-related genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes may also improve the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble morphologically and biochemically pluripotent stem cells and are typically isolated by morphological selection, doubling time, or 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, retroviral systems are used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, lentiviral systems are used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is 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 Sox15), certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof.

[0062] The term "progenitor cell" as used herein has its common and ordinary meaning as understood in light of the present specification and encompasses any cell that can be used in the methods described herein, during which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent stem cells (artificial or non-artificial), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, the progenitor cells can be derived from an embryo, an infant, a child, or an adult. In some embodiments, the progenitor cells can be somatic cells that are subjected to treatment to confer pluripotency via genetic engineering or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0063] In some embodiments, a step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of an early stage embryo, the blastocyst. Methods for deriving embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in this application, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0064] Additional stem cells that may be used in embodiments according to the present disclosure include, but are not limited to, those provided by or described in the National Stem Cell Bank (NSCB), databases hosted by the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC Cell Bank at the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, Calif.), Cellartis AB (Goteborg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion 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 may be used in embodiments according to the present disclosure include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (13), TE04 (14), TE06 (16), UCO1 (HSF1), UC06 (HSF6), WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0065] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" describes the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or modify the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0066] In some embodiments, adenoviruses can be used to deliver the four necessary genes, resulting in iPSCs that are virtually identical to embryonic stem cells. Adenoviruses do not combine their own genes with any of the targeted hosts, thus eliminating the risk of creating tumors. In some embodiments, non-viral based techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without any viral transfection system at all, albeit with very low efficiency. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modifications. In some embodiments, generation of mouse iPSCs is possible using a similar methodology: repeated treatment of cells with certain proteins delivered to the cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0067] The term "definitive endoderm" or "DE" as used herein has its common and usual meaning as understood in light of the present specification and refers to the developmental cell type that gives rise to the intestinal tract and the resulting gastrointestinal organs, including the esophagus, stomach, small intestine, colon, liver, and pancreas. The anterior DE forms the foregut and its associated organs, including the liver and pancreas, and the posterior DE forms the midgut and hindgut, which form the small and large intestines, and parts of the urogenital system. Markers of the DE include SOX17 and FOXA2. During development, Wnt and FGF signaling pathways establish regionalization between anterior and posterior patterning of the DE. Pluripotent stem cells can be differentiated into definitive endoderm by culturing the pluripotent stem cells with one or more transforming growth factor β (TGFβ) growth factor family members, such as activin A, activin B, or nodal. As previously investigated, definitive endoderm can be differentiated into three-dimensional organoid structures that resemble downstream gastrointestinal organs.

[0068] The term "feeder cells" as used herein has its common and usual meaning as understood in light of the present specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying them 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 radiation), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells are not necessarily 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, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, 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 forehead fibroblasts, human skin 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 instead of or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the expansion of target stem cells.

[0069] Intestinal and colonic organoids and methods for making them The intestinal and colonic organoids disclosed herein are produced by a differentiation process from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) or their intermediates (e.g., definitive endoderm), and include epithelial and mesenchymal cell types, as well as specialized cell types of the intestine or colon.Exemplary methods for making intestinal and colonic organoids can be found in U.S. Patent Nos. 9,719,068 and 10,174,289, and PCT International Publication Nos. WO2016 / 061464, WO2018 / 106628, WO2018 / 200481, WO2019 / 126626, WO2020 / 160371, and WO2021 / 030373, each of which is expressly incorporated herein by reference in its entirety.

[0070] In some embodiments, intestinal and colonic organoids are differentiated by culturing definitive endoderm cells.These definitive endoderm cells can be differentiated from pluripotent cells by contacting definitive endoderm with Nodal, Activin, and / or BMP subgroup of TGFβ superfamily of growth factors.In some embodiments, pluripotent stem cells are contacted with Nodal, Activin A, Activin B, BMP4, or any combination thereof to differentiate pluripotent stem cells into definitive endoderm.In some embodiments, pluripotent stem cells are contacted with Activin A to differentiate pluripotent stem cells into definitive endoderm.

[0071] The definitive endoderm can be further subjected to FGF / Wnt-induced posterior endoderm patterning towards hindgut specification.

[0072] In some embodiments, to produce intestinal and colonic organoids, first, definitive endoderm is contacted with Wnt signaling pathway activator and FGF signaling pathway activator to posteriorize definitive endoderm to hindgut endoderm.During this culture process, hindgut endoderm grows as a monolayer, but spontaneously buds as a cell aggregate called hindgut spheroid in suspension.In some embodiments, Wnt signaling pathway activator comprises Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16, or any combination thereof.In some embodiments, Wnt signaling pathway activator is Wnt3a. In some embodiments, the Wnt signaling pathway activator comprises a glycogen synthase kinase-3 (GSK3) inhibitor acting 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. The hindgut endoderm and hindgut spheroids produced contain CDX2+ polarized epithelium surrounded by CDX2+ mesenchyme and lack Alb and Pdx1, indicative of foregut endoderm.

[0073] After the formation of hindgut endoderm or hindgut spheroids, which can be engineered in suspension and embedded in basement membrane matrix (e.g., Matrigel) for three-dimensional culture, BMP signaling pathway regulates the formation of different regional types of intestine. Inhibition of BMP signaling after hindgut stage promotes proximal intestinal cell fate (duodenum / jejunum). Activation of BMP signaling after hindgut stage promotes more distal intestinal cell fate (cecum / colon). In some embodiments, hindgut endoderm is contacted with BMP signaling pathway activator to differentiate hindgut endoderm into intestinal organoid. In some embodiments, hindgut endoderm is contacted with BMP signaling pathway inhibitor to differentiate hindgut endoderm into colon 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.

[0074] Dissociated Organoid Composition Disclosed herein is a cell suspension comprising a dissociated cell population comprising epithelial and mesenchymal cell types. In some embodiments, the dissociated cell population is dissociated from intestinal and / or colon organoids, and the intestinal and / or colon organoids comprise epithelial and mesenchymal cell types. In some embodiments, the intestinal and / or colon organoids are derived from progenitor cells selected from embryonic stem cells, induced pluripotent stem cells, and definitive endoderm cells. In some embodiments, the cell suspension or the intestinal and / or colon organoids are allogeneic to the subject. In some embodiments, the cell suspension or the intestinal and / or colon organoids are derived from cells from the subject, and the intestinal and / or colon organoids are autologous to the subject. In some embodiments, the cell suspension or the intestinal and / or colon organoids are derived from induced pluripotent stem cells derived from cells isolated from the subject. In some embodiments, the dissociated cell population is prepared by enzymatic and / or mechanical dissociation of the intestinal and / or colon organoids. In some embodiments, enzymatic dissociation comprises dissociating intestinal and / or colonic organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof.In some embodiments, mechanical dissociation comprises passing intestinal and / or colonic organoids through successively narrower diameter channels.In some embodiments, dissociated cell population comprises MKI67+ proliferative cells. In some embodiments, the percentage of cells in the dissociated cell population that are mesenchymal cell types is, is about, is at least about, is less than, or is less than about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or is a percentage within a range defined by any two of the foregoing percentages, e.g., 10-50%, 40-80%, 70-95%, 85-95%, or 30-95%.In some embodiments, the percentage of cells in the dissociated cell population that are mesenchymal cell types is, is about, is at least about, is at most, is less than, or is less than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or any percentage within a range defined by any two of the foregoing percentages, e.g., 85-95%, 85-90%, 90-95%, or 88-92%. In some embodiments, the remaining percentage of cells in the dissociated cell population is comprised of epithelial cell types. In some embodiments, the percentage of cells in the dissociated cell population that are epithelial cell types is, is about, is at least about, is less than, or is less than about, 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, or is a percentage within a range defined by any two of the foregoing percentages, e.g., 0-75%, 0-25%, 0-15%, 5-25%, 5-15%, 10-50%, or 50-75%. In some embodiments, the percentage of cells in the dissociated cell population that are epithelial cell types is, is about, is at least about, is at most, is less than, or is less than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or any percentage within a range defined by any two of the foregoing percentages, e.g., 5-15%, 5-10%, 10-15%, or 8-12%. In some embodiments, the remaining percentage of cells in the dissociated cell population is comprised of mesenchymal cell types.

[0075] In some embodiments, the concentration of the dissociated cell population in the cell suspension is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 1011 or about, at least, at least about, or less than, or about, or less than, or any cell concentration within a range defined by any two of the foregoing concentrations, e.g., 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10 In some embodiments, the concentration of cells that are mesenchymal cell types in the dissociated cell population is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 or about, at least, at least about, or less than, or about, or less than, or any cell concentration within a range defined by any two of the foregoing concentrations, e.g., 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10 In some embodiments, the concentration of cells that are epithelial cell types in the dissociated cell population is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 or about, at least, at least about, or less than, or about, or less than, or any cell concentration within a range defined by any two of the foregoing concentrations, e.g., 10 5 ~10 11 , 10 5 ~10 8 , 10 9~10 11 , or 10 6 ~10 10 In cells / mL.

[0076] In some embodiments, the dissociated cell population is comprised of multicellular fragments that are, are about, are at least about, are less than, or are less than about, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or are any percentage within a range defined by any two of the foregoing percentages, e.g., 30-100%, 50-100%, 75-100%, 90-100%, 30-75%, or 50-95%, of the total cells in the dissociated cell population. In some embodiments, the dissociated cell population is in the form of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments. In some embodiments, the dissociated cell population is in the form of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments.

[0077] In some embodiments, the mesenchymal cell type of the dissociated cell population expresses vimentin (VIM) and / or elastin microfibril interfacer 1 (EMILIN1). In some embodiments, the epithelial cell type of the dissociated cell population expresses E-cadherin (CDH1) and / or caudal homeobox 2 (CDX2).

[0078] Also disclosed herein are pharmaceutical compositions comprising an effective amount of any of the cell suspensions and / or dissociated cell populations disclosed herein and at least one pharma- ceutically acceptable carrier, excipient, or diluent.

[0079] How to use In some embodiments, intestinal and colonic organoids, comprising epithelial and mesenchymal cell types, as disclosed herein or otherwise known in the art, are used in the methods of repairing intestinal damage disclosed herein.

[0080] As provided herein, the method is directed to treating intestinal injury. Treating intestinal injury includes restoring or alleviating the damaged intestinal tissue to a healthy state, or delaying, inhibiting, preventing, or arresting the progression or occurrence of intestinal injury. It also includes ameliorating one or more symptoms associated with intestinal injury. As used herein, intestinal injury may refer to the injured state of intestinal tissue, which may be, but is not necessarily, caused by mechanical and / or chemical insults, and may be, but is not necessarily, associated with the apoptotic and / or necrotic behavior of intestinal tissue. Other forms of intestinal injury and their symptoms are also envisioned.

[0081] Disclosed herein is a method for treating intestinal injury in a subject in need of such treatment. In some embodiments, the method comprises administering a dissociated cell population dissociated from intestinal and / or colonic organoid to the luminal wall of the intestine of the subject. In some embodiments, the dissociated cell population comprises epithelial cell type and mesenchymal cell type. In some embodiments, the mesenchymal cell type of the dissociated cell population expresses vimentin (VIM) and / or elastin microfibril interfacer 1 (EMILIN1). In some embodiments, the epithelial cell type of the dissociated cell population expresses E-cadherin (CDH1) and / or caudal homeobox 2 (CDX2). In some embodiments, the percentage of cells in the dissociated cell population that are mesenchymal cell types is, is about, is at least about, is less than, or is about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or is a percentage within a range defined by any two of the preceding percentages, e.g., 10-50%, 40-80%, 70-95%, 85%-95%, or 30-95%. In some embodiments, the percentage of cells in the dissociated cell population that are mesenchymal cell types is, is about, is at least about, is at most, is less than, or is less than about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or any percentage within a range defined by any two of the foregoing percentages, e.g., 85-95%, 85-90%, 90-95%, or 88-92%. In some embodiments, the remaining percentage of cells in the dissociated cell population is comprised of epithelial cell types.In some embodiments, the percentage of cells in the dissociated cell population that are epithelial cell types is, is about, is at least about, is less than, or is less than about, 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, or is a percentage within a range defined by any two of the foregoing percentages, e.g., 0-75%, 0-25%, 0-15%, 5-25%, 5-15%, 10-50%, or 50-75%. In some embodiments, the percentage of cells in the dissociated cell population that are epithelial cell types is, is about, is at least about, is at most ... 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 or about, at least, at least about, or less than, or about, or less than, or any cell concentration within a range defined by any two of the foregoing concentrations, e.g., 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10In some embodiments, the concentration of cells that are mesenchymal cell types in the dissociated cell population is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 or about, at least, at least about, or less than, or about, or less than, or any cell concentration within a range defined by any two of the foregoing concentrations, e.g., 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10 In some embodiments, the concentration of cells that are epithelial cell types in the dissociated cell population is 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 or about, at least, at least about, or less than, or about, or less than, or any cell concentration within a range defined by any two of the foregoing concentrations, e.g., 10 5 ~10 11 , 10 5 ~10 8 , 10 9 ~10 11 , or 10 6 ~10 10 In some embodiments, the intestine of a subject contemplated herein comprises the small intestine and / or colon. In some embodiments, administering the dissociated cell population to the luminal wall of the intestine of a subject comprises administering the cell population to a location of the lumen of the intestine affected by intestinal injury. In some embodiments, the location is directly adjacent to or near the intestine affected by intestinal injury. In some embodiments, the dissociated cell population is administered to the surface of the luminal wall.

[0082] In some embodiments of any of the methods disclosed herein, the dissociated cell population is administered to the luminal wall of the intestine of the subject as a cell suspension. In some embodiments, the suspension is in an isotonic solution, such as saline or lactated Ringer's solution. In some embodiments, administering the cell population to the luminal wall of the intestine of the subject comprises administering the cell population by a non-invasive or minimally invasive process. In some embodiments, the cell population is administered by an enteral catheter, a nasal catheter, or an enema. In some embodiments, the cell population is administered by direct intraluminal injection.

[0083] In some embodiments of any of the methods disclosed herein, the intestinal and / or colonic organoids are derived from progenitor cells selected from embryonic stem cells, induced pluripotent stem cells, and definitive endoderm cells. In the intestinal and / or colonic organoids, they are allogeneic to the subject. In some embodiments, the intestinal and / or colonic organoids are derived from cells isolated from the subject. In some embodiments, the intestinal and / or colonic organoids are autologous to the subject. In some embodiments, the intestinal and / or colonic organoids are derived from induced pluripotent stem cells derived from cells isolated from the subject. In some embodiments, the cells isolated from the subject can be any cells suitable for pluripotent reprogramming. Common cells suitable for pluripotent reprogramming used include skin fibroblasts or peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells isolated from the subject include skin fibroblasts or PBMCs from the subject.

[0084] In some embodiments of any of the methods disclosed herein, the dissociated cell population dissociated from intestinal and / or colonic organoids is prepared by enzymatic and / or mechanical dissociation of intestinal and / or colonic organoids.In some embodiments, the enzymatic dissociation comprises dissociating intestinal and / or colonic organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof, or any other cell dissociation enzyme or reagent otherwise known in the art.In some embodiments, the mechanical dissociation comprises passing intestinal and / or colonic organoids through a channel of successively narrower diameter.In some embodiments, the channel may be a needle, a microfluidic channel, a capillary tube, or a tube.In some embodiments, the channel of successively narrower diameter comprises 18 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, or 25 gauge channel, or any combination thereof. In some embodiments, the successively narrower diameter channels include 18 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, and 25 gauge channels, or a series of channels including or lacking any one, two, or three of the foregoing gauge channels. In some embodiments, the successively narrower diameter channels include and / or begin with an 18 gauge channel. In some embodiments, the successively narrower diameter channels include a 20 gauge channel. In some embodiments, the successively narrower diameter channels include and / or end with a 25 gauge channel. In some embodiments, the successively narrower diameter channels include, consist essentially of, or consist of 18 gauge, 20 gauge, and 25 gauge channels.

[0085] In some embodiments of any of the methods disclosed herein, the dissociated cell population dissociated from the intestinal and / or colon organoids is cultured at a concentration of 1000-15000 ng / mL for 1 mm of affected intestinal surface area. 2or 10,000 cells per mm, or within a range defined by any two of the foregoing values. 2 Any amount of cells per mm, e.g. 2 50-10,000 cells per 1 mm 2 50-5000 cells per 1mm 2 50-1000 cells per 1mm 2 5000-10000 cells per 1mm 2 2000-8000 cells per mm 2 The dissociated cell population is administered to the subject at a concentration of 500-5000 cells per 1000 cells. The affected intestinal surface area can be determined through conventional methods by those skilled in the art, for example, by macroscopically measuring either the outer or inner surface of the intestine, where the apparent increase in surface area due to villus processes can be ignored or taken into account. In some embodiments, the dissociated cell population is administered to the subject multiple times until amelioration of intestinal damage is observed. In some embodiments, the dissociated cell population is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or about, or at least about, or less than, or about.

[0086] In some embodiments of any of the methods disclosed herein, the cells of the dissociated cell population integrate into the mucosa and muscularis of the intestine of the subject. In some embodiments, the cells of the dissociated cell population integrated into the intestine of the subject maintain their intestinal and / or colonic regionality. In some embodiments, the cells of the dissociated cell population integrated into the intestine of the subject, or a subpopulation thereof, differentiate into smooth muscle actin (SMA) positive smooth muscle cell types. In some embodiments, the dissociated cell population includes proliferation marker KI67+ (MKI67+) proliferative cells that integrate into the intestine of the subject and promote healing of intestinal injury. In some embodiments, the dissociated cell population improves the intestinal barrier in the intestine of the subject after administration to the subject. In some embodiments, the dissociated cell population promotes the formation of an intact intestinal barrier in the intestine of the subject after administration to the subject. In some embodiments, the percentage of the dissociated cell population that incorporates into the intestine of a subject is, is about, is at least about, is at least about, or is a range defined by any two of the preceding values, e.g., 10-50%, 40-80%, 70-95%, or 30-95%. In some embodiments, the cells of the dissociated cell population are 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 120%, 122%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 140%, 141%, 142%, 1 %, 49%, or 50%, or about, at least, at least about, less than, or about less than, or any percentage of the surface area within a range defined by any two of the foregoing percentages, e.g., 10%-50% of the surface area, 10%-25% of the surface area, 25%-50% of the surface area, or 15%-35% of the surface area.In some embodiments, the percentage of the repaired intestinal tissue that is composed of cells from the dissociated cell population is, is about, is at least about, is less than, or is less than about, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or any percentage within a range defined by any two of the foregoing percentages, e.g., 50-99%, 50-75%, 50-60%, 75-99%, or 65-85%.

[0087] In some embodiments of any of the methods disclosed herein, the methods disclosed herein are used to treat intestinal injury in a subject in need thereof. In some embodiments, the intestinal injury comprises an intestinal ulcer. In some embodiments, the intestinal ulcer may be associated with leakage of blood or proteins into the lumen of the intestine. In some embodiments, the intestinal injury is chemical and / or mechanical. In some embodiments, the intestinal injury is associated with a gastrointestinal condition. In some embodiments, the gastrointestinal condition is selected from Crohn's disease, ulcerative colitis, intestinal disease associated with nonsteroidal anti-inflammatory drugs (NSAIDs) or other medications, radiation-induced enteropathy, and intestinal disease associated with a pathogenic infection such as tuberculosis.

[0088] In some embodiments of any of the methods disclosed herein, the intestinal and / or colon organoid or the dissociated cell population dissociated from the intestinal and / or colon organoid is mammalian.In some embodiments, the intestinal and / or colon organoid or the dissociated cell population dissociated from the intestinal and / or colon organoid is human.In some embodiments, the subject is a mammal.In some embodiments, the subject is human.

[0089] In some embodiments of any of the methods disclosed herein, the method further comprises generating intestinal and / or colonic organoids comprising epithelial and mesenchymal cell types, and / or dissociating the intestinal and / or colonic organoids to generate a dissociated cell population comprising epithelial and mesenchymal cell types. In some embodiments, the dissociated cell population is comprised of multicellular fragments that are, are about, are at least about, are less than, or are less than about, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or are any percentage within a range defined by any two of the foregoing percentages, e.g., 30-100%, 50-100%, 75-100%, 90-100%, 30-75%, or 50-95%, of the total cells in the dissociated cell population. In some embodiments, the dissociated cell population is in the form of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% multicellular fragments. In some embodiments, the dissociated cell population is in the form of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% multicellular fragments. In some embodiments, the intestinal and / or colonic organoids are produced from pluripotent stem cells. The intestinal and / or colonic organoids may be produced by the methods disclosed herein or otherwise known in the art.

[0090] Also disclosed herein are dissociated cell populations dissociated from intestinal and / or colon organoids, as described herein, for use in a method of treating a gastrointestinal disorder in a subject in need thereof, and pharmaceutical compositions thereof.

[0091] Some embodiments described herein relate to pharmaceutical compositions comprising, consisting essentially of, or consisting of an effective amount of a dissociated cell population or composition described herein and a pharma- ceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0092] Also disclosed herein is a cell suspension or pharmaceutical composition provided herein for use in treating intestinal injury. EXAMPLES

[0093] Some aspects of the embodiments discussed herein are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will appreciate that many other embodiments fall within the scope of the disclosure, as described and claimed herein.

[0094] Example 1. Administration of dissociated HIO for intestinal injury repair The generation of human intestinal organoids (HIOs) from pluripotent stem cells by manipulating growth factors to mimic early development and small intestinal specification has been described previously (Spence et al. Nature. (2011) 470:105-109; McCracken et al. Nat. Protoc. (2011) 6:1920-1928; Watson et al. Nat. Med. (2014) 20:1310-1314). Upon differentiation, HIOs give rise to a complex epithelial layer containing all major cell types as well as mesenchymal components that form functional laminated structures when transplanted into an immunocompromised host. Herein, the therapeutic potential of HIOs in vitro was evaluated. The H1 embryonic stem cell line, modified to constitutively express green fluorescent protein (GFP), was used to facilitate downstream xenograft analysis in preclinical models of injured intestine. However, it is envisioned that other pluripotent stem cells, such as other embryonic stem cells and induced pluripotent stem cells, may be used. It was confirmed that the constitutive expression of GFP in H1 cells does not interfere with their karyotype (Figure 3A). The modified cell lines were characterized prior to use and determined to pass quality control standards (Figures 3A-B). Immunocompromised Rag1 and Il2g knockout (RRG) rats were utilized as host subjects to surgically create blind segments of the distal small intestine, which were then subjected to chemical and mechanical injury during immediate reseeding with enteroids derived from dissociated HIOs or transplanted HIOs (Figure 1A). The surgical procedure performed resulted in a blind end-to-side "Y" segment or loop, which was tied with absorbable sutures so that the segment could drain distally into the host intestinal tract (Figure 4, panels A-E). The use of this absorbable suture resulted in a short window for HIO retention, epithelial restoration, and engraftment to occur. Subsequently, a more physiological condition of intestinal drainage and luminal content exposure occurred. The injury resulted in a segment of intestine that was primarily denuded of epithelium with exposed mesenchyme, as observed histologically and by topographic micrographs, when compared to healthy rat jejunum (Figure 5, panels A-B).Early death within 1 week was observed in some rats that underwent this procedure. In total, 68-70% of the rats survived until the 10-week postoperative time point (Figure 6A). This was likely due to the severity of injury in the loops, primarily caused by sepsis and unable to re-establish an effective epithelial barrier and translocation when exposed to luminal bacteria, as shown by veterinary necropsy.

[0095] At 10 weeks post-surgery, loops were harvested and evaluated (Figure 6B). In sham loops reseeded with medium alone, healing was observed histologically. Immunohistochemistry for the human-specific marker, Ku autoantigen, 80 kDa (KU80), was not detected in sham loops, indicating the absence of human cellular content (Figure 1H). The observed healing illustrated that the small intestine has an inherent ability to heal, and that epithelial restoration and re-establishment of barrier function can occur given time. However, in loops reseeded with dissociated GFP HIOs, areas expressing viable-GFP were observed, constituting an average of 16.93% of the loop's surface area (Figure 1B-C). Immunohistochemistry for KU80 revealed stable human cell incorporation within the dissected GFP+ areas of the reseeded loops (Figure 1D). KU80+ cells were observed throughout the entire thickness of the intestine. In loops reseeded with fragmented GFP enteroids, areas expressing viability-GFP were also observed, but they were significantly smaller, occupying on average only 1.68% of the loop surface area (Figure 1E-F). Again, human cell uptake was observed within the dissected GFP+ areas of loops reseeded with enteroid fragments, with stability reduced in accordance with viability-GFP expression observations (Figure 1G). Here, engraftment of KU80+ cells was restricted to the intestinal epithelium. In performing these experiments, we normalized the reseeded cell content to the total number of cells, rather than discriminating between epithelial cell numbers alone (Figure 10A-D). After comparing the efficacy of both HIOs and enteroids as a source of cell therapy, we continued to use only fragmented HIOs as a source of cells, due to their increased engraftment efficiency, their expanded contribution to intestinal recovery across all layers, and therefore their greater potential for clinical use.

[0096] To further demonstrate the extent of human cell contribution within loops reseeded with fragmented GFP HIOs, a tile scan was performed on a region of a GFP+ loop stained for the human marker KU80 (Figure 2A). Human cell contribution was observed within the mucosa and muscularis layers, which showed proper circular and longitudinal muscularis alignment (Figure 2B). Humanization of the muscularis layer was further highlighted by immunostaining for both GFP and actin alpha 2, smooth muscle (ACTA2), where fibers were shown to be perpendicular in both sham (loops reseeded with medium without cellular content) and reseeded loops, whereas the presence of GFP was only observed in the reseeded loops (Figure 2C). Clinically, this is an important feature as it broadens the application of potential cell therapy for conditions that result in damage beyond the mucosa (e.g., non-healing ulcers and fistulizing Crohn's disease).

[0097] The effect of engraftment of HIOs in the intestinal injury model on the enteric nervous system (ENS), which is responsible for various functions including host innervation and motility, was evaluated. Whether the myenteric plexus remained at the interface of two newly formed human muscle layers or whether it was replaced during the cell proliferation and remodeling process was evaluated. This was an important aspect to consider, since the effect on muscle layer humanization on the ENS when using epithelium-only or enteroid constructs, rather than the HIOs herein containing additional cell types including mesenchyme, was unknown. Immunostaining for the pan-neuronal marker tubulin beta 3 class III (TUBB3) confirmed that host innervation was not replaced after reconstitution with human cells, suggesting that its function was also preserved (Figure 2D).

[0098] Example 2. Reseeded HIOs undergo early engraftment during wound healing and maintain territoriality Since a significant contribution to intestinal healing by 10 weeks was observed by reseeded HIOs, additional experiments were performed at 7 days to evaluate earlier stages of engraftment. Immunohistochemistry for KU80 again revealed an absence of human cell contribution in sham loops, whereas human cell contribution was found in both epithelium and mesenchyme of reseeded loops at 7 days (Figure 7A). The level of epithelial structuring in sham loops was not as extensive as that observed in reseeded loops. Furthermore, immunostaining for the proliferation marker KI67 (MKI67) revealed that in sham surgical loops, the primary mechanism of healing at 7 days was due to migration with few cells that were actively cycling and MKI67+ (Figure 7B, left panel). However, in reseeded loops, MKI67+ cells were observed more extensively, indicating that HIO fragments play an active role during early engraftment and recovery of the intestine (Figure 7B, right panel).

[0099] We next determined by protein expression whether the regenerative process of engrafted HIOs results in late reconstitution of the epithelial stem / progenitor system for homeostatic regeneration. Immunostaining for MKI67 in the loops showed a familiar proliferative zonal distribution similar to human jejunum controls (Figure 7B). Expression of olfactomedin 4 (OLFM4), an anti-apoptotic protein associated with stem and progenitor cells in the intestine, was observed to be restricted within the crypt-like regions of the loops, similar to the expression pattern observed in human jejunum controls (Figure 7B). Subepithelial telocytes, an important source of niche signals to intestinal stem cells and epithelium, as marked by clotting factor III, tissue factor (F3), were also observed to be localized adjacent to the epithelium in the loops and human jejunum controls similarly (Figure 7B). Taken together, these data support the re-emergence of an epithelial stem cell compartment and niche in HIO-derived neoepithelium.

[0100] We then examined whether the intestinal fate of the HIO neoepithelium in the loops was preserved by protein expression. Immunostaining for the proximal region marker, GATA binding protein 4 (GATA4), revealed positive expression in the loops, similar to human jejunum controls (Fig. 8A, left panel). Paneth cells, specialized secretory cells localized in the small intestinal crypts, were detected as demonstrated by the expression of the antimicrobial peptide Defensin Alpha 5 (DEFA5), and were localized to crypt-like structures in the loops, as observed in human jejunum controls (Fig. 8A, center panel). Sucrase Isomaltase (S1), an enzyme involved in carbohydrate digestion in the small intestine, was also observed and localized to the brush border of the loops, as also seen in human jejunum controls (Fig. 8A, right panel). As further validation, we also investigated the expression of proteins restricted to the more distal small intestine and colon. Immunostaining for the distal region marker, DNA-binding protein SATB Homeobox 2 (SATB2), was not observed in the loops but was found to be present throughout the epithelium of human colonic controls (Figure 8B, left panel). Membrane Spanning 4-Domains A12 (MS4A12), a calcium channel localized primarily to the apical membrane of colonocytes, was not observed in the loops but was present throughout the epithelium of human colonic controls (Figure 8B, center panel). Mucin 5B (MUC5B), a gel-forming mucus throughout the colon, was also absent in the loops but was observed in goblet cells of human colonic controls (Figure 8B, right panel). Taken together, the positive protein expression of proximal intestinal markers along with the absence of distal markers suggests that the regionality of the HIO neoepithelium in the loops was maintained during engraftment and expansion of the HIO fragments.

[0101] Example 3. Engraftment of reseeded HIOs forms an intact epithelial barrier To gain functional insight, Ussing chamber assays were performed on the epithelium of viable-GFP-expressing loop regions and healthy rat jejunum 10 weeks postoperatively (proximal to the anastomosis site and outside the loop) (Figure 9A-B). The epithelium was exposed to a series of chemical stimuli while electrophysical properties were continuously recorded. Both the loop and rat jejunum epithelium were responsive to challenge with forskolin, 3-isobutyl-1-methylxanthine (IBMX), and bumetanide, as observed in real-time recordings (Figure 9C-D). The short-circuit current (I), a reflection of active ion transport, was measured. sc The changes in β-aminobutyric acid (mAb) and β-aminobutyric acid (mAb) were consistent with the known mode of action of each compound applied to both groups. This demonstrated that proper cyclic adenosine monophosphate regulation and function of ion channels in the neoepithelium was occurring. Furthermore, viable-GFP expressing areas of neoepithelium demonstrated higher sensitivity to both forskolin and IBMX when compared to healthy rat jejunum. Baseline readings of transepithelial electrical resistance (TEER) in rat jejunum were measured and observed within their established range (Figure 9F). Resistance values ​​without the muscularis and serosal layers ranged from 20 to 45 Ω for the rat small intestine. * cm 2 For the adult human small intestine, TEER values ​​have previously been shown to be between 50 and 100 Ω. * cm 2 It has been reported that TEER values ​​of the loops fall within the established TEER range for the adult human small intestine, whereas others were observed to be lower. This difference may be related to the known fetal state of the HIOs used for engraftment or the result of human epithelial interactions with the host luminal contents.

[0102] To further evaluate the integrity and stability of the neo-epithelium in the loops, paracellular permeability was assessed. Fluorescein isothiocyanate (FITC)-dextran was added to the apical chamber of each specimen, and samples were collected from the basolateral chamber every 30 min for evaluation of fluorescence intensity over time (Figure 9G). From this data set, FITC-dextran flux values ​​were calculated and no differences between groups were observed (Figure 9H). This indicates that the neo-epithelium in the loops established a sufficient barrier during the healing process.

[0103] Example 4. Biodistribution and proliferation potential of H1 cells We investigated the biodistribution and potential proliferation of H1 GFP cells in rats receiving fragmented HIO as cell therapy. Samples were collected from various off-target organs from rats 10 weeks post-surgery. Sections of brain, colon, heart, kidney, liver, lung, and small intestine were collected for histology and PCR. No gross abnormalities and tumor formation were observed in any of the harvested organs. When immunostaining for the human marker KU80 across serial sections spanning 2 mm tissue thickness, KU80+ cells were observed only in the transplanted HIO positive control (Figure 11A-D). To further investigate the presence of human cells, the gold standard of Alu-based real-time PCR was performed to distinguish human from rodent cells. To determine the significance threshold, titrations were performed against known human gDNA samples and related back to cell equivalents (Table 1). Experimental samples were then performed including the H1 GFP cell line and various off-target organs. No Ct data points were found below the Ct value threshold for the presence of human cells (FIG. 11B), suggesting little to no biodistribution of human cells within the experimental setting.

[0104] Example 4. Methodology Human Tissues: Collection of human tissues was performed with prior approval from the Institutional Review Board. Surgical samples of pathologically normal adult human small intestine and colon were obtained from patients aged 14-25 years undergoing bariatric or revision / resection procedures. Informed consent or assent was obtained from all patients and / or parents / legal guardians, as appropriate. Additional de-identified samples of pathologically normal colon were obtained via the Discover Together Biobank of Cincinnati Children's Hospital Medical Center. All human tissues were utilized in accordance with institutional ethical guidelines.

[0105] Rats: All animal procedures and experiments were performed with prior approval from the Institutional Animal Care and Use Committee. Both males and females were utilized for the experiments. Adult immunodeficient rats with Rag1 and Il2rg gene deletion (RRG), 3-6 months of age, were used for the mucosectomy experiments (founder, in-house breeding, Transgenesis Rat ImmunoPhenomic Platform, Nantes, France). Rats were housed in a barrier animal house 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 with water bottles supplemented with fluconazole (0.1 mg / mL, NorthStar Rx, LLC). Both food and water were provided ad libitum before and after surgery. 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 3 days post-operatively for signs of pain and distress and were given additional analgesia as needed.

[0106] Generation of H1-GFP cell line: CRISPR / Cas9 was used for introduction of green fluorescent protein (GFP) sequence into the AAVS1 safe harbor site of commercially available H1 human embryonic stem cells (hESCs, WiCell Research Institute, Inc.) using modified previously published reagents. Briefly, a single-stranded donor oligonucleotide (ssODN) encoding a validated guide RNA sequence (5'-GGGGCCACTAGGGACAGGAT-3', SEQ ID NO: 1) for targeting the AAVS1 locus was annealed and subcloned into PX458M-HF, a modified version of pSpCas9(BB)-2A-GFP (PX458M, Addgene#48138) generated by the Cincinnati Children's Medical Center Transgenic Core, which harbors an optimized single guide RNA. Transfections were performed on hESC-compatible Matrigel®-coated plates (Corning®) in mTeSR1® medium containing 10 μM Y-276323 using TransIT-LT1 transfection reagent according to the manufacturer's recommendations. Four hours after transfection, medium was removed and replaced with mouse embryonic fibroblast (MEF)-conditioned hESC medium (DMEM / F12, 20% knockout serum replacement (KOSR), 0.1 mM nonessential amino acid (NEAA), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng / mL bFGF) containing 10 μM Y-27632, and medium was replaced with MEF-conditioned hESC medium every day. Two days after transfection, a single cell suspension of cells was generated using Accutase® (StemCell Technologies®) and plated at approximately 10,000 cells / cm for Geneticin® (G418) selection. 2The cells were reseeded at a density of 100 μg / mL. Starting 3 days after transfection, G418 selection (100 μg / mL) was performed for 8 days, followed by daily feeding using mTeSR1. After 2 weeks, remaining G418-resistant colonies were picked using Accutase and seeded at cloning density in mTeSR® with CloneR® supplement (StemCell Technologies). Recovered clones were manually picked, grown in mTeSR1® medium, and subjected to genotyping. Precisely targeted insertion of the 2A-NeoR-CAG-GFP cassette at the AAVS1 locus was performed by PCR, Sanger sequencing, copy number analysis, and GFP expression. This cell line was used in all experiments. Monthly mycoplasma testing was performed on all cell cultures using the MycoAlert Plus Detection Kit and Control Set (Lonza#LT07-705 and LT07-518), with consistently negative results.

[0107] Generation of human intestinal organoids: Human intestinal organoids (HIOs) were generated and maintained as previously described (Watson et al. Nature Medicine (2014) 20: 1310-1314, Spence et al. Nature (2011) 470: 105-109, McCracken et al. Nature Protocols (2011) 6: 1920-1928). Briefly, H1-GFP cells were grown in feeder-free conditions in 6-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTeSR1 medium (StemCell Technologies). For definitive endoderm (DE) induction, cells were passaged as single cell suspensions generated using Accutase® (StemCell Technologies®) and seeded at a density of approximately 100,000 cells / well in 24-well Nunc® plates. Cells were grown in mTeSR1® medium for 2 days and then treated with 100ng / mL activin A for 3 days. DE were then treated with hindgut induction medium (RPMI1640, 100x NEAA, 2% dialyzed fetal calf serum (dFCS)) with 100ng / mL FGF4 (R&D Systems®) and 3μM CHIRON99021 (CHIR99021, Tocris®) to induce mid-hindgut spheroids. Spheroids were then seeded in Growth Factor Reduced (GFR) Matrigel® and maintained in Intestinal Growth Medium (Advanced DMEM / F12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng / mL EGF (R&D Systems) to generate HIOs. Medium was changed twice weekly and HIOs were reseeded in fresh Matrigel® on day 14. HIOs were utilized for surgical implantation between days 28-34.

[0108] Generation of enteroids from transplanted human intestinal organoids: Crypts were isolated from transplanted HIOs as previously described (40). Briefly, segments of HIO tissue were pinned onto SYLGARD184 (Dow)-coated Petri dishes, gently scraped to remove villi, washed with 2 mM chelation buffer, and then incubated in 2 mM chelation buffer for 30 min. The tissue was then gently scraped again to release the crypts. The chelation buffer containing the crypts was removed from the Petri dishes, filtered through a 150 μm 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 seeded onto Matrigel (Corning) and enteroids were generated using IntesiCult medium (STEMCELL Technologies). Medium was changed twice weekly and passages were performed every 7–10 days.

[0109] Mucosal resection surgical procedure: The mucosal resection procedure was optimized for our purposes from a previously published study (Avansino et al. Surgery (2006) 140:423-434).

[0110] Loop Creation: One day prior to the procedure, food was removed and rats were placed on GelDiet76A (ClearH2O) and continued for 7 days post-operatively before being returned to food. Rats were anesthetized with 2% inhaled isoflurane (Butler Schein) and their abdomens were shaved and prepared in a sterile fashion using swabs coated with isopropyl alcohol and povidone-iodine. An approximately 3 cm midline laparotomy was performed. A single dose of piperacillin and tazobactam (100 mg / kg) was administered intraperitoneally using an 18 G blunt fill needle attached to a 5 mL syringe. The cecum was then identified and the intestine was removed using saline warmed to 37°C to keep the tissue moist throughout the procedure. A suitable stretch of intestine (approximately 2-3 cm) was first identified for blind loop creation. To create the loop, the intestine and mesentery at the proximal end of what would become the loop were transfected using scissors and a bobby pen as necessary. The distal end of the blind loop was then partially cut using scissors. An anastomosis was performed in a simple knotted fashion using 7-0 silk suture (PERMA-HAND, Ethicon) between the open end of the fully transfected intestine and the partially cut distal end of the blind loop. This established complete continuity for the rat and maintained the distal connection of the blind loop.

[0111] Injury Creation: A bulldog clamp was applied to the distal end of the blind loop to block flow to the continuous intestine. First, chemical injury was induced. Using a 20 mL syringe with a cannula, the loop was flushed with saline warmed to 37° C. for 2 minutes. Using a 20 mL syringe with a cannula, the loop was then flushed with 1 mM dithiothreitol (DTT) in saline warmed to 37° C. for 2 minutes. The loop was then flushed again with warm saline, followed by a 10-minute flush with 5 mM isotonic ethylenediaminetetraacetic acid (EDTA) buffer warmed to 37° C. using a 20 mL syringe with a cannula. Approximately 50 mL of EDTA solution was used for the 10-minute flush. This series of flushes was repeated, and then a final flush was performed with saline warmed to 37° C. To induce mechanical injury, a dental interstitial brush flosser of appropriate diameter was inserted and removed three times with a slight twist during insertion and removal. After creation of the injury, the bulldog clamp was removed and the distal end of the blind loop was sutured using 4-0 absorbable ChromicGut suture (ETHILON, Ethicon).

[0112] Reseeding of loops: Loops were reseeded with medium without dissociated HIO or cellular contents. To fragment the constructs, HIOs were collected and pooled in their medium. HIOs were then drawn into a syringe fitted with an 18G blunt filled needle, the 18G needle was replaced with a 20G needle, and the contents of the syringe were expelled into a well of a 24-well plate. This process was repeated using successively smaller needles, terminating at 25G (Figure 10A). HIO fragments were then drawn into a 1 mL syringe with a cannula and placed into the prepared intestinal loop. The same process can also be performed with enteroids (Figure 10B). Figure 10C shows brightfield images of cells dissociated from HIOs or enteroids. The approximate number of cells constituting the original HIO or enteroid constructs used for dissociation is shown in Figure 10D. For reseeding with dissociated HIOs, approximately 100,000 cells were used per 4 mm of intestinal length. After reseeding, the proximal end of the loop was closed using 5-0 silk suture (PERMA-HAND, Ethicon) when the cannula was removed. The intestine was then carefully placed back into the abdominal cavity. To close the incision, the muscle was sutured in a continuous fashion using 4-0 coated absorbable suture (VICRYL RAPIDE, Ethicon). The skin was then closed again in a buried knot fashion 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 7 days postoperatively as an early time point during the healing process and 10 weeks postoperatively as a long-term recovery time point.

[0113] Quantification of HIO cells in vitro: Nuclei were isolated from HIOs using the Minute Detergent Free Nuclei Isolation Kit (Invent Biotechnologies, Inc.) according to the manufacturer's guidelines. To obtain sufficient cell quantities, constructs were pooled for isolation. Four day 28 (d28) HIOs were pooled for one round of nuclei isolation. Immediately after completion, nuclei were automatically quantified using a TC20 (Bio-Rad Laboratories, Inc.). The total number was then divided by four to obtain the number of cells per HIO.

[0114] Scanning electron microscopy: Segments of healthy and freshly injured rat jejunum were fixed overnight in 3% glutaraldehyde in 0.175M sodium cacodylate buffer pH 7.4. Samples were then rinsed in buffer and post-fixed in 1% osmium tetroxide in 0.175M cacodylate buffer for 1 h at 4°C. After rinsing in another buffer, samples were passed through a graded ethanol series (25%, 50%, 75%, 95%, 3x100%) for dehydration. Specimens were then critical point dried in an EM CPD300 (Leica®), stub mounted, and sputter coated with 60 / 40 gold / palladium to a thickness of 10 nm using an EM ACE600 (Leica®). Samples were imaged using a SU8010 transmission electron microscope (Hitachi®).

[0115] Tissue processing and immunostaining: Samples were harvested, fixed overnight in 4% paraformaldehyde (PFA), processed, and embedded in paraffin blocks. Sections were deparaffinized and either immediately stained with hematoxylin and eosin or subjected to antigen retrieval and antibody staining. Antibody incubations were performed overnight at 4°C in 1% bovine serum albumin in phosphate buffered saline (PBS). The antibodies and their respective dilutions are listed in Table 3. The Vectastain ABC system was used for amplification and a diaminobenzidine substrate kit was used for signal detection (Vector Laboratories®). Lilly-Meyer hematoxylin (Agilent Technologies®) was used as a counterstain. For biodistribution, serial sections were made and every 10th slide was stained over 2 mm tissue thickness.

[0116] [Table 1]

[0117] The titration of human DNA content relative to the Ct value and the equivalent amount of cells present used for tumorigenicity.

[0118] [Table 2]

[0119] Titration of human DNA content and their associated Ct values ​​in the H1 GFP cell line used for generation of HIOs. Quantification of human DNA content by PCR throughout major organs of rats undergoing a mucosal resection procedure with fragmented HIOs used as reseed material. Undetermined Ct values ​​are above 40 according to the sensitivity of the thermocycler. No values ​​were found that were below the determined Ct threshold of 29, therefore, no presence of human cells was found within the samples.

[0120] [Table 3]

[0121] [Table 4]

[0122] Image Acquisition: Surgical images were acquired using an M80 microscope equipped with an MC170HD camera (Leica Microsystems®). Overall images of harvested structures were acquired using a V40 ThinQ (LG Electronics®). Harvesting was performed using an M165FC microscope equipped with a DCF7000 T camera (Leica Microsystems®). Slides were imaged using an Eclipse Ti microscope (Nikon Corporation®) and subsequent analysis was performed using Nikon Element Imaging Software (Nikon Corporation®).

[0123] Ex vivo epithelial characteristics and permeability: Epithelium from freshly harvested healthy jejunum and reseeded loops was carefully dissected using a technique similar to seromuscular stripping as previously reported (Clarke, Am. J. Physiol. J. Gastrointest. Liver Physiol. (2009) 296:G1151-1166; Giles et al. Nat. Med. (2017) 23:829-838; Poling et al. Nat. Biomed. Eng. (2018) 2:429-442). Samples were opened and dissected in ice-cold Krebs buffer (117 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, 11 mM glucose). The full thickness tissue segments were then pinned into a dish containing hardened Sylgard (Electron Microscopy Sciences). The reseeded loops were confirmed to be GFP positive prior to further dissection and use in Ussing chamber assays. The seromuscular tissue was then microdissected from the epithelium using Dumont #5 and #7 forceps along with Vannas scissors (Fine Science Tools, Inc.). Overall tissue integrity was assessed using stereoscope bottom illumination for uniformity of appearance and any damaged areas were removed. After dissection, some residual subepithelial mucosa remained. With minimal handling, the central portion of the epithelium was then positioned for attachment between the hemi-chambers of an Ussing apparatus (Physiologic Instruments). 0.031 cm 2 The tissues were exposed to 5 mL of oxygenated Krebs buffer at 37°C throughout the assay. The transepithelial potential difference was detected with two pairs of electrodes fixed to a salt bridge containing 3.75% agar in 3 M KCl. The electrodes were connected to a VVC MC8 voltage clamp amplifier (Physiologic Instruments). The electrode potential difference and fluid resistance values ​​were corrected to zero just before the slider was attached between the chambers. Equilibration was allowed to establish for 30 min. The tissues were then voltage clamped at 0 mV and the short circuit current (I scChemical stimuli (10 μM forskolin, 100 μM IBMX, and 100 μM bumetanide) were applied while continuously measuring the concentration of ATP. For FITC-dextran permeability, 2.2 mg / mL FITC-dextran was added to the apical side and samples were taken from the basolateral side every 30 min for 3 h, replacing an equal amount of fresh modified Krebs buffer on the basolateral side to maintain pressure across the sample. Once all aqueous samples were collected, they were quantified using a plate reader (Synergy2, BioTek).

[0124] Human-specific Alu PCR primers and probe: Detection of human DNA was performed using primers and probes previously described. Briefly, Alu PCR was performed on gDNA extracted from various organs from reseeded mucosectomized rats and the H1 GFP cell line. The forward primer was designed to anneal upstream of the human-specific Alu sequence (5'-TGGTGGCTCTCTCCTGTAAT-3', SEQ ID NO:2) and the reverse primer was designed to anneal primarily within the human-specific Alu sequence (5'-GATCTCGGCTCACTGCAAC-3', SEQ ID NO:3), resulting in a 96 base pair amplicon. The probe was designed to bind between the two primers (5'-TGAGGCAGGAGAATCGCTTGAACC-3', SEQ ID NO:4) quencher-MGB-6FAM upstream of the hAlu-specific sequence. Primers and probes were custom ordered from Integrated DNA Technologies.

[0125] Alu PCR: Quantitative real-time PCR was performed on 200 ng of target template gDNA using TaqMan Universal PCR Master Mix (Applied Biosystems). Each sample was sequenced in triplicate using a OneStep thermocycler (Applied Biosystems). A standard curve was generated by adding 10-fold serial dilutions (200 ng to 0 ng) of hDNA (Millipore Sigma) and H1 GFP cells to each PCR plate. The crossing threshold (Ct) values ​​for the presence of human cells were calculated based on the standard values ​​using QunatoStudio software (Applied Biosystems).

[0126] Data representation, statistics, and reproducibility: For bar growth, data are presented as mean ± standard deviation, and all individual data points are represented. For violin plots, dashed lines indicate the mean and interquartile range, and all individual data points are represented. For statistics comparing two groups of paired data, the Wilcoxon signed rank test was performed. For statistics comparing unpaired data, the Student's t test was performed. The cutoff for statistical significance was p<0.05, with confidence intervals of 95%.

[0127] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the present subject matter, as defined by the appended claims.

[0128] With respect to the use of substantially all plural and / or singular terms herein, those of skill in the art may translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, the various singular / plural permutations may be expressly set forth herein.

[0129] Those of skill in the art will understand that the terms used in this specification, in general, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" is typically interpreted as "including but not limited to," the term "having" is typically interpreted as "having at least," the term "include" is typically interpreted as "including but not limited to," etc.). Those of skill in the art will further understand that, where a specific number of introduced claim recitations are intended, such intent will be expressly set forth in the claims, and, in the absence of such a recitation, no such intent is present. For example, as an aid to understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitations. However, the use of such phrases is typically interpreted as meaning that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to an embodiment that includes only one such recitation, even when the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" are typically interpreted to mean "at least one" or "one or more"), and the same applies to the use of a definite article used to introduce a claim recitation. In addition, those skilled in the art will recognize that even if a particular number of introduced claim recitations is explicitly recited, such recitation is typically interpreted to mean at least the recited number (e.g., the mere recitation of "two recitations" without other modifiers means at least two recitations or more than two recitations).Furthermore, where a convention similar to "such as at least one of A, B, and C" is used, such syntax generally is intended to mean what one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where a convention similar to "such as at least one of A, B, or C" is used, such syntax generally is intended to mean what one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, will typically be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0130] In addition, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0131] As would be understood by one of ordinary skill in the art, for all purposes, including in terms of being written, all ranges disclosed herein encompass all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least 2, 3, 4, 5, 10, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As would be understood by one of ordinary skill in the art, all words such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the numbers recited and that may be subsequently broken down into subranges as discussed herein. Finally, as would be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5 items, and so forth.

[0132] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0133] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification supersedes and / or is intended to take precedence over such conflicting material.

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Claims

1. 1. A dissociated cell population dissociated from intestinal and / or colon organoids for use in a method for treating intestinal damage in a subject in need thereof, wherein the cell population dissociated from the intestinal and / or colon organoids is administered to the luminal wall of the intestine of the subject, and the dissociated cell population comprises epithelial and mesenchymal cell types, and wherein the intestine of the subject comprises the small intestine and / or colon.

2. 2. The dissociated cell population of claim 1, wherein the dissociated cell population is administered to a location of the lumen of the intestine affected by the intestinal injury, optionally the location is directly adjacent to or near the intestine affected by the intestinal injury, and optionally administered to a surface of the luminal wall, and optionally wherein administration of the dissociated cell population to the luminal wall of the intestine of the subject is by enteral catheter, nasal catheter, or enema.

3. 3. The dissociated cell population of claim 1 or 2, wherein the dissociated cell population is administered to the luminal wall of the intestine of the subject as a cell suspension.

4. 3. The dissociated cell population of claim 1 or 2, wherein the intestinal and / or colonic organoids are derived from progenitor cells selected from embryonic stem cells, induced pluripotent stem cells, and definitive endoderm cells.

5. 3. The dissociated cell population of claim 1 or 2, wherein the intestinal and / or colon organoids are allogeneic to the subject.

6. 3. The dissociated cell population of claim 1 or 2, wherein the intestinal and / or colon organoids are derived from cells isolated from the subject, wherein the intestinal and / or colon organoids are autologous to the subject, and optionally the intestinal and / or colon organoids are derived from induced pluripotent stem cells derived from the cells isolated from the subject, and optionally the cells isolated from the subject comprise skin fibroblasts or peripheral blood mononuclear cells (PBMCs) from the subject.

7. 3. The dissociated cell population of claim 1 or 2, wherein the dissociated cell population is prepared by enzymatic and / or mechanical dissociation of the intestinal and / or colon organoids, optionally wherein the enzymatic dissociation comprises dissociating the intestinal and / or colon organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof, and / or optionally wherein the mechanical dissociation comprises passing the intestinal and / or colon organoids through channels of successively narrower diameters.

8. 3. The dissociated cell population of claim 1 or 2, wherein the percentage of cells in said dissociated cell population that are mesenchymal cell type is, or is about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or any percentage within a range defined by any two of the foregoing percentages; and / or optionally the percentage of cells in said dissociated cell population that are epithelial cell type is, or is about, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or any percentage within a range defined by any two of the foregoing percentages.

9. The dissociated cell population is measured over 1 mm of the affected intestinal surface area. ２ a concentration that is at or about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 cells per mm, or within a range defined by any two of the foregoing values ２ The dissociated cell population of claim 1 or 2, administered in any amount of cells per 100 ml.

10. 3. The dissociated cell population of claim 1 or 2, wherein the dissociated cell population is administered to the subject multiple times until improvement of the intestinal damage is observed, optionally the dissociated cell population is administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

11. 3. The dissociated cell population of claim 1 or 2, wherein cells of the dissociated cell population integrate into the mucosa and muscularis of the intestine of the subject, optionally wherein the cells of the dissociated cell population integrated into the intestine of the subject maintain their intestinal and / or colonic regionality, and optionally wherein the cells of the dissociated cell population integrated into the intestine of the subject, or a subpopulation thereof, differentiate into smooth muscle actin (SMA) positive smooth muscle cell types.

12. 3. The dissociated cell population of claim 1 or 2, wherein the dissociated cell population comprises proliferation marker KI67+ (MKI67+) proliferative cells that are incorporated into the intestine of the subject and promote healing of the intestinal injury, and / or the dissociated cell population promotes the formation of an intact intestinal barrier after administration.

13. 3. The dissociated cell population of claim 1 or 2, wherein the dissociated cell population incorporates at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the surface area of ​​the luminal wall of the intestine of the subject affected by the intestinal injury, or any percentage within a range defined by any two of the foregoing percentages.

14. 3. The dissociated cell population of claim 1 or 2, wherein the intestinal damage comprises an intestinal ulcer, and / or the intestinal damage is chemical and / or mechanical, and / or the intestinal damage is associated with a gastrointestinal disease, optionally selected from Crohn's disease, ulcerative colitis, enteropathy associated with nonsteroidal anti-inflammatory drugs (NSAIDs) or other medications, radiation-induced enteropathy, and enteropathy associated with a pathogenic infection such as tuberculosis.

15. 3. The dissociated cell population of claim 1 or 2, wherein the intestinal and / or colon organoids are mammalian, optionally the intestinal and / or colon organoids are human, and / or the subject is a mammal, optionally the subject is human.

16. The dissociated cell population of claim 1 or 2, wherein the dissociated cell population is produced by dissociating the intestinal and / or colonic organoids, and optionally the dissociated cell population is in the form of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments, or is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% multicellular fragments.

17. 3. The dissociated cell population of claim 1 or 2, further comprising producing said intestinal and / or colonic organoids in vitro, optionally from pluripotent stem cells.

18. A cell suspension comprising a dissociated cell population comprising epithelial and mesenchymal cell types.

19. 19. The cell suspension of claim 18, wherein the mesenchymal cell type expresses vimentin (VIM) and / or elastin microfibril interfacer 1 (EMILIN1) and the epithelial cell type expresses E-cadherin (CDH1) and / or caudal homeobox 2 (CDX2), and optionally the dissociated cell population comprises MKI67+ proliferative cells.

20. 20. The cell suspension of claim 18 or 19, wherein the dissociated cell population is dissociated from intestinal and / or colonic organoids, the intestinal and / or colonic organoids comprising epithelial and mesenchymal cell types, and optionally the intestinal and / or colonic organoids are derived from progenitor cells selected from embryonic stem cells, induced pluripotent stem cells, and definitive endoderm cells.

21. 20. The cell suspension of claim 18 or 19, wherein the cell suspension or the intestinal and / or colon organoids are allogeneic to the subject.

22. 20. The cell suspension of claim 18 or 19, wherein the cell suspension or the intestinal and / or colon organoids are derived from cells from a subject, and the intestinal and / or colon organoids are autologous to the subject, and optionally the cell suspension or the intestinal and / or colon organoids are derived from induced pluripotent stem cells derived from the cells isolated from the subject.

23. 20. The cell suspension of claim 18 or 19, wherein the dissociated cell population is prepared by enzymatic and / or mechanical dissociation of the intestinal and / or colon organoids, and optionally, the enzymatic dissociation comprises dissociating the intestinal and / or colon organoids with trypsin, chymotrypsin, collagenase, papain, hyaluronidase, elastase, thermolysin, neutral protease, or any combination thereof, and / or the mechanical dissociation comprises passing the intestinal and / or colon organoids through channels of successively narrower diameters.

24. 20. The cell suspension of claim 18 or 19, wherein the percentage of cells in the dissociated cell population that are mesenchymal cell type is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or a percentage within a range defined by any two of the foregoing percentages, and / or the percentage of cells in the dissociated cell population that are epithelial cell type is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or a percentage within a range defined by any two of the foregoing percentages.

25. The concentration of the dissociated cell population in the cell suspension is about 10 ５ , 10 ６ , 10 ７ , 10 ８ , 10 ９ , 10 １０ , or 10 １１ 20. The cell suspension of claim 18 or 19, wherein the concentration of cells of mesenchymal cell type in the dissociated cell population is about 10, 10, 10, 10, 10, 10, 10, 10, or 10 cells / mL, or any cell concentration within a range defined by any two of the aforementioned concentrations, and / or the dissociated cell population is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in the form of multicellular fragments, or at least such multicellular fragments, or any percentage within a range defined by any two of the aforementioned percentages.

26. 20. A pharmaceutical composition comprising an effective amount of the cell suspension of claim 18 or 19 and at least one pharmaceutically acceptable carrier, excipient, or diluent.

27. 20. A cell suspension according to claim 18 or 19 for use in the treatment of intestinal damage.

28. The pharmaceutical composition of claim 26 for use in the treatment of intestinal damage.

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