Organoid Recombination

Abstract

Disclosed herein is the organoid composition that has heterogeneous combination of epithelial and mesenchymal components, and the method for producing it by dissociating and combining the epithelial and mesenchymal components from different sources.These epithelial and mesenchymal components can be derived from the same or different cell types or organoid types.These organoid compositions can exhibit advantageous properties, such as enhanced in vivo engraftment.

Description

[Technical Field]

[0001] Statement on federally sponsored research and development This invention was made with government support under DK103117, awarded by the National Institutes of Health. The government has certain rights to this invention. The technical field to which the invention pertains.

[0002] The aspects of this disclosure generally relate to organoid compositions and methods for producing them, which include the dissociation and combination of epithelial and mesenchymal components of cell compositions such as organoids. [Background technology]

[0003] Organoids, particularly those derived from pluripotent stem cells, closely resemble in vivo tissues and have been shown to have great potential in many applications, including drug screening, transplantation, and personalized medicine. However, organoids produced by existing methods are still limited compared to in vivo tissues in certain characteristics, such as the epithelial-to-mesenchymal ratio observed in differentiated organoids. The applicant's initial research identified a method that enables pluripotent stem cells to differentiate into endoderm and mesenchyme, supporting in vivo engraftment. These patterned structures reflect the proximal small intestine and are called human intestinal organoids (HIOs). Recent methods for patterning the foregut (e.g., human gastric organoids (HGOs)) and hindgut (e.g., human colonic organoids (HCOs)) in vitro result in heterogeneity in the epithelial-to-mesenchymal ratio. Organoids with improved epithelial / mesenchymal ratios and morphologies, as well as methods for producing them, are now needed. The following are prior art documents related to the invention of this application (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries): (Prior art document) (Patent Document) (Patent Document 1) International Publication No. 2020 / 023245 (Patent Document 2) International Publication No. 2019 / 126626 (Non-patent literature) (Non-patent document 1) CHUA et al.,Single luminal epithelial progenitors can generate prostate organoids in culture,Nat Cell Biol.October 2014,Vol 16,No 10,pp 951-4.Entire document,especially abstract; p.951,col 2,para 3 to p.952,col 1,para 2; p.952,col 2,para 6 [Overview of the project]

[0004] One aspect of the present disclosure is a method for producing a composite organoid. In some embodiments, the method comprises obtaining mono-dissociated mesenchymal cells isolated from one or more organoids; obtaining epithelial structures isolated from organoids or enteroids; combining the mono-dissociated mesenchymal cells and epithelial structures; and culturing the combined mono-dissociated mesenchymal cells and epithelial structures to form a composite organoid. In some embodiments, the mono-dissociated mesenchymal cells, or the epithelial structures, or both, are isolated by mechanical dissociation and filtration. In some embodiments, the mono-dissociated mesenchymal cells and epithelial structures are combined by centrifugation. In some embodiments, the number of mesenchymal cells in the composite organoid is greater than the original number of mesenchymal cells in the organoid or enteroid from which the epithelial structures are isolated, and as a result, the composite organoid has concentrated mesenchyme. In some embodiments, 1) one or more organoids from which a single dissociated mesenchymal cell is isolated, and 2) organoids or enteroids from which epithelial structures are isolated each include a tissue type selected from esophageal, gastric, liver, intestinal, or colonic tissue types, or any combination thereof. In some embodiments, the tissue types of one or more organoids and the tissue types of organoids or enteroids are different. In some embodiments, unlike the tissue types of one or more organoids from which a single dissociated mesenchymal cell is isolated and the tissue types of organoids or enteroids from which epithelial structures are isolated, repatterning of the epithelial structures by the single dissociated mesenchymal cell does not occur, and as a result, the epithelium of the composite organoid maintains the tissue type of the organoid or enteroid from which the epithelial structures are isolated. In some embodiments, if the epithelial structure is derived from organoids that are at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or from organoids that are 14-30, 15-30, 18-30, 15-20, or 15-25 days old, repatterning of the epithelial structure does not occur. In some embodiments, if the epithelial structure is derived from enteroids, repatterning of the epithelial structure does not occur. In some embodiments, the enteroids are derived from adult tissue.In some embodiments, the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated differs from the histological type of organoid or enteroid from which the epithelial structure is isolated. Re-patterning of the epithelial structure by the single dissociated mesenchymal cell occurs, and as a result, the epithelium of the composite organoid exhibits the characteristics of the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated. In some embodiments, re-patterning of the epithelial structure by the single dissociated mesenchymal cell can occur when the epithelial structure is derived from organoids aged 8, 9, 10, 11, 12, or 13 days or younger, or from organoids aged 8-13, 8-10, or 10-13 days. In some embodiments, the enteroids are derived from adult tissue with distinct patterning, and the epithelial structure derived from the enteroids maintains its histological type even when recombined with a single dissociated mesenchymal cell. In some embodiments, the histological type of one or more organoids and the histological type of organoid or enteroid are the same. In some embodiments, one or more organoids and each organoid or enteroid contains only one of the esophageal, gastric, liver, intestinal, or colonic tissue types, and the resulting composite organoid is an homogeneous organoid containing one tissue type. In some embodiments, one or more organoids are derived from pluripotent stem cells (PSCs) from a first subject, and the organoid or enteroid is derived from PSCs from a second subject or isolated from gastrointestinal tissue from a second subject. In some embodiments, the first and second subjects are mammals. In some embodiments, the first and second subjects are humans. In some embodiments, the first and second subjects are the same individual.

[0005] In any embodiment of the methods disclosed herein, the method further includes implanting a composite organoid into a recipient subject. In some embodiments, the recipient subject is a first subject or a second subject. In some embodiments, the composite organoid exhibits greater engraftment and growth in the recipient subject compared to an equivalent non-composite organoid or enteroid.

[0006] Composite organoids are also disclosed herein. A composite organoid comprises mesenchyme, which includes mesenchymal cells isolated as single dissociated cells from a first organoid, and epithelium, which includes epithelial cells isolated as an epithelial structure from a second organoid or enteroid. In some embodiments, the ratio of mesenchymal cells to epithelial cells in the composite organoid is greater than the ratio in the second organoid or enteroid. In some embodiments, the first organoid and the second organoid or enteroid each include a tissue type selected from esophageal, gastric, liver, intestinal, or colonic tissue types, or any combination thereof. In some embodiments, the tissue types of the first organoid and the second organoid or enteroid are the same. In some embodiments, the tissue types of the first organoid and the second organoid or enteroid are different. In some embodiments, the composite organoid is an organoid produced by any one of the methods described herein.

[0007] Also disclosed herein are composite organoids comprising a mesenchyme containing a first tissue type selected from the esophageal, gastric, hepatic, intestinal, or colonic tissue type, or any combination thereof, and an epithelium containing a second tissue type selected from the esophageal, gastric, hepatic, intestinal, or colonic tissue type, or any combination thereof. In some embodiments, the first and second tissue types have at least one difference between them.

[0008] The use of any one of the organoids disclosed herein for treating gastrointestinal disorders in subjects requiring such treatment is also disclosed herein.

[0009] Also disclosed herein is a method of screening a candidate therapeutic agent, comprising contacting any one of the organoids disclosed herein with the candidate therapeutic agent, and determining an effect of the candidate therapeutic agent on the organoid.

[0010] Embodiments of the present disclosure provided herein are described by the following numbered alternatives.

[0011] 1. A method of producing a composite gastrointestinal organoid, comprising: a) isolating singly dissociated mesenchymal cells from one or more gastrointestinal organoids of a first tissue type; b) isolating an epithelial structure from a gastrointestinal organoid or enteroid of a second tissue type; c) combining the singly dissociated mesenchymal cells and the epithelial structure; d) culturing the combined singly dissociated mesenchymal cells and epithelial structure to form the composite gastrointestinal organoid, wherein the number of the singly dissociated mesenchymal cells is greater than the original number of mesenchymal cells in the gastrointestinal organoid of the second type, and the composite gastrointestinal organoid has an enriched mesenchyme.

[0012] 2. The method of Alternative 1, wherein the singly dissociated mesenchymal cells, the epithelial structure, or both are isolated by mechanical dissociation and filtration.

[0013] 3. The method according to any one of the preceding alternatives, wherein the singly dissociated mesenchymal cells and the epithelial structure are combined by centrifugation.

[0014] 4. The method according to any one of the preceding alternatives, wherein the first tissue type and the second tissue type are each independently selected from the group of tissue types consisting of esophagus, stomach, liver, intestine, colon, or a tissue type that is any combination of any of the foregoing.

[0015] 5. The method according to any one of the aforementioned alternatives, wherein the first and second histological types are the same, and the resulting composite gastrointestinal organoid histological type is homogeneous.

[0016] 6. The method according to any one of Alternatives 1 to 4, wherein the first and second histological types are different, and the resulting composite gastrointestinal organoid histological type is heterogeneous.

[0017] 7. The method according to any one of the above alternatives, wherein one or more gastrointestinal organoids of type 1 are derived from pluripotent stem cells (PSCs) from a first subject, and the gastrointestinal organoids or enteroids are derived from PSCs from a second subject or isolated from gastrointestinal tissue from a second subject.

[0018] 8. The method of Alternative 7, wherein the first and second subjects are mammals.

[0019] 9. The method of alternative 7 or 8, wherein the first and second subjects are human.

[0020] 10. The method according to any one of alternatives 7-9, wherein the first subject and the second subject are the same individual.

[0021] 11. A method according to any one of the above alternatives, further comprising transplanting a composite gastrointestinal organoid into a recipient.

[0022] 12. The method of alternative 11, wherein the recipient is either the first or second subject.

[0023] 13. The method according to Alternative 11 or 12, wherein the composite gastrointestinal organoid exhibits greater engraftment and growth in the recipient subject compared to an equivalent non-composite organoid of one or more gastrointestinal organoids of type 1, or an equivalent non-composite organoid or enteroid of a gastrointestinal organoid or enteroid of type 2, or both.

[0024] 14. A composite gastrointestinal organoid, Mesenchyme containing cells from the first source of gastrointestinal organoids, A second source of epithelium from gastrointestinal organoids or enteroids, A method wherein the ratio of mesenchymal cells to epithelial cells in a composite gastrointestinal organoid is greater than the ratio in a second type of organoid or enteroid, and / or the histological type of the first source is different from that of the second source.

[0025] 15. The gastrointestinal organoid according to Alternative 14, wherein the first gastrointestinal type and the second gastrointestinal type each include an esophageal type, a gastric type, a hepatic type, an intestinal type, or a colonic type, or any combination thereof.

[0026] 16. The gastrointestinal organoid according to alternative 14 or 15, wherein the first gastrointestinal type and the second gastrointestinal type are the same, and the composite gastrointestinal organoid is a homogeneous organoid.

[0027] 17. The gastrointestinal organoid according to alternative 14 or 15, wherein the first gastrointestinal type and the second gastrointestinal type are different, and the composite gastrointestinal organoid is a heterogeneous organoid.

[0028] 18. A composite gastrointestinal organoid produced by the method described in any one of Alternatives 1-13.

[0029] 19. A gastrointestinal organoid described in any one of Alternatives 14-18, for use in treating a gastrointestinal disorder in a patient requiring treatment.

[0030] 20. A method for producing a composite organoid, a) Isolating a single dissociated mesenchymal cell from one or more organoids, b) Isolating epithelial structures from organoids or enteroids, c) Combining a single dissociated mesenchymal cell with an epithelial structure, d) A method comprising culturing combined single dissociated mesenchymal cells and epithelial structures to form a composite organoid.

[0031] 21. The method according to alternative 20, wherein a single dissociated mesenchymal cell, or an epithelial structure, or both, is isolated by mechanical dissociation and filtration.

[0032] 22. A method according to one of the above alternatives, wherein a single dissociated mesenchymal cell and epithelial structure are combined by centrifugation.

[0033] 23. The method according to any one of the aforementioned alternatives, wherein the number of mesenchymal cells in the composite organoid is greater than the original number of organoids or enteroids from which the epithelial structure is isolated, and as a result the composite organoid has concentrated mesenchyme.

[0034] 24. The method according to any one of the above alternatives, wherein one or more organoids from which a single dissociated mesenchymal cell is isolated, and the organoid or enteroid from which an epithelial structure is isolated, each include a histological type selected from the esophageal, gastric, hepatic, intestinal, or colonic histological type, or any combination thereof.

[0035] 25. The method according to alternative 24, wherein one or more organoid tissue types and organoid or enteroid tissue types are different.

[0036] 26. The method according to alternative 24, wherein one or more organoid tissue types and organoid or enteroid tissue types are the same.

[0037] 27. The method according to Alternative 26, wherein one or more organoids and organoids or enteroids each contain only one of the esophageal, gastric, liver, intestinal, or colonic tissue types, and the resulting composite organoid is an allogeneic organoid containing one tissue type.

[0038] 28. The method according to any one of the above alternatives, wherein one or more organoids are derived from pluripotent stem cells (PSCs) from a first subject, and the organoids or enteroids are derived from PSCs from a second subject or isolated from gastrointestinal tissue from a second subject.

[0039] 29. The method of alternative 28, wherein the first and second subjects are mammals.

[0040] 30. The method of alternative 28 or 29, wherein the first and second subjects are human.

[0041] 31. The method according to any one of alternatives 28-30, wherein the first subject and the second subject are the same individual.

[0042] 32. A method according to any one of the above alternatives, further comprising transplanting a composite organoid into a recipient.

[0043] 33. The method according to alternative 32, wherein the recipient is either the first or second target.

[0044] 34. The method according to alternative 32 or 33, wherein the composite organoid exhibits greater engraftment and growth in the recipient compared to an equivalent non-composite organoid or enteroid.

[0045] 35. A composite organoid, A mesenchyme containing a single dissociated mesenchymal cell isolated from the first organoid, A composite organoid comprising epithelium containing an epithelial structure isolated from a second organoid or enteroid.

[0046] 36. The organoid according to Alternative 35, wherein the ratio of single dissociated mesenchymal cells to epithelial cells in the composite organoid is greater than the ratio in the second organoid or enteroid.

[0047] 37. The organoid according to alternative 35 or 36, wherein the first organoid and the second organoid or enteroid each include a tissue type selected from the esophageal, gastric, liver, intestinal, or colonic tissue type, or any combination thereof.

[0048] 38. The organoid according to alternative 37, wherein the tissue type of the first organoid and the tissue type of the second organoid or enteroid are the same.

[0049] 39. The organoid described in Alternative 37, wherein the tissue type of the first organoid and the tissue type of the second organoid or enteroid are different.

[0050] 40. An organoid produced by the method described in any one of Alternatives 20-34.

[0051] 41. An organoid described in any one of Alternatives 35-40 for use in treating gastrointestinal disorders in subjects requiring treatment.

[0052] Additional embodiments of the present disclosure provided herein are described by the following additionally numbered alternatives.

[0053] A method for producing composite organoids, a) Obtaining a single dissociated mesenchymal cell isolated from one or more organoids, b) Obtaining epithelial structures isolated from organoids or enteroids, c) Combining a single dissociated mesenchymal cell with an epithelial structure, d) A method comprising culturing combined single dissociated mesenchymal cells and epithelial structures to form a composite organoid.

[0054] The method according to Alternative 1, wherein obtaining a single dissociated mesenchymal cell involves isolating a single dissociated mesenchymal cell from one or more organoids.

[0055] The method according to Alternative 1 or 2, wherein obtaining an epithelial structure comprises isolating an epithelial structure from an organoid or enteroid.

[0056] The method according to any one of Alternatives 1 to 3, wherein a single dissociated mesenchymal cell, or an epithelial structure, or both, is isolated by mechanical dissociation and filtration.

[0057] A method according to one of the aforementioned alternatives, wherein a single dissociated mesenchymal cell and epithelial structure are combined by centrifugation.

[0058] The method according to any one of the aforementioned alternatives, wherein the number of mesenchymal cells in the composite organoid is greater than the original number of organoids or enteroids from which the epithelial structure is isolated, and as a result the composite organoid has concentrated mesenchyme.

[0059] The method according to any one of the above alternatives, wherein 1) one or more organoids from which a single dissociated mesenchymal cell is isolated, and 2) organoids or enteroids from which an epithelial structure is isolated each include a histological type selected from esophageal, gastric, hepatic, intestinal, or colonic histological types, or any combination thereof.

[0060] The method according to any one of the aforementioned alternatives, wherein 1) one or more organoids from which a single dissociated mesenchymal cell is isolated include an intestinal tissue type, and 2) organoids or enteroids from which an epithelial structure is isolated include a tissue type selected from esophageal, gastric, hepatic, intestinal, or colonic tissue types, or any combination thereof.

[0061] The method according to any one of the aforementioned alternatives, wherein one or more organoids from which a single dissociated mesenchymal cell is isolated are small intestinal organoids, optionally human intestinal organoids (HIOs).

[0062] The method according to any one of embodiments 7 to 9, wherein the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated is different from the histological type of organoid or enteroid from which an epithelial structure is isolated.

[0063] The histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated differs from the histological type of the organoid or enteroid from which the epithelial structure is isolated. Furthermore, repatterning of the epithelial structure by the single dissociated mesenchymal cell does not occur, and as a result, the epithelium of the composite organoid maintains the histological type of the organoid or enteroid. If an organoid or enteroid from which an epithelial structure is isolated is an organoid, and the organoid from which the epithelial structure is isolated is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or 14-30, 15-30, 18-30, 15-20, or 15-25 days old, Alternatively, the method according to alternative 10, wherein the organoid or enteroid from which the epithelial structure is isolated is an enteroid, and the enteroid is derived from adult tissue.

[0064] The method according to Alternative 10, wherein the organoid or enteroid from which the epithelial structure is isolated is an organoid, the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated is different from the histological type of the organoid from which the epithelial structure is isolated, repatterning of the epithelial structure by the single dissociated mesenchymal cell occurs, and as a result the epithelium of the composite organoid exhibits the characteristics of one or more organoid histological types, and optionally the organoid from which the epithelial structure is isolated is 8, 9, 10, 11, 12, or 13 days old or younger, or 8-13, 8-10, or 10-13 days old.

[0065] The method according to any one of Alternatives 7-9, wherein the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated is the same as the histological type of organoid or enteroid from which an epithelial structure is isolated.

[0066] The method according to Alternative 13, wherein one or more organoids, and each organoid or enteroid, comprises only one of the esophageal, gastric, liver, intestinal, or colonic tissue types, and the resulting composite organoid is an allogeneic organoid comprising one tissue type.

[0067] The method according to any of the aforementioned alternatives, wherein one or more organoids are derived from pluripotent stem cells (PSCs) from a first subject, and the organoids or enteroids are derived from PSCs from a second subject or isolated from gastrointestinal tissue from a second subject.

[0068] The method of alternative 15, wherein the first and second subjects are mammals.

[0069] The method of alternative 15 or 16, wherein the first and second subjects are human.

[0070] A method according to one of alternatives 15-17, wherein the first subject and the second subject are the same individual.

[0071] A method according to any one of the aforementioned alternatives, further comprising transplanting a composite organoid into a recipient.

[0072] The method of alternative 19, wherein the recipient is either the first or second target.

[0073] The method according to Alternative 19 or 20, wherein the composite organoid exhibits greater engraftment and growth in the recipient compared to an equivalent non-composite organoid or enteroid.

[0074] The method according to any one of the aforementioned alternatives, wherein 1) one or more organoids from which a single dissociated mesenchymal cell is isolated, or 2) an organoid or enteroid, or both, from which an epithelial structure is isolated, is manipulated to contain one or more exogenous nucleic acids or proteins.

[0075] The method according to any one of the aforementioned alternatives, wherein 1) one or more organoids from which a single dissociated mesenchymal cell is isolated, or 2) an organoid or enteroid, or both, from which an epithelial structure is isolated, contains a gene mutation, and optionally the gene mutation is associated with a disease state or a model of a disease state.

[0076] It is a composite organoid, Mesenchyme containing mesenchymal cells isolated as single dissociated cells from the first organoid, A composite organoid comprising epithelium containing epithelial cells isolated as an epithelial structure from a second organoid or enteroid.

[0077] The composite organoid according to Alternative 24, wherein the ratio of mesenchymal cells to epithelial cells in the composite organoid is greater than the ratio in the second organoid or enteroid.

[0078] The composite organoid according to alternative 24 or 25, wherein the first organoid and the second organoid or enteroid each include a tissue type selected from the esophageal, gastric, liver, intestinal, or colon tissue type, or any combination thereof.

[0079] A composite organoid according to alternative 26, wherein the tissue type of the first organoid and the tissue type of the second organoid or enteroid are the same.

[0080] A composite organoid according to alternative 26, wherein the tissue type of the first organoid and the tissue type of the second organoid or enteroid are different.

[0081] The tissue type of the first organoid from which mesenchymal cells are isolated differs from the tissue type of the second organoid or enteroid from which epithelial cells are isolated. As a result, repatterning of epithelial cells by mesenchymal cells does not occur, and the epithelium of the composite organoid maintains the tissue type of the second organoid or enteroid from which epithelial cells are isolated. Optionally, the second organoid or enteroid is the second organoid, and the second organoid is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or 14-30, 15-30, 18-30, 15-20, or 15-25 days old, or The composite organoid according to alternative 28, wherein, optionally, the second organoid or enteroid is a second enteroid, and the second enteroid is derived from adult tissue.

[0082] The composite organoid according to Alternative 28, wherein the second organoid or enteroid is a second organoid, and the histological type of the first organoid from which mesenchymal cells are isolated is different from the histological type of the second organoid from which epithelial cells are isolated, and repatterning of epithelial cells by mesenchymal cells occurs, resulting in the epithelium of the composite organoid exhibiting the characteristics of the histological type of the first organoid from which mesenchymal cells are isolated, and optionally the second organoid is 8, 9, 10, 11, 12, or 13 days old or younger, or 8-13, 8-10, or 10-13 days old.

[0083] It is a composite organoid, A mesenchyme containing a first histological type selected from the esophageal, gastric, hepatic, intestinal, or colonic histological type, or any combination thereof, Epithelium comprising an epithelium having an esophageal, gastric, hepatic, intestinal, or colonic tissue type, or a second tissue type selected from any combination thereof, A composite organoid in which the first tissue type and the second tissue type have differences of at least one tissue type.

[0084] A composite organoid according to any one of Alternatives 24-31, comprising one or more exogenous nucleic acids or proteins.

[0085] A composite organoid according to any one of Alternatives 24-32, wherein the composite organoid has a gene mutation or has been manipulated to include a gene mutation, and optionally the gene mutation is associated with a disease state or a model of a disease state.

[0086] An organoid produced by the method described in any one of Alternatives 1-23.

[0087] An organoid described in one of Alternatives 24-34 for use in treating gastrointestinal disorders in patients requiring treatment.

[0088] A method for screening candidate therapeutic agents, comprising: contacting an organoid described in any one of alternatives 24 to 34 with a candidate therapeutic agent; and determining the effect of the candidate therapeutic agent on the organoid.

[0089] The method according to Alternative 36, wherein an organoid is genetically modified, and optionally genetically modified to exhibit a disease or a model thereof.

[0090] The method according to alternative 36 or 37, wherein the mesenchyme and / or epithelium of an organoid is genetically modified, and optionally, to exhibit a disease or a model thereof. [Brief explanation of the drawing]

[0091] In addition to the features described herein, additional features and variations will be readily apparent from the following drawings and descriptions of exemplary embodiments. It should be understood that these drawings depict embodiments and are not intended to limit the scope. [Figure 1] Representative diagrams illustrating key steps of the dissociation / recombination protocol are shown. Representative in vitro organoids before (top) and after (bottom) manual dissociation from HIO on day 14 (Panel A). Images of 96-well plate wells containing a single dissociated mesenchyme and one epithelial structure after centrifugation (Panel B). Images of composite organoids 24 hours after recombination before plating in Matrigel (Panel C). [Figure 2A] This illustrates an embodiment of HIO-mesenchymal / HCO-epithelial xenolytic organoid formation. The images show the organization of the xenolytic organoids 48 hours and 9 days after recombination. HCO-epithelium is positive for GFP fluorescence. [Figure 2B] This document demonstrates the engraftment of HIO-mesenchymal / HCO-epithelial xenogeneic organoids into the renal capsule tissue of a mouse model. The xenogeneic organoids are comparable to HIO organoids and exhibit greater maturation and engraftment than HCO organoids. [Figure 2C]This image shows an example of immunofluorescence imaging in which HIO-mesenchymal / HCO-epithelial heterogeneous organoids derived from source organoids on day 18 were positive for colon-specific special AT-rich sequence binding protein 2 (SATB2) and negative for small intestine-specific sucrase-isomaltase (SI), demonstrating that the epithelial structure derived from HCO at this age maintains its colonic identity. [Figure 2D] This image shows an embodiment of immunofluorescence imaging in which HIO-mesenchymal / HCO-epithelial heterogeneous organoids derived from source organoids on day 11 are positive for small intestine-specific GATA-binding protein 4 (GATA4) and negative for colon-specific SATB2 (compared to source organoids on day 18), demonstrating that epithelial structures derived from HCO at this age can be reprogrammed by the surrounding mesenchyme (e.g., changing colonic epithelium to small intestinal identity). [Figure 2E] This illustrates the formation of HIO-mesenchymal / HGO-epithelial xenolytic organoids. The images show the organization of xenolytic organoids 4 and 11 days after recombination. Immunofluorescence staining confirms the maturation of GFP-positive intestinal mesenchyme and CDH1-positive gastric epithelium. [Figure 2F] This illustrates an embodiment of HIO-mesenchymal / enteroid organoid formation. The images show the organization of heterologous organoids 10, 21, and 31 days after recombination. The enteroids are positive for GFP fluorescence. [Figure 3]The images illustrate the formation of different types of heterogeneous organoids and their engraftment into the renal capsule of a mouse model. HIO-mesenchymal / HIO-epithelial (Panel A), HIO-mesenchymal / HCO-epithelial (Panel B), and HIO-mesenchymal / HAGO-epithelial (Panel C) heterogeneous organoids were tested. HIO-mesenchymal-enteroid (Panel D), HAGO-mesenchymal / HAGO-epithelial (Panel E), and HCO-mesenchymal / HCO-epithelial (Panel F) organoids were also formed. HAGO and HCO allogeneic organoids were prepared by enriching HAGO or HCO mesenchymal cells from multiple source organoids and recombining these mesenchymal cells with epithelial structures. [Figure 4A] This document shows an embodiment of images taken 48 hours after in vitro recombination between GFP-positive HIO mesenchyme and GFP-negative HEO epithelium. [Figure 4B] This image shows an example of a recombinant HIO / HEO graft 8 weeks after transplantation into the renal capsule of NSG mice. [Figure 4C] This shows an example of H&E staining of transplanted HIO / HEO tissue. [Figure 4D] Examples of GFP and CDH1 staining demonstrating the purity of recombination after engraftment are shown. [Figure 4E] This document describes the immunohistochemical staining of transplanted HIO / HEO tissue to reveal the development and maturation of esophageal epithelium, including the basal layer (KRT5 and KRT14) and the suprabasal layer (KRT13 and IVL), as well as positivity for the esophageal-specific transcription factor p63. [Modes for carrying out the invention]

[0092] Recent methods for patterning the foregut (e.g., human gastric organoids [HGO]) and hindgut (e.g., human colon organoids [HCO]) in vitro result in heterogeneity in the epithelial-to-mesenchymal ratio, which prevents or limits the in vivo engraftment ability of these structures. Hereinafter, the applicants have shown that the lack of robust mesenchyme reduces the success rate of engraftment in animal models and disclose methods and compositions having improved mesenchyme to address one or more of the limitations of conventional organoids.

[0093] Disclosed herein are composite organoids and compositions thereof, as well as methods for producing them, comprising dissociating and reassociating (recombining) epithelial and mesenchymal components to form organoids having improved epithelial-to-mesenchymal ratios and morphologies. These organoids can be used for purposes such as drug screening or personalized medicine, and are suitable, for example, for autologous or allogeneic transplantation into subjects such as humans or other mammals, or for xenotransplantation into immunocompromised animals. In some embodiments, the composite organoid is a composite gastrointestinal organoid. In other embodiments, the composite organoid is a composite organoid of other tissue types, such as brain, nerve, muscle, thyroid, heart, lung, kidney, or pancreatic organoids. In some embodiments, the epithelial and mesenchymal components are derived from donor organoids derived from pluripotent stem cells (PSCs) using a stepwise approach that mimics embryonic intestinal development, or from enteroids isolated from gastrointestinal tissue derived from PSCs or from donor subjects. In some embodiments, the organoid has a three-dimensional structure including polarized epithelium surrounded by supporting mesenchymal cells. In some embodiments, the composite organoid or donor organoid, or both, includes intestinal organoids, colon organoids, gastric organoids, antral gastric organoids, gastric fundus organoids, liver organoids, or pancreatic organoids, or any combination thereof. In some embodiments, the epithelial component is derived from patient-derived enteroids. In some embodiments, the composite organoid or donor organoid, or both, are human. Methods for producing organoids or enteroids can be found in U.S. Patents 9,719,068 and 10,174,289, and PCT International Publications 2016 / 061464, 2017 / 192997, 2018 / 106628, 2018 / 200481, 2018 / 085615, 2018 / 085622, 2018 / 085623, 2018 / 226267, 2019 / 074793, and 2020 / 023245, each of which is expressly incorporated herein in whole by reference.

[0094] The following detailed description refers to the accompanying drawings, which form part of it. In the drawings, unless otherwise indicated in the context, similar symbols typically identify similar components. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. The aspects of this disclosure generally described herein and shown in the drawings may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0095] term Unless otherwise specified, the technical and scientific terms used herein have the same meanings as they would be generally understood by those skilled in the art when this disclosure is read in light of it. For the purposes of this disclosure, the following terms are defined below:

[0096] The articles "a" and "an" are used herein to refer to one or more (e.g., at least one) grammatical objects of the article. For example, "element" means one or more elements.

[0097] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by approximately 10% relative to the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length being referenced.

[0098] Throughout this specification, unless otherwise required by context, the words “comprise,” “comprises,” and “comprise” mean to include the described step or element or group of steps or elements, but not to exclude any other step or element or group of steps or elements. “Consisting of” means to include and be limited to what follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the enumerated elements are necessary or essential, and other elements may not be present. “Consisting essentially of” means to include all elements enumerated before this phrase, and other elements are limited to those that do not interfere with or contribute to the activity or action expressed in this disclosure with respect to the enumerated elements. Therefore, the phrase "essentially consists of" indicates that the enumerated elements are required or mandatory, while the other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0099] As used herein, the terms “individual,” “subject,” or “patient” have their general and ordinary meanings as understood in light of this specification and mean human or non-human mammals, e.g., dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates, or birds, e.g., chickens, and other vertebrates or invertebrates. The term “mammal” is used in its ordinary biological sense. This includes, specifically, primates, including monkeys (chimpanzees, apes, and primates) and humans, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like.

[0100] As used herein, the terms “effective dose” or “effective amount” have their general and ordinary meanings as understood in light of the specification and refer to the amount of the described composition or compound that produces an observable effect. The actual dose levels of the active ingredient in the active composition of the subject currently disclosed may be varied to administer an amount of the active composition or compound that is effective in achieving a desired response for a particular subject and / or use. The selected dose 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, if a minimum dose is administered and there is no dose-limiting toxicity, the dose is increased to the minimum effective dose. This specification is intended to evaluate the determination and adjustment of effective doses, as well as when and how such adjustments should be made.

[0101] As used herein, the terms “function” and “functional” have their obvious and ordinary meanings as understood in light of this specification, and refer to biological, enzymatic, or therapeutic functions.

[0102] As used herein, the term “inhibit” has its general and ordinary meaning as understood in light of this specification and can mean a reduction or prevention of biological activity. The reduction may be a percentage that is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or about those, at least those, at least about those, or less than or about those, or within the range defined by any two of the aforementioned values. As used herein, the term “delay” has its general and ordinary meaning as understood in light of this specification and can mean a delay, postponement, or delay of a biological event to a later time than would otherwise be expected. The delay may be a percentage that is approximately 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or approximately one of those percentages, at least one of those percentages, at least one of those percentages, or less than or approximately one of those percentages, or within the range defined by any two of the aforementioned values. The terms inhibition and delay do not necessarily imply 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0103] As used herein, the term “isolated” has the general and ordinary meaning as understood in light of the specification and means a substance and / or entity that (1) when it was first produced (in nature and / or in a laboratory environment) it was separated from at least some of the components to which it was associated, and / or (2) when it was produced, prepared and / or manufactured by human hands it was separated from at least some of the components to which it was associated. An isolated substance and / or entity may be separated from 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 equal to 100%, about the aforementioned values, at least the aforementioned values, at least about the aforementioned values, less than or equal to the aforementioned values, or less than or equal to the aforementioned values ​​(or a range including and / or spanning the aforementioned values). In some embodiments, the isolated agent is approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, substantially 100%, or equivalent to 100% pure, approximately the aforementioned values, at least the aforementioned values, at least approximately the aforementioned values, less than or equal to the aforementioned values, or less than or equal to the aforementioned values ​​(or a range including and / or spanning the aforementioned values). As used herein, “isolated” substance can be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multicellular organism or tissue.

[0104] As used herein, “in vivo” is given its general and ordinary meaning as understood in light herein, and refers to the implementation of the method in living organisms, typically animals, mammals including humans, and plants, as opposed to tissue extracts or dead organisms.

[0105] As used herein, “exvivo” is given its general and ordinary meaning as understood in light of the specification and refers to the execution of a method in vitro with little alteration of natural conditions.

[0106] As used herein, “in vitro” is given its general and ordinary meaning as understood in light of the specification and refers to the execution of the method outside of biological conditions, for example, in a petri dish or test tube.

[0107] As used herein, the terms “nucleic acid” or “nucleic acid molecule” have their general and ordinary meanings as understood herein, and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that occur naturally in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of the following: ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have changes in the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications may include, for example, substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or functionalization of sugars as ethers or esters. Furthermore, the entire sugar moiety can be replaced with a stereochemically and electronically similar structure, such as aza sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoranilideates, or phosphoramidates. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which include naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. “Oligocyte” can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.Nucleic acids (or nucleic acids) may be contained in nucleic acid vectors or constructs (e.g., plasmids, viruses, retroviruses, lentiviruses, bacteriophages, cosmids, fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or human artificial chromosomes (HACs)) that can be used for amplification and / or expression of nucleic acids (or nucleic acids) in various biological systems. Typically, vectors or constructs may also contain elements such as promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeted sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0108] A nucleic acid or nucleic acid molecule may contain one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences may be contiguous within the same nucleic acid or nucleic acid molecule, or with extra nucleic acids between linker, repeat, or restriction enzyme sites, for example, or with any other sequence of any length 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 long, about that length, at least that length, at least about that length, less than or equal to that length, or about that length, or any length within the range defined by any two of the aforementioned lengths. As used herein, the term “downstream” with respect to nucleic acids has its general and ordinary meaning as understood in light of the specification, and, in the case of a double-stranded nucleic acid, refers to the sequence following the 3' end of the sequence preceding the sequence on the strand containing the coding sequence (sense strand). As used herein, the term “upstream” with respect to nucleic acids has its general and ordinary meaning as understood in light of the specification, and, in the case of a double-stranded nucleic acid, refers to the sequence preceding the 5' end of the sequence following the sequence on the strand containing the coding sequence (sense strand).As used herein, the term “grouping” with respect to nucleic acids has its general and ordinary meaning as understood in light of this specification and refers to two or more sequences that occur in close proximity to any other sequence that is, for example, an extra nucleic acid between linkers, repeats, or restriction enzyme sites, or that is, about, at least, less than, less than, or less than, or less than, or any length within the range defined by any two of the aforementioned lengths, but does not occur with any sequence in between that encodes a functional or catalytic polypeptide, protein, or protein domain.

[0109] The nucleic acids described herein include nucleic acid bases. Primary, standard, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleic acid bases include, but are not limited to, purines, pyrimidines, modified nucleic acid bases, 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.

[0110] As used herein, the terms “peptide,” “polypeptide,” and “protein” have their general and ordinary meanings as understood herein and refer to macromolecules composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structural, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, though not always, produced biologically by ribosome complexes using nucleic acid templates, but chemosynthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations can be performed, such as substitution, deletion, shortening, addition, replication, or fusion of two or more peptides, polypeptides, and proteins. These fusions of two or more peptides, polypeptides, or proteins can be joined adjacent to each other within the same molecule, or to any other sequence of any length that is, for example, an extra amino acid between linkers, repeats, epitopes, or tags, or of a length of 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, or about that length, at least that length, at least about that length, less than or equal to that length, or about that length, or any length within the range defined by any two of the aforementioned lengths. The term “downstream” in relation to polypeptides as used herein has its general and ordinary meaning as understood in light of the specification and refers to the sequence following the C-terminus of the preceding sequence. As used herein, the term “upstream” in relation to polypeptides has its general and ordinary meaning as understood in light of the specification, and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0111] In some embodiments described herein, the present specification relates to pharmaceutical compositions comprising, essentially, or consisting of, an effective amount of a cell composition and a pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0112] As used herein, “pharmaceutically acceptable” means carriers, excipients, and / or stabilizers that have their obvious and ordinary meaning as understood in light of this specification and are non-toxic to cells or mammals to which cells or mammals are exposed at the doses and concentrations used, or that have an acceptable level of toxicity. As used herein, “pharmaceutically acceptable,” “diluents,” “excipients,” and / or “carriers” are intended to include all kinds of solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that have their obvious and ordinary meanings as understood in light of this specification and are suitable for administration to human, cat, dog, or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are approved by federal, state, or other regulatory authorities for use in animals, including humans and non-human mammals such as cats and dogs, or are listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The terms diluent, excipient, and / or “carrier” may refer to the diluent, adjuvant, excipient, or vehicle through which the pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin. Water, physiological 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, wheat flour, chalk, silica gel, sodium stearate, glyceryl 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 a pH-buffered aqueous solution.Physiologically acceptable carriers may also contain one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming inhibitors such as sodium; nonionic surfactants such as TWEEN® and polyethylene glycol (PEG); and PLURONICS®. The compositions may also contain small amounts of wetting agents, fillers, emulsifiers, or pH buffers as needed. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulations are typically adapted to the method of administration.

[0113] Cryotherapy agents are cell composition additives used to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryotherapy agents 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. Cryotherapy agents may be used as part of a cryopreservation medium that also contains other components such as nutrients to enhance cell viability after thawing (e.g., albumin, serum, bovine serum, fetal bovine serum [FCS]). In these cryopreservation media, at least one antifreeze agent may be found in concentrations of 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 about those, at least those, at least about those, less than or about those, or about those less than or about those, or any percentage within the range defined by any two of the aforementioned numbers.

[0114] 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 sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may include, but are not limited to, serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof, as residues or contaminants from the manufacturing process. The amount of excipients may be found in the composition in any weight percentage within the range defined by 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 about those, at least those, at least about those, less than or about those, or about that than, or any two of the aforementioned numbers.

[0115] The term "pharmaceutically acceptable salt" has its plain and ordinary meaning as understood in light 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, and other materials. 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, and p-toluenesulfonic acid. Examples of inorganic bases suitable for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such a class of 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; and trihydroxymethylaminoethane.

[0116] The appropriate formulation will vary depending on the chosen route of administration. The formulations and techniques for administering the compounds described herein are known to those skilled in the art. Multiple techniques for administering the compounds exist in the art, including, but are not limited to, enteral, oral, rectal, topical, sublingual, oral, intraocular, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intra-arterial, intra-intravenous), intra-portal, intra-articular, intradermal, peritoneal, intrathecal, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections. Pharmaceutical compositions will generally be formulated to suit a specific intended route of administration.

[0117] As used herein, “carrier” means 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 organs of the body, in its plain and ordinary sense as understood in light of this specification.

[0118] As used herein, “diluent” has its obvious and ordinary meaning as understood in light of the specification and refers to a component in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically 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 may also be a liquid for dissolving a drug administered by injection, ingestion, or inhalation. Common forms of diluents in the art are buffered aqueous solutions, such as phosphate-buffered saline which mimics the composition of human blood, but are not limited thereto.

[0119] The term “purity” used herein for any given substance, compound, or material has its general and ordinary meaning as understood in light of the specifications and refers to the actual amount of the substance, compound, or material relative to the expected amount. For example, a substance, compound, or material is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, a 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, mycoplasmas, pyrogens, bacterial endotoxins, and exogenous infectious agents. Purity can be measured using techniques such as electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-Vis spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric analysis, or titration, or any combination thereof.

[0120] The term “yield” for any given substance, compound, or material used herein has its general and ordinary meaning as understood in light of the specifications and refers to the actual total amount of the substance, compound, or material relative to the expected abundance. For example, the yield of a substance, compound, or material may be 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, or about that, at least that, at least about that, less than or equal to that, or about less than or equal to that, including all decimals in between. The yield may be affected by the efficiency of the reaction or process, undesirable side reactions, decomposition, the quality of the input substances, compounds, or materials, or the loss of the desired substance, compound, or material at any step of production.

[0121] As used herein, the term “single dissociation” has its general and ordinary meaning as understood in light of this specification and refers to a preparation of a single-cell suspension. These single-cell suspensions can be prepared, for example, by processing multicellular tissue or structure by conventional mechanical and / or chemical (e.g., enzymatic) means. When used in the methods and compositions disclosed herein, single dissociation may apply to mesenchymal cells processed from multicellular tissue or structure such as organoids into a single-cell suspension. While “single dissociation” refers to a population of single cells, it should be understood that a preparation of single-dissociated cells does not necessarily have to contain only single cells, and a preparation of single-dissociated cells may include populations such as undissociated cell structures, cell aggregates, aggregated cells, and cell fragments. Similarly, in some embodiments, a composition of a particular type of single-dissociated cell (e.g., mesenchymal) may contain another type of cell (e.g., epithelial).

[0122] As used herein, the term “epithelial structure” has the general and ordinary meaning as understood in light of this specification, and refers, for example, to a multicellular tissue or fragment thereof composed of intact epithelial cells derived from differentiated organoids or enteroids (although other sources of epithelial cells are also assumed). When used in the methods disclosed herein, these epithelial structures are aggregates of epithelial cells that can be isolated, for example, using a suitable cell filter that allows single cells to pass through but retains these larger epithelial structures.

[0123] As used herein, the term “composite organoid” has its general and ordinary meaning as understood in light of this specification and refers to a cellular organoid that includes both mesenchyme and epithelium, which are combined as described herein to form a composite organoid. As assumed herein, these two cell populations may originate from the same type of organoid or organoid having the same histological type (for example, both mesenchyme and epithelium originate from gastric organoids, but at least some of the gastric organoids used to isolate the mesenchyme and the gastric organoids used to isolate the epithelium are not the same organoid), or they may originate from different types of organoids or organoids having different histological types (for example, mesenchyme originates from intestinal organoids and epithelium originates from gastric organoids). In some embodiments, organoids from which mesenchyme and epithelium originate may have several differences other than histological type, such as exhibiting genetic modifications or disease phenotypes, even if they have the same histological type. The combination of mesenchyme and epithelium for forming complex organoids is distinctly different from organoids produced from pluripotent stem cells, as previously disclosed, because the mesenchyme and epithelium of these organoids arise sequentially, for example, in the case of gastrointestinal organoids, through the process of cell differentiation from pluripotent stem cells to endoderm and then to enteroderm.

[0124] As used herein, the terms “w / w%” or “weight / weight%” have their general and ordinary meanings as understood herein, and refer to a percentage expressed in relation to the weight of an ingredient or drug relative to the total weight of the composition multiplied by 100. As used herein, the terms “v / v%” or “volume / volume%” have their general and ordinary meanings as understood herein, and refer to a percentage expressed in relation to the liquid volume of a compound, substance, ingredient or drug relative to the total liquid volume of the composition multiplied by 100.

[0125] This disclosure uses positive language to describe many embodiments. This disclosure also includes embodiments in which subject matter such as substances or materials, method steps and conditions, protocols or procedures are completely or partially excluded.

[0126] stem cells As used herein, the term “totipotent stem cell” (also known as omnipotent stem cell) has its general and ordinary meaning as understood in light of this specification and refers to a stem cell capable of differentiating into embryonic and extraembryonic cell types. Such cells can construct a complete and viable organism. These cells are produced from the fusion of an egg and a sperm cell. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0127] As used herein, the term “embryonic stem cell (ESC)” is also commonly abbreviated as ES cell and, as used herein, refers to pluripotent cells derived from the inner cell mass of a blastocyst, which is an early embryo, having its obvious and ordinary meaning as understood in light of this specification. For the purposes of this disclosure, the term “ESC” may be used more broadly to encompass embryonic germ cells.

[0128] As used herein, the term “pluripotent stem cells (PSCs)” has its obvious and ordinary meaning as understood herein and encompasses any cell that can differentiate into any of the three germ layers (embryonic epithelium), including nearly all cell types of the body, namely, cells derived from any of the three germ layers, namely the endoderm (endoderm, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), and ectoderm (epidermal tissue and nervous system). PSCs may be descendants of inner cell mass cells of a preimplantation blastocyst, or may be obtained by induction of non-pluripotent stem cells, such as adult somatic cells, by forcing the expression of specific genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including humans, rodents, pigs, and cattle.

[0129] As used herein, the term “induced pluripotent stem cell (iPSC)” has its obvious and common meaning as understood in light of this specification, and is commonly abbreviated as iPS cell. It refers to a type of pluripotent stem cell artificially induced from normally non-pluripotent cells, such as adult somatic cells, by inducing the “forced” expression of specific genes. hiPSC refers to human iPSCs. In some methods known in the art, iPSCs can be induced by transfection of non-pluripotent cells, such as adult fibroblasts, with specific stem cell-related genes. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcription factors Oct-3 / 4 (POU5F1) and Sox2, but other genes may also improve the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically, 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 critical 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), specific members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), specific members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), specific 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.

[0130] As used herein, the term “progenitor cell” has its obvious and ordinary meaning as understood in light of the specification and encompasses any cell that can be used in the methods herein, through which one or more progenitor cells acquire the ability to regenerate themselves or to differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or capable of becoming pluripotent. In some embodiments, progenitor cells are subjected to treatment with an extrinsic factor (e.g., a growth factor) to acquire pluripotency. In some embodiments, progenitor cells may be totipotent (or omnipotent) stem cells, pluripotent stem cells (artificial or unartificial), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells may be derived from an embryo, infant, child, or adult. In some embodiments, progenitor cells may be somatic cells subjected to treatment such that pluripotency is conferred via genetic engineering or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0131] In some embodiments, one 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 totipotent cells of an early mammalian embryo and are capable of unlimited undifferentiated growth in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, which is an early stage embryo. Methods for inducing embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells (e.g., H1, H7, or H9 ESC lines) are used in the exemplary embodiments described herein, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0132] Additional stem cells that may be used in embodiments of this disclosure include, but are not limited to, those provided by or listed in the National Stem Cell Bank (NSCB), 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, California), Cellartis AB (Goteborg, Sweden), ES Cell International Pte Ltd (Singapore), Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Databases hosted by Princeton University and the University of Pennsylvania. Examples of embryonic stem cells that can be used in embodiments of this 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 (H1), WA07 (H7), WA09 (H9), WA13 (H13), and 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.

[0133] 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 a specific specialized target cell type. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or alter the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0134] In some embodiments, adenoviruses can be used to transport the four required genes, resulting in iPSCs substantially identical to embryonic stem cells. Since adenoviruses do not combine their own genes with any of the target hosts, the risk of tumor formation is eliminated. In some embodiments, non-viral-based techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system, albeit with very low efficiency. In other embodiments, iPSCs are generated using direct protein delivery, thus eliminating the need for viruses or gene modification. In some embodiments, mouse iPSC generation is possible using similar methodologies. Repeated treatment of cells with specific proteins delivered to cells via polyarginine anchors has been sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0135] As used herein, the term “feeder cell” has its general and ordinary meaning as understood herein and refers to cells that support the growth of pluripotent stem cells by secreting growth factors into the culture medium or displaying them on the cell surface, etc. Feeder cells are generally adherent cells and may cease to grow. For example, feeder cells may cease to grow by irradiation (e.g., gamma rays), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily cease to grow. 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 heterogeneous 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 precutaneous fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal myocytes, human fetal fibroblasts, or human adult Fallopian tube epithelial cells. In some embodiments, a conditioned medium prepared from the feeder cells is used instead of, or in combination with, the feeder cell co-culture. In some embodiments, the feeder cells are not used during the growth of the target stem cells.

[0136] PSC differentiation In some embodiments, PSCs, such as ESCs and iPSCs, undergo stepwise directed differentiation, first into the definitive endoderm (DE), then into the foregut or hindgut lineage, and then into tissues such as gastrointestinal tissue or any other biological tissue. In some embodiments, PSCs undergo unstepwise directed differentiation, with the simultaneous addition of molecules (e.g., growth factors, ligands) to promote DE formation and molecules for subsequent tissue formation. In some embodiments, directed differentiation is achieved by selectively activating specific signaling pathways in iPSCs and / or DE cells. In some embodiments, signaling pathways include, but are not limited to, the Wnt signaling pathway, the Wnt / APC signaling pathway, the FGF signaling pathway, the TGF-beta signaling pathway, the BMP signaling pathway, the Notch signaling pathway, the Hedgehog signaling pathway, the LKB signaling pathway, and the Par polarity signaling pathway.

[0137] The endoderm of the embryo gives rise to the intestinal tract. The anterior DE forms the foregut and its associated organs, including the esophagus, lungs, stomach, liver, and pancreas, while the posterior DE forms the midgut and hindgut, which form the small and large intestines, as well as part of the genitourinary system. Studies using mouse, chicken, and frog embryos suggest that establishing an anterior-posterior pattern in the DE during gastrulation is a prerequisite for subsequent foregut and hindgut development. Wnt and FGF signaling pathways are important for promoting the final outcome of either posterior endoderm / hindgut or anterior endoderm / foregut. In the hindgut, the simple cuboidal epithelium first develops into pseudostratified columnar epithelium, then into polarized columnar epithelium and villi containing a growth zone at the base of the villi corresponding to the putative progenitor region.

[0138] Any method for producing endoderm from pluripotent cells (e.g., iPSCs or ESCs) can be used in the methods herein. In some embodiments, the pluripotent cells are derived from a morula. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the inner cell mass of an embryo or the gonadal ridge of an embryo. Embryonic stem cells or germ cells may arise from a variety of animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce endoderm. In some embodiments, human embryonic germ cells are used to produce endoderm. In some embodiments, iPSCs are used to produce endoderm. In some embodiments, human iPSCs (hiPSCs) are used to produce endoderm. In some embodiments, PSCs are first modified before differentiating into endoderm. In some embodiments, PSCs are genetically modified to express, for example, exogenous nucleic acids or proteins before differentiating into endoderm.

[0139] In some embodiments, embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a period of time of 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any time within the range defined by any two of the aforementioned times, for example, 6 hours to 300 hours, 24 hours to 120 hours, 48 ​​hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the two or more small molecule compounds, activators, inhibitors, or growth factors may be added simultaneously or separately.

[0140] In some embodiments, embryonic stem cells or germ cells or iPSCs are at concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL, or 15000 ng / mL, or approximately these, or at least these. The patient is treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration that is, at least about, or less than, or about less than, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors changes during the course of treatment. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the concentrations of the two or more small molecule compounds, activators, inhibitors, or growth factors may be different.

[0141] In some embodiments, ESCs, germ cells, or iPSCs are cultured in a growth medium that supports stem cell growth. In some embodiments, the stem cell growth medium is RPMI 1640, DMEM, DMEM / F12, Mini-Intestinal Medium, mTeSR 1, or mTeSR Plus Medium. In some embodiments, the stem cell growth medium contains fetal bovine serum (FBS). In some embodiments, the stem cell growth medium contains FBS in concentrations of 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or about those, at least those, at least about those, less than or about those, or less than or about those, or any percentage within the range defined by any two of the aforementioned concentrations, for example, 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%. In some embodiments, the stem cell growth medium does not contain heterogeneous components. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0142] In some embodiments, a population of cells enriched with endoderm cells is used. In some embodiments, the endoderm cells are isolated or substantially purified. In some embodiments, the isolated or substantially purified endoderm cells express one or more of the SOX17, FOXA2, or CXRC4 markers (e.g., at least one, three) in greater amounts than one or more of the OCT4, AFP, TM, SPARC, or SOX7 markers (e.g., at least one, three, five).

[0143] In some embodiments, endoderm cells and hESCs are treated with one or more growth factors. Such growth factors may include growth factors from the TGF-β superfamily. In some embodiments, one or more growth factors include the Nodal / activin and / or BMP subgroup of the TGF-β superfamily of growth factors. In some embodiments, one or more growth factors are selected from the group consisting of Nodal, activin A, activin B, BMP4, Wnt proteins, or any combination of these growth factors. For example, in humans, Wnt proteins include, but are not limited to, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.

[0144] In some embodiments, activin-induced endoderm (DE) may further undergo FGF / Wnt-induced anterior or posterior endoderm patterning, foregut or hindgut identification and morphogenesis, and ultimately gastrointestinal growth, morphogenesis, and cell differentiation into functional gastrointestinal cell types. In some embodiments, PSCs are efficiently directed to differentiate in vitro into gastrointestinal epithelium or mesenchyme, including secretory, endocrine, and absorptive cell types. It will be understood that molecules such as growth factors may be added at some developmental stage to promote the formation of specific types of intestinal tissue.

[0145] In vitro human gastrointestinal development occurs at stages close to fetal intestinal development, endoderm formation, anterior or posterior endoderm pattern formation, foregut or hindgut morphogenesis, fetal intestinal development, epithelial morphogenesis, formation of putative progenitor domains, and differentiation into functional cell types.

[0146] Those skilled in the art will understand that altering the concentration, expression, or function of one or more Wnt signaling proteins, in combination with altering the concentration, expression, or function of one or more FGF proteins, can result in directed differentiation according to this disclosure. In some embodiments, inhibition or activation of the Wnt and / or FGF signaling pathway can be brought about by using cellular components associated with the Wnt and / or FGF signaling pathway, such as native inhibitors, antagonists, activators, or agonists of the pathway. In some embodiments, these pathways can be inhibited or activated by using siRNA and / or shRNA that target cellular components associated with the Wnt and / or FGF signaling pathway.

[0147] Fibroblast growth factor (FGF) is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGF is a heparin-binding protein, and its interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. FGF plays a crucial role in the growth and differentiation of a wide variety of cells and tissues. In humans, 22 members of the FGF family have been identified, all of which are structurally related signaling molecules. Members FGF1-FGF10 all bind to the fibroblast growth factor receptor (FGFR). FGF1 is also known as acidic fibroblast growth factor, and FGF2 is also known as basic fibroblast growth factor (bFGF). Members FGF11, FGF12, FGF13, and FGF14, also known as FGF homologs 1-4 (FHF1-FHF4), have been shown to have distinctly different functional characteristics compared to FGF. These factors possess remarkably similar sequence homology, but they do not bind to FGFR and are involved in intracellular processes unrelated to FGF. This group is also known as "iFGF". Members FGF15-FGF23 are newer and less characterized. FGF15 is a mouse ortholog of human FGF19 (therefore, there is no human FGF15). Human FGF20 was identified based on homology with African clawed frog (Xenopus) FGF-20 (XFGF-20). In contrast to the local activity of other FGFs, FGF15 / FGF19, FGF21, and FGF23 have more systemic effects.

[0148] It will be understood by those skilled in the art that in some embodiments, any of the FGFs can be used in combination with proteins from the Wnt signaling pathway. In some embodiments, the FGFs used are one or more of 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, and FGF23.

[0149] The differentiation of PSCs into DE cultures and subsequently into various intermediate mature gastrointestinal cell types can be determined by the presence of stage-specific cell markers. In some embodiments, the expression of representative cell components is used to determine DE formation. Representative cell components include CMKOR1, CXCR4, GPR37, RTN4RL1, SLC5A9, SLC40A1, TRPA1, AGPAT3, APOA2, C20orf56, C21orf129, CALCR, CCL2, CER1, CMKOR1, CRIP1, CXCR4, CXorf1, DIO3, DIO30S, EB-1, EHHADH, ELOVL2, EPSTI1, FGF17, FLJ10970, FLJ21195, FLJ22471, FLJ23514, and FOXA. 2, FOXQ1, GATA4, GPR37, GSC, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SOX17, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706, or AW772192, or any combination thereof, are included but not limited to these. In some embodiments, the absence of cellular components such as the foregut marker Pdx1 and albumin may be used to indicate directed hindgut formation. In some embodiments, one or more (e.g., at least one, two, or three) intestinal transcription factors CDX2, KLF5, or SOX9 may be used to represent intestinal development. In some embodiments, intestinal development can be represented using one or more of the GATA4 or GATA6 protein expressions.

[0150] In some embodiments, morphological changes can be used to represent the progression of directed differentiation. In some embodiments, spheroids (e.g., mid-hindgut, hindgut, anterior foregut, or posterior foregut spheroids) are subjected to three-dimensional culture conditions for maturation. In some embodiments, gastrointestinal organoids mature in 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, or 40 days, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any number of days defined by any two of the aforementioned number of days, for example, 1 to 40 days, 20 to 30 days, 30 to 40 days, or 1 to 20 days. In some embodiments, a highly complex epithelium surrounded by mesenchymal cells may be observed following spheroid formation. In some embodiments, gastrointestinal organoids, epithelium, polarized columnar epithelium, mesenchyme, nerve cells, or smooth muscle cells may be observed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, or 40 days, or about those days, at least those days, at least about those days, less than or about those days, or less than or about those days, or any number of days within the range defined by any two of the aforementioned number of days, for example, 1 to 40 days, 20 to 30 days, 30 to 40 days, or 1 to 20 days.

[0151] In some embodiments, pluripotent stem cells are converted to gastrointestinal cell types by a "one-step" process. For example, one or more molecules that can differentiate pluripotent stem cells into DE cultures (e.g., activin A) are combined with additional molecules that can promote directed differentiation of DE cultures (e.g., Wnt3a and FGF4) to directly process the pluripotent stem cells.

[0152] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, pluripotent stem cells are prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, PBMCs are grown on 0.1% gelatin.

[0153] In some embodiments, pluripotent stem cells are prepared from PBMCs by viral transduction. In some embodiments, PBMCs are transduced with Sendai virus, lentivirus, adenovirus, or adeno-associated virus, or any combination thereof. In some embodiments, PBMCs are transduced with Sendai virus containing expression vectors for Oct3 / 4, Sox2, Klf4, or L-Myc, or any combination thereof. In some embodiments, PBMCs are transduced with one or more viruses having MOIs of 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, approximately, at least, at least about, less than or equal to, or approximately less than or equal to, or any MOI within the range defined by any two of the aforementioned MOIs, for example, MOIs of 0-5.0, 1.0-4.0, 2.0-3.0, 0-3.0, or 1.0-5.0. In some embodiments, after transduction, PBMCs express stem cell reprogramming factors. In some embodiments, after transduction, PBMCs are reprogrammed into iPSCs. In some embodiments, iPSCs grow on feeder cell substrates. In some embodiments, iPSCs grow on MEF feeder cell substrates. In some embodiments, iPSCs grow on irradiated MEF feeder cell substrates. In some embodiments, iPSCs are grown on 0.1% gelatin. In some embodiments, iPSCs are grown on RPMI 1640, DMEM, DMEM / F12, Mini Intestinal Medium, mTeSR1, or mTeSR Plus Medium.

[0154] In some embodiments, iPSCs are expanded in cell culture. In some embodiments, iPSCs are expanded in extracellular matrix or its mimics or derivatives. In some embodiments, iPSCs are expanded in Matrigel. In some embodiments, iPSCs are expanded in cell culture medium containing a ROCK inhibitor (e.g., Y-27632). In some embodiments, iPSCs are expanded to 80-95% confluence. In some embodiments, iPSCs differentiate into endoderm cells. In some embodiments, iPSCs differentiate into endoderm cells by contacting them with activin A. In some embodiments, iPSCs are further contacted with BMP4. In some embodiments, the iPSC comes into contact with BMP4 at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, about those, at least those, at least about those, less than or equal to those, or about those concentrations.

[0155] In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids. In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids by contacting them with one or more (e.g., at least one or two) of the GSK3 inhibitor or FGF4. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, FGF4 is recombinant FGF4. In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids without contacting them with one or more (e.g., at least one or two) of the GSK3 inhibitor or FGF4. In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids without contacting them with CHIR99021 or FGF4, or both. In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids by contacting them with epidermal growth factor (EGF). In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids by contacting them with a BMP inhibitor. In some embodiments, the BMP inhibitor is noggin. In some embodiments, endoderm cells differentiate into foregut or hindgut spheroids by contacting them with retinoic acid.

[0156] In some embodiments, the foregut or hindgut spheroids are embedded in a basement membrane or basement membrane mimic. In some embodiments, the foregut or hindgut spheroids are embedded in Matrigel. In some embodiments, the foregut or hindgut spheroids are cultured in basal gut medium (e.g., mini gut medium). In some embodiments, the foregut or hindgut spheroids are cultured in basal gut medium to differentiate them into gastrointestinal organoids. In some embodiments, the basal gut medium contains one or more of the following: advanced DMEM-F12, N2 supplement, B27 supplement, HEPES, L-glutamine, penicillin-streptomycin, epidermal growth factor (EGF), or ROCK inhibitor (e.g., Y-27632), or any combination thereof. In some embodiments, the basal gut medium contains EGF.

[0157] In some embodiments, endoderm cells differentiate into spheroids. In some embodiments, endoderm cells differentiate into spheroids by contacting them with one or more of the following (e.g., at least one, two, three, or four): a GSK3 inhibitor, FGF, a BMP inhibitor, or retinoic acid (RA). In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the FGF is FGF4. In some embodiments, the FGF4 is recombinant FGF4. In some embodiments, the BMP inhibitor is noggin. In some embodiments, endoderm cells differentiate into spheroids without contacting them with one or more of the following (e.g., at least one, two, three, or four): a GSK3 inhibitor, FGF4, a BMP inhibitor, or RA, or any combination thereof. In some embodiments, endoderm cells differentiate into spheroids without contacting the endoderm with CHIR99021, FGF4, noggin, or RA, or any combination thereof. In some embodiments, endoderm cells differentiate into spheroids by contacting the endoderm cells with epidermal growth factor (EGF).

[0158] Preparation of composite organoids Disclosed herein are composite organoids and methods for producing them. In some embodiments, the method comprises obtaining a single dissociated mesenchymal cell isolated from one or more organoids; obtaining an epithelial structure isolated from an organoid or enteroid; combining the single dissociated mesenchymal cell and the epithelial structure; and culturing the combined single dissociated mesenchymal cell and epithelial structure to form a composite organoid. In some embodiments, obtaining a single dissociated mesenchymal cell comprises isolating a single dissociated mesenchymal cell from one or more organoids. In some embodiments, obtaining an epithelial structure comprises isolating an epithelial structure from an organoid or enteroid. The process of combining a single dissociated mesenchymal cell from two or more organoids (or enteroids) with an epithelial structure allows for the formation of an organoid containing a greater number of mesenchymal cells than is typically achievable using conventional organoid differentiation methods known in the art. By carrying out these methods, a desired organoid lacking mesenchyme, such as a patient-derived organoid or enteroid, can be produced and cultured in a more closely resembling biological tissue. In some embodiments, single dissociated mesenchymal cells can be isolated from organoids of the same tissue type and combined with epithelial structures isolated from organoids of the same type to enrich the organoid mesenchymal population. In some embodiments, the increase in the number of mesenchymal cells improves growth and maturation capacity both in vitro and in vivo if engrafted, thereby overcoming problems commonly encountered with traditionally produced organoids.

[0159] In some embodiments, the methods provided herein enable the formation of hybrid organoids containing cell types of different organo-tissue types (e.g., organoids exhibiting both gastric and colonic cells). In some embodiments, a single dissociated mesenchymal cell is isolated from organoids of different tissue types, while the epithelial structure can be isolated from organoids of the same tissue type as one of the organoids used for mesenchymal cell isolation, or from a tissue type different from all of the other organoids. These hybrid organoids can be used in research in personalized medicine, drug screening, or developmental biology, including high-throughput studies. Studies of different aspects of cancer, such as migration, metastasis, angiogenesis, and immune evasion, using hybrid organoids containing both normal and malignant cell types are also conceivable.

[0160] Also disclosed herein are composite organoids and compositions thereof, for example, those produced by the methods disclosed herein. In some embodiments, the composite organoid comprises mesenchyme and epithelium. In some embodiments, the mesenchyme comprises a single dissociated mesenchymal cell isolated from the first organoid. In some embodiments, the epithelium comprises an epithelial structure isolated from the second organoid or enteroid. In some embodiments, the composite organoid comprises a number of mesenchymal cells greater than the number of mesenchymal cells in the first organoid, or the second organoid or enteroid, or both. In some embodiments, the ratio of single dissociated mesenchymal cells to epithelial cells in the composite organoid is greater than the ratio in the first organoid, or the second organoid or enteroid, or both. In some embodiments, the histological type of the first organoid and the histological type of the second organoid or enteroid are the same. In some embodiments, the composite organoid is a homogeneous organoid comprising one histological type. In some embodiments, the histological type of the first organoid and the histological type of the second organoid or enteroid are different. In some embodiments, the histological type of the first organoid from which mesenchymal cells are isolated differs from the histological type of the second organoid or enteroid from which epithelial structures are isolated, and no repatterning of epithelial cells by mesenchymal cells occurs, and as a result, the epithelium of the composite organoid maintains the histological type of the second organoid or enteroid from which the epithelial structures are isolated. In some embodiments, if the epithelial structures are derived from organoids that are at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or from organoids that are 14-30, 15-30, 18-30, 15-20, or 15-25 days old, no repatterning of epithelial cells occurs. In some embodiments, when the epithelial structure originates from enteroids, repatterning of epithelial cells does not occur. In some embodiments, the enteroids originate from adult tissue.In some embodiments, unlike the first organoid tissue type from which mesenchymal cells are isolated and the second organoid or enteroid tissue type from which epithelial structures are isolated, repatterning of epithelial cells by mesenchymal cells occurs, and as a result, the epithelium of the composite organoid exhibits the characteristics of the first organoid tissue type from which mesenchymal cells are isolated. In some embodiments, repatterning of epithelial cells by mesenchymal cells may occur when the epithelial structures are derived from organoids that are 8, 9, 10, 11, 12, or 13 days old or younger, or from organoids that are 8-13, 8-10, or 10-13 days old. In some embodiments, the enteroids are derived from adult tissue with distinct patterning, and the epithelial structures derived from the enteroids maintain their tissue type even when recombined with mesenchymal cells. In some embodiments, the composite organoid is a heterogeneous organoid comprising two or more tissue types. In some embodiments, the liver organoid is a composite organoid produced by any one of the methods described herein.

[0161] In some embodiments, the composite organoids disclosed herein may comprise one or more exogenous nucleic acids or proteins. For example, these one or more exogenous nucleic acids or proteins may be used as reporters or markers.

[0162] In some embodiments, the composite organoids disclosed herein may include gene mutations. In some embodiments, the gene mutations may be associated with a desired organ function or reporter function. In some embodiments, the gene mutations may be associated with a disease state or a model of a disease state. In some embodiments, the disease state or a model of a disease state may be induced in the composite organoids disclosed herein by other means, such as treatment with a composition that induces the disease state or a model of a disease state.

[0163] The following describes various embodiments that are typically included in, but not necessarily included in, preparations of composite organoids.

[0164] Sources of mesenchymal and epithelial cells In any of the methods disclosed herein, 1) one or more organoids from which a single dissociated mesenchymal cell is isolated, and 2) organoids or enteroids from which epithelial structures are isolated may be organoids of any tissue type, e.g., organoids or tissue types of the brain, nerve, muscle, thyroid, heart, lung, kidney, bladder, testis, pancreas, gastrointestinal tract, esophagus, stomach, liver, intestine, or colon, or enteroids produced from intestinal or colonic (also called colonoid) epithelial tissue. These donor organoids or enteroids of any tissue type can be produced according to any applicable method known in the art. When produced by conventional methods, these organoids may exhibit little to no mesenchyma compared to biological tissue, which is counterproductive for long-term tissue culture and / or engraftment in a subject and may not represent how the corresponding organ functions. In the case of enteroids, they lack any mesenchyma and are unsuitable for culture. Therefore, in some embodiments of the methods provided herein, either of these organoids or enteroids can be enriched with respect to the mesenchyme to improve their characteristics. It should be understood that the “tissue type” of an organoid is the type of tissue to which the organoid closely resembles, based on considerations such as the phenotype of the cells present in the organoid (e.g., gene expression, protein expression, morphology, etc.). An organoid does not need to be identical in all embodiments to the corresponding tissue in order to constitute an organoid of its “tissue type.”

[0165] Methods for producing these donor organoids or enteroids can be found, for example, in U.S. Patents 9,719,068 and 10,174,289, and PCT International Publications 2011 / 140411, 2015 / 183920, 2016 / 061464, 2017 / 192997, 2018 / 106628, 2018 / 200481, 2018 / 085615, 2018 / 085622, 2018 / 085623, 2018 / 226267, 2019 / 074793, and 2020 / 023245, each of which is expressly incorporated herein by reference in its entirety.

[0166] In some of the methods for producing donor organoids or enteroids provided herein or known in the art, the donor organoids or enteroids are derived from a defined source. In some embodiments, one or more organoids from which a single dissociated mesenchymal cell is isolated are derived from PSCs from a first subject. In some embodiments, organoids or enteroids from which an epithelial structure is isolated are derived from PSCs from a first subject, isolated from a first subject, or isolated from a second subject that is not a first subject, e.g., gastrointestinal tissue. In some embodiments, the PSCs are induced pluripotent stem cells. In some embodiments, the PSCs are obtained from a first subject and / or a second subject by reprogramming somatic cells or adult stem cells isolated from the first subject and / or a second subject. In some embodiments, the somatic cells or adult stem cells include myeloid cells, peripheral cells, mobilized peripheral cells, or any other somatic cells. The somatic cells or adult stem cells can be reprogrammed into PSCs according to any applicable method conventionally known in the art. In some embodiments, the first and second subjects are mammals. In some embodiments, the first and second subjects are humans. In some embodiments, the first and second subjects are the same individual. In some embodiments, the first and / or second subjects have, have previously had, or are at risk of contracting the disease.

[0167] In some embodiments, the donor organoid or enteroid is first cultured for a number of days that is, is about, is at least, is at least about, is no more than, or is no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 days, or any number of days within a range defined by any two of the foregoing numbers of days, for example, for 1 to 50 days, 10 to 30 days, 20 to 40 days, 1 to 30 days, or 20 to 50 days.

[0168] In some embodiments, the one or more organoids from which singly dissociated mesenchymal cells are isolated is, is about, is at least, is at least about, is no more than, or is no more than about 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 organoids, or any number of organoids within a range defined by any two of the foregoing numbers of organoids, for example, 1 to 10 9 organoids, 10 2 to 10 7 organoids, 10 4 to 10 6 organoids, 1 to 10 4 organoids, or 10 4 to 10 9 organoids.

[0169] In some embodiments, organoids or enteroids are prepared in an extracellular matrix, or its mimics or derivatives. Some examples of extracellular matrix, or its mimics or derivatives, include, but are not limited to, cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, polylysine, polyornithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, basement membranes, Matrigel, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. In some embodiments, the extracellular matrix, or its mimic or derivative, is or contains Matrigel. In some embodiments, the organoids or enteroids are released from the extracellular matrix, or its mimic or derivative, when ready for use. In some embodiments, the organoids or enteroids are released by depolymerizing the extracellular matrix, or its mimic or derivative. In some embodiments, organoids or enteroids are released using a cell recovery solution (Corning). In some embodiments, if organoids or enteroids of different tissue types are used, the different tissue types are processed simultaneously. In some embodiments, the organoids or enteroids are cryopreserved in advance. Cryopreserving organoids or enteroids allows for simultaneous processing, for example, if the organoids or enteroids grow at different rates or are isolated and / or differentiated at different times.

[0170] In some embodiments, the organoids or enteroids are of two or more tissue types. In some embodiments, the organoids or enteroids of each tissue type may be cultured separately, for example, under conditions optimized for each tissue type. In other embodiments, the organoids or enteroids of each tissue type may be cultured together. In some embodiments, the organoids or enteroids of two or more tissue types include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tissue types, or about them, at least them, at least about them, less than or equal to them, or about less than or equal to them.

[0171] In some embodiments, either a donor organoid or enteroid, or a progenitor cell, is engineered to contain one or more exogenous nucleic acids or proteins, or both. In some embodiments, the one or more exogenous nucleic acids or proteins may include nucleic acids or proteins having a desired organ function or reporter function, such as a fluorescent or luminescent protein, or a nucleic acid encoding a fluorescent or luminescent protein. In some embodiments, the donor organoid or enteroid is engineered directly to contain one or more exogenous nucleic acids or proteins. In some embodiments, the PSC or gastrointestinal tissue used to induce the donor organoid or enteroid is engineered to contain one or more exogenous nucleic acids or proteins before the organoid or enteroid is formed.

[0172] In some embodiments, the donor organoid or enteroid, or progenitor cells, either have or are manipulated to include a gene mutation. In some embodiments, the gene mutation may be associated with a desired organ function or reporter function. In some embodiments, the gene mutation may be associated with a disease state or a model of a disease state. In some embodiments, the use of one or more of these donor organoids or enteroids in the methods or composite organoid compositions disclosed herein may result in a disease state or a model of a disease state, or a composite organoid exhibiting all or some of its symptoms. The presence of gene mutations in the donor organoid or enteroid also generally applies to separate cellular components constituting the donor organoid or enteroid, including mesenchymal cell populations and epithelial cell populations. In some embodiments, the donor organoid or enteroid having a gene mutation may be derived from a patient, for example, a patient with or susceptible to the disease. In some embodiments, the disease state or a model of a disease state may be induced in the donor organoid or enteroid by other means, such as treatment with a composition that induces the disease state or a model of a disease state.

[0173] Dissociation of organoids into mesenchymal cells and epithelial structures The donor organoids or enteroids produced according to any applicable method correspond to one or more organoids from which a single dissociated mesenchymal cell is isolated and / or organoids or enteroids from which an epithelial structure is isolated. Each of the donor organoids or enteroids is destroyed or dissociated to release a single dissociated mesenchymal cell or epithelial structure, which refers to a fragment of organoid or enteroid epithelium prepared as an intact group of epithelial cells, although not completely dissociated into single cells. In some embodiments, the epithelial structure may contain additional cell types (e.g., mesenchymal cells).

[0174] In any of the methods disclosed herein, one or more organoids from which a single dissociated mesenchymal cell is isolated, and an organoid or enteroid from which an epithelial structure is isolated, each comprises a histological type (brain, nerve, muscle, thyroid, heart, lung, kidney, bladder, testis, pancreas, gastrointestinal tract, esophagus, stomach, liver, intestine, or colon histological type, or any combination thereof). In some embodiments, the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated is different from the histological type of the organoid or enteroid. In some embodiments, the histological type of one or more organoids and the histological type of the organoid or enteroid are the same.

[0175] Organoids or enteroids can be destroyed or dissociated using any applicable method conventionally known in the art, such as mechanical or enzymatic dissociation. In some embodiments, single dissociated mesenchymal cells, or epithelial structures, or both, are isolated by mechanical dissociation and filtration. In some embodiments, organoids are mechanically dissociated using a pipette, microchannel, or other device having a hole or channel of appropriate size for mechanically shearing a group of cells without destroying individual cells. In some embodiments, the pipette is a conventional 5 mL pipette. In some embodiments, a hole or channel of appropriate size includes diameter. In some embodiments, the diameter is 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mm, or about those, at least those, at least about those, less than or equal to those, or about that or equal to any diameter within the range defined by any two of the aforementioned diameters. In some embodiments, the mesenchymal and epithelial components are further separated using a cell filter. In some embodiments, the cell filter has a mesh size of about 40 μm, about 70 μm, or about 100 μm, or about those, at least those, at least about those, less than or equal to those, or less than or equal to those, or any mesh size within the range defined by any two of the aforementioned mesh sizes. In some embodiments, the cell filter allows a single dissociated mesenchymal cell to pass through while retaining the epithelial structure. The separated epithelial structure and single dissociated mesenchymal cell can then be collected for further use.

[0176] In some embodiments, after dissociation, the collected single dissociated mesenchymal cells or epithelial structures, or both, are cultured in a growth medium. In some embodiments, the growth medium is any medium disclosed herein or otherwise known in the art for supporting mesenchymal cells or epithelial cells, or both. In some embodiments, the growth medium comprises EGF, a ROCK inhibitor (e.g., Y-27632), or both. In some embodiments, the growth medium is a mini-intestinal medium supplemented with hEGF or Y-27632, or both. In some embodiments, the culture of single dissociated mesenchymal cells or epithelial structures, or both, allows for the growth and / or expansion of each cell to obtain a larger number of cells. In other embodiments, single dissociated mesenchymal cells or epithelial structures, or both, are used immediately after dissociation.

[0177] In some embodiments, isolated single dissociated mesenchymal cells or epithelial structures, or both, are cryopreserved after dissociation. In some embodiments, single dissociated mesenchymal cells or epithelial structures, or both, are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 4 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days, 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, 9 It is frozen for 3, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 days, or about those days, at least those days, at least about those days, less than or about those days, or less than or about those days, or any number of days within the range defined by any two of the aforementioned times, for example, 1 to 120 days, 1 to 60 days, 10 to 120 days, 10 to 90 days, 10 to 60 days, 10 to 50 days, 20 to 30 days, 30 to 60 days, 60 to 120 days, 1 to 30 days, or 20 to 60 days. In some embodiments, cryopreservation of a single dissociated mesenchymal cell or epithelial structure, or both, allows for the formation of a composite organoid at a later point in time. In some embodiments, the composite organoid comprises mesenchymal cells from multiple organoids and / or epithelial structures from organoids that are not identical to the isolated organoid, with at least a portion of the single dissociated mesenchymal cells being the same.

[0178] In some embodiments, single dissociated mesenchymal cells or epithelial structures isolated by any of the methods described herein each originate from one or more tissue types of organoids or enteroids. In some embodiments, single dissociated mesenchymal cells or epithelial structures derived from two or more tissue types of organoids or enteroids may be isolated from each of the organoids or enteroids of each tissue type after being cultured separately. In some embodiments, single dissociated mesenchymal cells isolated from separate organoids of each tissue type can then be pooled to provide a population of single dissociated mesenchymal cells from two or more tissue types. In some embodiments, epithelial structures isolated from separate organoids or enteroids of each tissue type can then be pooled to provide a population of epithelial structures from two or more tissue types. In other embodiments, when organoids or enteroids of each tissue type are cultured together, the dissociation of the organoids or enteroids results in a population of single dissociated mesenchymal cells and epithelial structures already composed of two or more tissue types. In some embodiments, single dissociated mesenchymal cells or epithelial structures may be dissociated from separate populations of organoids that are cryopreserved and combined. In some embodiments, single dissociated mesenchymal cells or epithelial structures of a certain tissue type may be cryopreserved and subsequently used to pool together with one or more other, optionally selected, cryopreserved populations of single dissociated mesenchymal cells or epithelial structures. In other embodiments, single dissociated mesenchymal cells or epithelial structures of multiple tissue types may be pooled before cryopreservation. In some embodiments, each of the pooled single dissociated mesenchymal cells or epithelial structures comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tissue types, or about them, at least them, at least about them, less than or about them, or less than or about them.

[0179] The dissociation steps provided herein are typically performed to isolate a single dissociated mesenchymal cell or epithelial structure, or both, from a donor organoid or enteroid. However, intact donor organoids and / or enteroids can also be used in subsequent processes. In some embodiments, organoids containing both mesenchymal and epithelial tissue, and / or enteroids lacking mesenchymal tissue, can be used instead of epithelial structures.

[0180] Recombination of mesenchymal and epithelial cells After isolation of a single dissociated mesenchymal cell and epithelial structure from a donor organoid or enteroid, and after optional pooling and / or cryopreservation, the single dissociated mesenchymal cell and epithelial structure (or a suitable substitute, e.g., an intact organoid having mesenchyme and epithelium) is combined.

[0181] In a single use, mesenchyme (i.e., the number of mesenchymal cells) can be enriched for any organoid. For organoid types that do not produce much mesenchyme when differentiated from conventional in vitro methods (or produce less mesenchyme compared to in vivo tissue), culturing and growing such organoids can be relatively difficult. By carrying out the methods provided herein, organoids with a reliable amount of mesenchyme can be produced. In some embodiments, the number of mesenchymal cells in a composite organoid is greater than the original number of mesenchymal cells in one or more organoids from which a single dissociated mesenchymal cell is isolated, or in an organoid or enteroid from which an epithelial structure is isolated, or in both. In some embodiments, the number of single dissociated mesenchymal cells is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 times the original number of organoids, or organoids, or enteroids, or both mesenchymal cells, or any number within the range defined by any two of the aforementioned multiplicities, e.g., 1 to 500 times, 10 to 400 times, 50 to 200 times, 1 to 100 times, or 100 to 500 times. In some embodiments, the ratio of single dissociated mesenchymal cells to epithelial cells in a composite organoid is greater than the ratio of one or more organoids in which single dissociated mesenchymal cells are isolated, or the ratio of organoids or enteroids in which epithelial structures are isolated, or both. In some embodiments, the ratio of single dissociated mesenchymal cells to epithelial cells in a composite organoid is 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9n is a ratio of mesenchymal cells to epithelial cells within the range defined by 10, about 10, at least 10, at least about 10, less than or equal to 10, or about 10 or less, or any ratio of mesenchymal cells to epithelial cells within the range defined by any two of the aforementioned numbers. In some embodiments, the total number of single dissociated mesenchymal cells per composite organoid is 10 4 , 10 5 , 10 6 , or 10 7 The number of cells is any single dissociated mesenchymal cells, or about any number of such cells, or at least any number of such cells, or at least about any number of such cells, or at least about any number of such cells, or at least any number of such cells within the range defined by any two of the aforementioned single dissociated mesenchymal cell counts per composite organoid. The resulting composite organoid has concentrated mesenchyme.

[0182] In some embodiments, a combination of a single dissociated mesenchymal cell and an epithelial structure can result in either an allogeneic or heterogeneic complex organoid. In some embodiments, the single dissociated mesenchymal cell comprises only one histological type, and / or the one or more organoids from which the single dissociated mesenchymal cell is isolated comprises only one histological type. In some embodiments, the epithelial structure comprises only one histological type, and / or the organoid or enteroid from which the epithelial structure is isolated comprises only one histological type. In some embodiments, the single dissociated mesenchymal cell and one histological type of the epithelial structure are the same, and as a result, the resulting complex organoid is an allogeneic organoid comprising one histological type. In some embodiments, the single dissociated mesenchymal cell comprises one or more histological types, and / or the one or more organoids from which the single dissociated mesenchymal cell is isolated comprises one or more histological types. In some embodiments, the epithelial structure comprises one or more histological types, and / or the organoid or enteroid from which the epithelial structure is isolated comprises one or more histological types. In some embodiments, the histological types of a single dissociated mesenchymal cell and one or more epithelial structures are different. In some embodiments, the histological types of the single dissociated mesenchymal cell and the epithelial structures are different, and repatterning of the epithelial structures by the single dissociated mesenchymal cell does not occur, and as a result, the epithelium of the composite organoid maintains the histological type of the organoid or enteroid from which the epithelial structures are isolated. In some embodiments, if the epithelial structures are derived from organoids that are at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or from organoids that are 14-30, 15-30, 18-30, 15-20, or 15-25 days old, repatterning of the epithelial structures does not occur. In some embodiments, if the epithelial structures are derived from enteroids, repatterning of the epithelial structures does not occur. In some embodiments, the enteroids are derived from adult tissue.In some embodiments, the histological type of a single dissociated mesenchymal cell and the histological type of the epithelial structure differ, and repatterning of the epithelial structure by the single dissociated mesenchymal cell occurs, resulting in the epithelium of the composite organoid exhibiting the characteristics of one or more organoids from which the single dissociated mesenchymal cell is isolated. In some embodiments, repatterning of the epithelial structure by a single dissociated mesenchymal cell can occur when the epithelial structure is derived from organoids aged 8, 9, 10, 11, 12, or 13 days or younger, or from organoids or enteroids between 8 and 13 days, 8 and 10, or 10 and 13 days. In some embodiments, the enteroid is derived from adult tissue with distinct patterning, and the epithelial structure derived from the enteroid maintains its histological type even when recombined with a single dissociated mesenchymal cell. In some embodiments, the resulting composite organoid is a heterogeneous organoid containing two or more histological types. Allogeneic or heterogeneous composite organoids exhibiting characteristics of various organ types can be used in drug screening and in studies of the biologically relevant functions and interactions of different organ tissues.

[0183] In some embodiments, intact organoids containing both mesenchymal and epithelial tissues are used instead of epithelial structures. Intact organoids containing a certain histological type are combined with single dissociated mesenchymal cells containing one or more histological types to produce composite organoids containing the histological types of both the intact organoid and the single dissociated mesenchymal cell. Therefore, composite organoids produced using intact organoids may be either homogeneous or heterogeneous organoids.

[0184] In some embodiments, enteroids containing only epithelial cells and little to no mesenchymal cells are used in place of or as a source of isolated epithelial structures. In some embodiments, an enteroid or an epithelial structure derived therefrom is combined with a single dissociated mesenchymal cell. In some embodiments, the histological type of both the enteroid and the single dissociated mesenchymal cell is either intestinal tissue or colonic tissue, or both. In other embodiments, the histological type of the single dissociated mesenchymal cell includes other histological types other than intestinal or colonic tissue, thereby forming a composite organoid from an enteroid having the properties of the other histological type. In some embodiments, the enteroid may be derived from intestinal or colonic tissue from a subject. In some embodiments, the enteroid is derived from intestinal or colonic tissue obtained from a biopsy. It is assumed that the formation of a composite organoid from the process herein using an enteroid derived from intestinal or colonic tissue may be faster, easier, more cost-effective, or less destructive to the subject (e.g., if a biopsy has already been performed) compared to a similar organoid produced from a subject-derived PSC.

[0185] Any of the uses and / or embodiments provided herein may be used in combination with any other uses and / or embodiments to produce combined single dissociated mesenchymal cells and epithelial structures, and the resulting composite organoids.

[0186] Combination of single dissociated mesenchymal cells and epithelial structures In some embodiments, a single dissociated mesenchymal cell and an epithelial structure (or a suitable substitute) are combined by centrifugation. In some embodiments, suitable centrifugation parameters (e.g., rate and duration) for combining a single dissociated mesenchymal cell and an epithelial structure are 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 × g, or about those, less than those, about less than those, at least those, or at least about those, or a range defined by any two of the aforementioned values, e.g., 50-600xg, 100-300xg, 150-500xg, 200-400xg, 50-300xg, 50-35 A rate of 0xg or 250-600xg, and / or a duration of centrifugation selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or about those, less than or equal to those, about less than or equal to those, at least those, or at least about those, or a range defined by any two of the aforementioned values, e.g., 1-30 minutes, 1-5 minutes, 1-10 minutes, 10-20 minutes, 20-30 minutes, or 5-15 minutes. In other embodiments, single dissociated mesenchymal cells and epithelial structures are combined by any other suitable conventional method for agglutinating cells, including sedimentation by gravity.

[0187] Maturation of composite organoids After combining a single dissociated mesenchymal cell and an epithelial structure (or any suitable substitute or variant discussed herein), the combined single dissociated mesenchymal cell and epithelial structure are cultured to form, grow, and mature a composite organoid. The combined cells can be cultured in vitro or transplanted into a compatible organism (e.g., a human, mouse, rat, dog, cat, monkey, or any other mammal that may be optionally immunodeficient) to mature.

[0188] In some embodiments, the combined single dissociated mesenchymal cells and epithelial structures are cultured under growth conditions suitable for supporting the resulting composite organoid. In some embodiments, the combined single dissociated mesenchymal cells and epithelial structures are cultured under the conditions described herein or otherwise known in the art. These conditions may involve contacting the combined cells with one or more signaling pathway activators, signaling pathway inhibitors, or any other growth factors. For example, the combined cells are contacted with a ROCK inhibitor to improve cell viability. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor is at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, e.g., 1–20 μM, 1–10 μM, 5–15 μM, or 10–20 μM. In some embodiments, where the composite organoid includes two or more tissue types, the conditions for culturing the combined cells may be optimized for one of the tissue types, or a combination of conditions optimized for two or more tissue types.

[0189] In some embodiments, the composite organoid is embedded in an extracellular matrix or its mimetic or derivative for further culture and growth. In some embodiments, the composite organoid is cultured for a desired time. In some embodiments, the composite organoid is cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, or 40 days, or any number of days within the range defined by any two of the aforementioned number of days, for example, 1 to 40 days, 1 to 10 days, 10 to 20 days, 20 to 30 days, 30 to 40 days, 1 to 2 days, 1 to 30 days, or 10 to 40 days.

[0190] In some embodiments, the composite organoid is transplanted into an appropriate region of the recipient subject. In some embodiments, the recipient subject is one of either a donor organoid or an enteroid (and therefore a single dissociated mesenchymal cell and / or epithelial structure). In other embodiments, the recipient subject is not a donor organoid or an enteroid. In some embodiments, the composite organoid may be transplanted after culturing the composite organoid for a certain period of time, or immediately after combining the single dissociated mesenchymal cell and the epithelial structure.

[0191] In some embodiments, transplantation is performed after culturing the composite organoid for 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 days, or about those days, at least those days, at least about those days, less than or about those days, or less than or about those days, or any number of days within the range defined by any two of the aforementioned number of days, for example, 1 to 50 days, 10 to 40 days, 20 to 30 days, 1 to 30 days, or 20 to 50 days. In some embodiments, the composite organoid is sufficiently mature for transplantation and / or study several days before organoids prepared by other methods known in the art reach the same or similar state of maturity, the number of days being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any number of days within the range defined by any two of the aforementioned number of days, e.g., 1 to 20 days, 5 to 15 days, 10 to 15 days, 1 to 15 days, or 10 to 20 days.

[0192] In some embodiments, the composite organoid is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 4 in the recipient object. 0, 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, 8 Growing over 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 days, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any number of days within the range defined by any two of the aforementioned number of days, for example, 1 to 120 days, 1 to 60 days, 10 to 120 days, 10 to 90 days, 10 to 60 days, 10 to 30 days, 30 to 60 days, or 60 to 120 days. In some embodiments, the composite organoid exhibits integration with the recipient tissue. In some embodiments, the composite organoid comprises a single dissociated mesenchymal cell and a tissue-type cell lineage of epithelial structures as its components. In some embodiments, the composite organoid spontaneously develops cell lineages after engraftment.

[0193] In some embodiments, composite organoids exhibit greater engraftment and growth in recipient tissue compared to equivalent non-composite organoids or enteroids. In some embodiments, equivalent non-composite organoids or enteroids fail to engraft and / or grow in recipient tissue, while composite organoids successfully engraft and / or grow in recipient tissue. This can be used for mesenchymal cell enrichment in composite organoids, which is lacking in equivalent non-composite organoids. In some embodiments, equivalent non-composite organoids or enteroids are organoids cultured in vitro according to conventional methods known in the art or applicable as described herein (e.g., used as donor organoids or enteroids) and transplanted into the same or similar recipient tissue. In some embodiments, greater engraftment and growth can be measured in terms of the time required for complete maturation after engraftment. In some embodiments, the composite organoid requires a length of time that is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the length of time it takes for an applicable conventional organoid or enteroid to mature after engraftment, or about those, at least those, at least about those, less than those, or about those less than those, or any percentage of time within the range defined by any two of the aforementioned percentages of time, e.g., 5% to 100%, 10% to 50%, 50% to 90%, or 30% to 50%. In some embodiments, greater engraftment and growth can be measured in terms of the relative size of the composite organoid.In some embodiments, the composite organoid is 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% of the size of an applicable conventional organoid (with respect to any dimension including, but not limited to, length, width, depth, radius, diameter, circumference, volume, or surface area), or about that, at least that, at least about that, less than that, or about that, or any percentage within the range defined by any two of the aforementioned percentages, e.g., 100% to 500%, 100% to 200%, 200% to 500%, or 300% to 500%.

[0194] Gastrointestinal organoids One application of the composite organoids disclosed herein is for use relating to the gastrointestinal organs. In some embodiments, the composite organoids disclosed herein are gastrointestinal organoids. In some embodiments, the gastrointestinal organoids are esophageal organoids, gastric organoids, gastric fundus organoids, vestibulostomy organoids, liver organoids, intestinal organoids, or colon organoids, or any combination thereof. In some embodiments, the gastrointestinal organoids used in any of the methods disclosed herein (e.g., to isolate a single dissociated mesenchymal cell and / or epithelial structure) are produced according to the methods described herein or otherwise known in the art.

[0195] In some embodiments, 1) one or more organoids from which a single dissociated mesenchymal cell is isolated, and 2) organoids or enteroids from which epithelial structures are isolated each include a tissue type selected from esophageal, gastric, hepatic, intestinal, or colonic tissue types, or any combination thereof. In some embodiments, one or more organoids, or organoids or enteroids, or both, are human organoids or enteroids. In some embodiments, one or more organoids, or organoids or enteroids, or both, include human esophageal organoids (HEO), human gastric organoids (HGO), human fundic gastric organoids (HFGO), human antral gastric organoids (HAGO), human hepatic organoids (HHO), human intestinal organoids (HIO), or human colonic organoids (HCO), or any combination thereof.

[0196] In some embodiments, 1) one or more organoids from which a single dissociated mesenchymal cell is isolated include an intestinal tissue type, and 2) organoids or enteroids from which epithelial structures are isolated include esophageal, gastric, hepatic, intestinal, or colonic tissue types, or any combination thereof. In some embodiments, one or more organoids from which a single dissociated mesenchymal cell is isolated are small intestinal organoids, optionally HIO. The use of single dissociated mesenchymal cells isolated from intestinal organoids may offer some clearly different advantages, as currently known processes for differentiating intestinal organoids from pluripotent stem cells produce a considerable amount of mesenchymal cells compared to differentiation protocols for organoids of other tissue types, which can result in a reduction in the number of mesenchymal cells. In some embodiments, the methods for producing composite organoids disclosed herein are intended to compensate for this reduction in mesenchymal cell differentiation in other organoid protocols. In some embodiments, a large number of mesenchymal cells produced from intestinal organoid differentiation can be used to supplement organoids of other tissue types. However, it should be noted that the methods disclosed herein are not limited to the use of intestinal organoids solely as a source of single dissociated mesenchymal cells. Furthermore, in some embodiments, the use of single dissociated mesenchymal cells as disclosed herein has additional / other advantages, such as producing composite organoids of a single tissue type.

[0197] In some embodiments, the tissue types of one or more organoids and the tissue types of organoids or enteroids are different. In some embodiments, the resulting gastrointestinal organoids are heterogeneous organoids. When applied to the composite organoids disclosed herein, a composite organoid may include a mesenchyme comprising a first tissue type selected from the esophageal, gastric, hepatic, intestinal, or colonic tissue types, or any combination thereof, and an epithelium comprising a second tissue type selected from the esophageal, gastric, hepatic, intestinal, or colonic tissue types, or any combination thereof. In some embodiments, the first and second tissue types have at least one tissue type difference. In some embodiments, some non-limiting examples of composite gastrointestinal organoids are intestinal mesenchymal / intestinal epithelial organoids, intestinal mesenchymal / colonic epithelial organoids, intestinal mesenchymal / gastric epithelial organoids, intestinal mesenchymal / enteroid epithelial organoids, gastric mesenchymal / gastric epithelial organoids, and colonic mesenchymal / colonic epithelial organoids. In some embodiments, the tissue type of one or more organoids from which a single dissociated mesenchymal cell is isolated differs from the tissue type of organoid or enteroid from which the epithelial structure is isolated. Re-patterning of the epithelial structure by the single dissociated mesenchymal cell does not occur, and as a result, the epithelium of the composite organoid maintains the tissue type of the organoid or enteroid from which the epithelial structure is isolated. In some embodiments, if the epithelial structure is derived from an organoid that is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or from an organoid that is 14-30, 15-30, 18-30, 15-20, or 15-25 days old, re-patterning of the epithelial structure does not occur. In some embodiments, if the epithelial structure is derived from an enteroid, re-patterning of the epithelial structure does not occur. In some embodiments, the enteroid is derived from adult tissue.In some embodiments, the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated differs from the histological type of organoid or enteroid from which the epithelial structure is isolated. Re-patterning of the epithelial structure by the single dissociated mesenchymal cell occurs, and as a result, the epithelium of the composite organoid exhibits the characteristics of the histological type of one or more organoids from which a single dissociated mesenchymal cell is isolated. In some embodiments, re-patterning of the epithelial structure by the single dissociated mesenchymal cell may occur when the epithelial structure is derived from organoids aged 8, 9, 10, 11, 12, or 13 days or younger, or from organoids aged 8-13, 8-10, or 10-13 days. In some embodiments, the enteroids are derived from adult tissue with distinct patterning, and the epithelial structure derived from the enteroids maintains its histological type even when recombined with a single dissociated mesenchymal cell.

[0198] In some embodiments, the tissue type of one or more organoids and the tissue type of an organoid or enteroid are the same. In some embodiments, one or more organoids and the organoid or enteroid each include only one of the esophageal, gastric, liver, intestinal, or colonic tissue types. In some embodiments, the resulting composite gastrointestinal organoid is a homogeneous organoid containing only one tissue type.

[0199] In some embodiments, the combined single dissociated mesenchymal cells and epithelial structures are cultured under conditions optimized for esophageal, gastric, hepatic, intestinal, or colonic organoid growth. In some embodiments, the combined single dissociated mesenchymal cells and epithelial structures are cultured in mini-intestinal medium. In some embodiments, the mini-intestinal medium comprises one or more of the following: advanced DMEM / F12 medium, glutamine, HEPES, penicillin, streptomycin, N2 supplement, B27 supplement, epidermal growth factor (EGF), or ROCK inhibitor, or any combination thereof. In some embodiments, the EGF is human EGF (hEGF). In some embodiments, EGF is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or about those concentrations, at least those concentrations, at least about those concentrations, or less than or about those concentrations, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 10-200 ng / mL, 50-150 ng / mL, 80-120 ng / mL, 10-100 ng / mL, or 100-200 ng / mL. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor is a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about those, at least those, at least about those, less than or about those, or about less than or about those, or any concentration within the range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM.

[0200] In some embodiments, the composite gastrointestinal organoid includes a lumen. In some embodiments, the composite gastrointestinal organoid includes a lumen that accounts for a percentage of the total volume of the composite gastrointestinal organoid. In some embodiments, the lumen accounts for 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 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%, 38%, 39%, or 40% of the total volume of the composite gastrointestinal organoid. A percentage of the total volume of composite gastrointestinal organoids that is %, approximately %, at least %, at least approximately %, less than or approximately %, or less than or equal to %, or any percentage within the range defined by any two of the aforementioned percentages, e.g., 1% to 40%, 10% to 30%, 15% to 20%, 1% to 20%, or 10% to 40%.

[0201] In non-limited cases of HIO-mesenchymal / HCO-epithelial xenogeneic organoids, the xenogeneic organoids express sucrase-isomaltase (SI), which is specific to the epithelium of the small intestine, and do not express SATB2, which is specific to the epithelium of the large intestine.

[0202] In non-limited cases of HIO mesenchymal / HEO epithelial xenogeneic organoids, the xenogeneic organoids express KRT5, KRT14, KRT13, IVL, p63, and CDH1.

[0203] Use of composite organoids Composite organoids disclosed herein, or produced by any of the methods disclosed herein, may be used for a variety of purposes, including but not limited to providing a source of tissue for transplantation, drug screening, studying organ function, neuronal function, microbiome interactions, or any combination thereof.

[0204] In some embodiments, the methods disclosed herein include an additional step of transplanting one of the composite organoids disclosed herein into a recipient subject not only to mature the organoids described herein, but also in addition to or as an alternative to restoring, repairing, or improving organ function in the recipient subject. These methods may be used to treat subjects with impaired organ function, or to do so in subjects requiring the improvement, inhibition, or treatment of adverse organ damage. In some embodiments, the recipient subject is one or more organoids from which a single dissociated mesenchymal cell is isolated, or an organoid or enteroid from which an epithelial structure is isolated, or both are isolated. In some embodiments, the composite organoid or its components are derived from PSCs isolated from the recipient subject. In some embodiments, the recipient subject is a mammal. In some embodiments, the recipient subject is an immunodeficient mammal. In some embodiments, the recipient subject is an immunodeficient mouse. In some embodiments, the recipient subject is a monkey, cat, dog, hamster, or rat. In some embodiments, the recipient is an immunodeficient monkey, cat, dog, hamster, or rat. In some embodiments, the recipient is a human. In some embodiments, the recipient is an immunodeficient human. In some embodiments, the recipient is an immunocompetent human. In some embodiments, the recipient is an immunocompetent human treated with an immunosuppressant. In some embodiments, the recipient is an immunocompetent human and the composite organoid is autologous to the recipient. In some embodiments, the recipient is an immunocompetent human and the composite organoid is allogeneic to the recipient. In some embodiments, the recipient is a mammal in need of organ transplantation. In some embodiments, the recipient is a human in need of organ transplantation.

[0205] Composite organoids for use in treating diseases in subjects requiring such treatment are also described herein. In some embodiments, the composite organoid is a composite organoid described herein. In some embodiments, the liver organoid is a liver organoid produced by any one of the methods described herein.

[0206] In any of the therapeutic methods or uses provided herein, the composite organoid may be a composite gastrointestinal organoid. Therefore, any of the methods provided herein are applicable to treating subjects with impaired gastrointestinal function, or to doing so in subjects requiring improvement, inhibition, or treatment of adverse gastrointestinal disorders. In some embodiments, the method involves implanting or engrafting a composite gastrointestinal organoid in a subject. In some embodiments, the composite gastrointestinal organoid is an esophageal organoid, gastric organoid, gastric fundus organoid, vestibulogastric organoid, hepatic organoid, small intestine (intestinal) organoid, or large intestine (colon) organoid. In some embodiments, the subject requires gastrointestinal transplantation. In some embodiments, the gastrointestinal organoid is implanted or engrafted as a whole gastrointestinal organoid. In some embodiments, the transplantation site is gastrointestinal tissue. Composite gastrointestinal organoids can also be used to do so in subjects requiring treatment of gastrointestinal diseases.

[0207] Methods for screening candidate therapeutic agents are also disclosed herein. In some embodiments, the method includes contacting one of the organoids disclosed herein with a candidate therapeutic agent and determining the effect of the candidate therapeutic agent on the organoid. In some embodiments, the organoid is genetically modified. In some embodiments, the organoid is genetically modified to exhibit a disease or a model thereof. In some embodiments, the mesenchyme and / or epithelium of the organoid is genetically modified. In some embodiments, the mesenchyme and / or epithelium of the organoid is genetically modified to exhibit a disease or a model thereof.

[0208] Examples Some aspects of the embodiments discussed herein are further disclosed in the following examples, and these are not intended to limit the scope of this disclosure. Those skilled in the art will understand that many other embodiments, as described herein and in the claims, are also within the scope of this disclosure.

[0209] Example 1. Production of a composite organoid composition Human pluripotent stem cells were cultured and induced to differentiate into endoderm cells. Subsequently, the endoderm cells were differentiated into human intestinal organoids (HIO), human colon organoids (HCO), or human vestibulostomy organoids (HAGO). Organoids cultured for 10–30 days could be used in the following steps. Alternatively, mesenchymal enteroids could be derived from intestinal or colon tissue from the subject. Methods for producing these organoids or enteroids can be found, for example, in PCT International Publications 2011 / 140441, 2015 / 183920, 2016 / 061464, 2017 / 192997, and 2018 / 106628, each of which is expressly incorporated herein in whole by reference.

[0210] Organoids or enteroids were prepared in extracellular matrix (e.g., Matrigel Growth Factor Reduced [Corning]). The organoids or enteroids were washed with an appropriate volume of Dulbecco's PBS (DPBS; e.g., 500 μL per 24-well plate), the DPBS was removed, and an appropriate volume of ice-cold cell recovery solution (Corning; e.g., 500 μL) was added to separate the extracellular matrix droplets from the culture plate. The organoids or enteroids and extracellular matrix were transferred to 15 mL tubes and incubated at 4°C for 30 minutes with gentle agitation to induce depolymerization of the extracellular matrix. Up to 24 wells can be pooled in the same 15 mL tube. For heterorecombination purposes, different types of organoids or enteroids are typically processed simultaneously in separate tubes. The tubes were centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and 5 mL of fresh ice-cold cell recovery solution was added. The mesenchyme and epithelium of organoids (or enteroid epithelium) were mechanically dissociated by pipetting up and down using a 5 mL serum pipette. The dissociation process was periodically monitored under a microscope. Complete dissociation was achieved when intact epithelial structures were free from surrounding mesenchyme (Figure 1, Panel A). To facilitate the separation of mesenchyme from epithelium and prevent the epithelial structures from becoming fragmented, manual dissociation could be alternated with incubation at 4°C for 10 minutes with gentle agitation. The dissociated solution was filtered using a 40 μm mesh cell filter placed upside down on top of a 50 mL tube to separate single dissociated mesenchyme from the epithelial structures. The cell filter was rinsed with 5 mL of fresh, cold DPBS. Using forceps, the cell filter, holding the epithelial structures, was moved to the right into a 6-well plate containing 5 mL of DPBS. The cell filter was immersed in the solution several times to separate the epithelial structures from the cell filter mesh into the wells. To prevent epithelial structures from remaining on the cell filter mesh and adhering to the bottom of the wells, the cell filter can be immersed in a PBS solution of 0.5% BSA in the wells for 30 minutes before use.A 50 mL tube containing a single dissociated mesenchymal cell was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and 2 mL of mini-intestinal medium (high-grade DMEM / F12 medium supplemented with 2 mM glutamine, 10 mM HEPES, 100 U / mL penicillin, 100 μM streptomycin, 1 × N2 supplement, and 1 × B27 supplement) was added, supplemented with 100 ng / mL human epidermal growth factor (hEGF) and 10 μM Y-27632 (or equivalent ROCK inhibitor). The number of mesenchymal cells was counted using trypan blue to eliminate any dead cells. If epithelial cell contamination was observed in the mesenchymal cell solution, the solution in the tube was transferred to a plate and incubated at room temperature for several minutes to allow the epithelial cells to adhere to the bottom of the plate. Non-adherent mesenchymal cells were then slowly aspirated and could be used in subsequent steps.

[0211] Seed 150,000 mesenchymal cells per well into a very low-adhesion round-bottom 96-well plate (Corning). Under a horizontal laminar flow hood, pick up epithelial structures with a micropipette. Add one epithelial structure to each well containing mesenchymal cells. Depending on the purpose of the experiment, the epithelial structure may be from the same type of organoid as the mesenchymal cells, a different type of organoid, or an enteroid. If the epithelial structure is larger than the tip of the micropipette tip, the tip pore can be enlarged by cutting the pore using sterile scissors. Centrifuge the plate at 300 × g for 2 minutes to aggregate the epithelial structures and mesenchymal cells (Figure 1, Panel B). Incubate the plate overnight at 37°C so that the combined cells form organoid morphology (Figure 1, Panel C). Collect each composite organoid using a micropipette with an appropriately sized pore and plate it in 50 μL of Matrigel droplets in a 24-well plate. Add 500 μL of mini-intestinal medium supplemented with 100 ng / mL of human EGF to the plate. Maintain the composite organoid in the culture for the desired time. In some embodiments, the composite organoid is cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, or 40 days, or any number of days within the range defined by any two of the aforementioned number of days, for example, 1 to 40 days, 1 to 10 days, 10 to 20 days, 20 to 30 days, 30 to 40 days, 1 to 2 days, 1 to 30 days, or 10 to 40 days.

[0212] Example 2. Recombination of epithelial and mesenchymal tissue from different sources reliably produces functional xenoorganoids. Human intestinal organoids (HIO) and human colon organoids (HCO) were prepared separately. HCO was differentiated from GFP-expressing iPSCs. Single dissociated mesenchymal HIO and epithelial structures of HCO were prepared and recombined according to Example 1 (using organoids from either day 11 or day 18) to form HIO-mesenchymal / HCO-epithelial heteroorganoids. These organoids exhibited reliable recombination and growth in vitro when examined 48 hours and 9 days after recombination, with the intestinal mesenchyme enclosing GFP-positive colon epithelium (Figure 2A). After engraftment onto the renal capsule of immunodeficient mice, the heteroorganoids prepared from day 18 source organoids matured and formed luminal structures and distinctly different microvilli (Figure 2B). Microvilli arising from the progenitor colon were positive for GFP and E-cadherin (E-CAD; CDH1). Heterogeneous organoids prepared from source organoids on day 18 also expressed special AT-rich sequence-binding protein 2 (SATB2) in the epithelial layer, but were negative for small intestine-specific sucrase-isomaltase (SI), suggesting that the epithelial structures from these HCOs retained their distal features (Figure 2C).

[0213] Heterogeneous organoids prepared from source organoids on day 11 expressed small intestine-specific GATA-binding protein 4 (GATA4) but were negative for colon-specific SATB2, demonstrating an interesting property that younger and more immature epithelial structures can be reprogrammed to exhibit properties mediated by the surrounding mesenchyme (in this case, HCO-derived epithelium exhibits small intestinal properties (Figure 2D)).

[0214] HIO and human gastric organoids (HGO) were prepared separately. Single dissociated mesenchymal HIO and epithelial structures of HGO were prepared and recombined according to Example 1 to form HIO-mesenchymal / HGO-epithelial heterogeneous organoids. These organoids showed reliable recombination and growth in vitro when examined on days 4 and 11 after recombination (Figure 2E).

[0215] HIO and human enteroids were prepared separately. Since the enteroids lacked mesenchyme, no further processing was necessary. Single dissociated mesenchyme of HIO and enteroids were recombined according to Example 1 to form HIO-mesenchymal / enteroid heterogeneous organoids. These organoids exhibited reliable recombination and growth in vitro when examined 10, 21, and 31 days after recombination (Figure 2F).

[0216] Example 3. Engraftment of composite organoids We evaluated the engraftment ability of various heterologous organoids in an immunodeficient mouse model. Human organoids differentiated from iPSCs into different organ types have been observed to possess varying amounts of mesenchyme. For example, HIO produced according to known methods has abundant mesenchymal cells that support epithelial and organoid maturation, while HGO and HCO significantly reduce the number of mesenchymal cells, and enteroids completely lack mesenchyme, making engraftment of these organoid types difficult. The process of recombining mesenchymal and epithelial components from separate organoid sources with a mesenchymal-to-epithelial ratio that closely resembles in vivo tissue leads to greater success in organoid engraftment and growth when transplanted into the mouse kidney capsule (Figure 3). HIO-mesenchymal / HIO-epithelial, HIO-mesenchymal / HCO-epithelial, and HIO-mesenchymal / HAGO-epithelial heterologous organoids all exhibited successful engraftment and maturation. HIO-mesenchymal / enteric organoids were also engraftable. Furthermore, mesenchymal cells were isolated from several iPSC-differentiated HAGO or HCO organoids, and these mesenchymal cells were recombined with HAGO or HCO epithelial structures to prepare HAGO mesenchyme / HAGO epithelium and HCO mesenchyme / HCO epithelium allogeneic organoids, thereby enriching the number of available supporting mesenchymal cells in each organoid. Compared to control HAGO and HCO organoids that did not mature and grow after engraftment, or showed limited maturation and growth, the mesenchymal-enriched HAGO and HCO organoids grew more reliably on recipient renal capsule tissue.

[0217] Example 4. Production of composite esophageal organoids Similar to HGO and HCO, human esophageal organoids (HEOs) produced by previous methods have a reduced number of mesenchymal cells compared to intestinal organoids. A method for producing esophageal organoids can be found, for example, in PCT International Publication 2019 / 074793, which is expressly incorporated herein in its entirety by reference.

[0218] On day 12, single dissociated HIO mesenchyme (approximately 50,000 cells) and intact HEO epithelium (one or two structures) were recombined in low-adhesion 96-well plates using the protocol described herein. The following day, the recombinant HIO / HEO was transferred to Matrigel and cultured in vitro until transplantation into the renal capsule of NSG mice on day 28. The transplanted tissue was harvested 8 weeks after engraftment, fixed in 4% paraformaldehyde, processed, and embedded in paraffin for imaging. Figure 4A shows an image of the combined organoid structure consisting of HIO mesenchyme (GFP-positive) and HEO epithelium (GFP-negative) 48 hours after the recombination procedure. Figure 4B shows an image of the HIO-mesenchymal / HEO-epithelial organoid 8 weeks after transplantation into the mouse renal capsule. Figure 4C shows hematoxylin / eosin staining of the transplanted HIO-mesenchymal / HEO-epithelial organoid. Figure 4D shows images of GFP (indicating HIO mesenchyme) and E-cadherin (CDH1; indicating HEO epithelium), showing clearly different isolations of the two layers. Figure 4E shows immunofluorescence images showing the expression of keratin 5 (KRT5), keratin 14 (KRT14), keratin 13 (KRT13), involucrin (IVL), p63, and CDH1 in mature esophageal epithelium.

[0219] In at least some of the embodiments described above, one or more elements used in the embodiment may be interchangeably used in another embodiment unless such substitution is not technically feasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter as defined by the appended claims.

[0220] With regard to substantially all use of plural and / or singular terms herein, those skilled in the art can paraphrase from plural to singular and / or singular to plural as appropriate to the context and / or use. For clarity, various singular / plural substitutions can be clearly described herein.

[0221] A person skilled in the art will understand that, in general, the terms used herein, and in particular in the appended claims (e.g., the text of the appended claims), are intended to be generally "open" terms (for example, the term "including" is typically interpreted as "including but not limited to," the term "having" is typically interpreted as "having at least," and the term "include" is typically interpreted as "including but not limited to"). A person skilled in the art will further understand that where there is an intended specific number of claim details to be introduced, such intent is explicitly detailed in this claim, and where there is no such detail, such intent does not exist. For example, for the sake of understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce claim details. However, the use of such phrases is typically interpreted as meaning that the introduction of a claim detail by the indefinite article "a" or "an" limits any particular claim containing such introduced detail to only one embodiment containing such detail, even if the same claim contains the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (for example, "a" and / or "an" are typically interpreted as meaning "at least one" or "one or more"). The same applies to the use of the definite article used to introduce a claim detail. In addition, even if a particular number of introduced claim details is explicitly detailed, a person skilled in the art will recognize that such details are typically interpreted as meaning at least the number of details (for example, the mere detail "two details" without other modifying phrases means at least two details or two or more details).Furthermore, where a convention similar to "at least one of A, B, and C, etc." is used, such a construction is usually intended to mean what a person skilled in the art would understand (for example, "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or systems having A, B, and C together, etc.). Where a convention similar to "at least one of A, B, or C, etc." is used, such a construction is usually intended to mean what a person skilled in the art would understand (for example, "a system having at least one of A, B, or C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or systems having A, B, and C together, etc.). A person skilled in the art will further understand that substantially any disjunct words and / or disjunct phrases presenting two or more alternative terms in the specification, claims, or drawings are typically understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B".

[0222] In addition, if any feature or aspect of the present disclosure is described by the Markush Group, a person skilled in the art will recognize that the present disclosure may also be described by any individual member or subgroup of a member of the Markush Group.

[0223] For all purposes, including the standpoint of documenting the subject matter in a manner that can be understood by those skilled in the art, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. It is readily apparent that any scope enumerated may be adequately described and made possible to decompose the same scope into at least two, three, four, five, ten, etc., subscopes. As a non-limiting example, each scope considered herein may be readily decomposed into a lower third, a middle third, an upper third, etc. As can be understood by those skilled in the art, all terms such as “maximum,” “at least,” “greater than,” and “less than” include the numbers detailed and refer to scopes that may later be decomposed into subscopes as considered herein. Finally, as can be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0224] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be obvious to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to limit, and the true scope and spirit are set forth in the following claims.

[0225] All references cited herein, including but not limited to published and unpublished applications, patents, and documents, are incorporated herein by reference in their entirety and become part of this Specification. To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained in this Specification, this Specification is intended to take precedence over and / or supersede such conflicting material.

[0226] References K.W.McCracken,Mechanisms of endoderm patterning and directed differentiation of human stem cells into foregut tissues,PhD Thesis,University of Cincinnati,2014. J.O.Munera,N.Sundaram,S.A.Rankin,D.Hill,C.Watson,M.Mahe,J.E.Vallance,N.F.Shroyer,K.L.Sinagoga,A.Zarzoso-Lacoste,J.R.Hudson,J.C.Howell,P.Chatuvedi,J.R.Spence,J.M.Shannon,A.M.Zorn,M.A.Helmrath,J.M.Wells,Differentiation of Human Pluripotent Stem Cells into Colonic Organoids via Transient Activation of BMP Signaling,Cell Stem Cell.21(2017)51-64.e6.https: / / doi.org / 10.1016 / j.stem.2017.05.020. C.L.Watson,M.M.Mahe,J.Munera,J.C.Howell,N.Sundaram,H.M.Poling,J.I.Schweitzer,J.E.Vallance,C.N.Mayhew,Y.Sun,G.Grabowski,S.Finkbeiner,J.R.Spence,N.F.Shroyer,J.M.Wells,M.A.Helmrath,An in vivo model of human small intestine using pluripotent stem cells,Nat.Med.20(2014)1310-1314.https: / / doi.org / 10.1038 / nm.3737. M.M.Mahe,N.Sundaram,C.L.Watson,N.F.Shroyer,M.A.Helmrath,Establishment of human epithelial enteroids and colonoids from whole tissue and biopsy,JoVE J.Vis.Exp.(2015)e52483. M.M.Mahe,N.E.Brown,H.M.Poling,M.A.Helmrath,In vivo model of small intestine,in:Organ Regen.,Springer,2017:pp.229-245.

Claims

1. An in vitro method for producing complex organoids, a) Obtaining single, dissociated intestinal mesenchymal cells isolated from one or more intestinal organoids, b) Obtaining epithelial structures isolated from organoids or enteroids, c) Combining the single dissociated intestinal mesenchymal cell and the epithelial structure in vitro, d) A method comprising culturing the combined single dissociated intestinal mesenchymal cells and the epithelial structure in vitro for at least one day to form the composite organoid.

2. The method according to claim 1, wherein obtaining the single dissociated intestinal mesenchymal cell comprises isolating the single dissociated intestinal mesenchymal cell from one or more intestinal organoids, and / or obtaining the epithelial structure comprises isolating the epithelial structure from the organoid or enteroid.

3. The method according to any one of claims 1 to 2, wherein the single dissociated intestinal mesenchymal cell, or the epithelial structure, or both, is isolated by mechanical dissociation and filtration, and / or the single dissociated intestinal mesenchymal cell and the epithelial structure are combined by centrifugation.

4. The method according to any one of claims 1 to 3, wherein the number of mesenchymal cells in the composite organoid is greater than the original number of mesenchymal cells in the organoid or enteroid from which the epithelial structure is isolated, and as a result the composite organoid has concentrated mesenchyme.

5. The method according to any one of claims 1 to 4, wherein 1) one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated, and 2) the organoid or enteroid from which the epithelial structure is isolated each comprises a tissue type selected from esophageal, gastric, liver, intestinal, or colonic tissue types, or any combination thereof.

6. The method according to any one of claims 1 to 5, wherein 1) the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated include an intestinal tissue type, and 2) the organoid or enteroid from which the epithelial structure is isolated includes a tissue type selected from esophageal, gastric, liver, intestinal, or colonic tissue types, or any combination thereof, and / or the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated is a small intestinal organoid.

7. The histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cells are isolated, and the histological type of the organoid or enteroid from which the epithelial structure is isolated, are different, or The histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated differs from the histological type of the organoid or enteroid from which the epithelial structure is isolated, and the re-patterning of the epithelial structure by the single dissociated intestinal mesenchymal cell does not occur, and as a result the epithelium of the composite organoid maintains the histological type of the organoid or enteroid, and / or The histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated is different from the histological type of the organoid or enteroid from which the epithelial structure is isolated, and the organoid or enteroid from which the epithelial structure is isolated is an organoid, and the organoid from which the epithelial structure is isolated is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or 14-30, 15-30, 18-30, 15-20, or 15-25 days old, and / or The method according to any one of claims 5 to 6, wherein the histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated is different from the histological type of the organoid or enteroid from which the epithelial structure is isolated, and the organoid or enteroid from which the epithelial structure is isolated is an enteroid, and the enteroid is derived from adult tissue.

8. The method according to claim 7, wherein the organoid or enteroid from which the epithelial structure is isolated is an organoid, and the histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated is different from the histological type of the organoid from which the epithelial structure is isolated, and repatterning of the epithelial structure by the single dissociated intestinal mesenchymal cell occurs, and as a result the epithelium of the composite organoid exhibits the characteristics of the histological type of the one or more organoids.

9. The method according to any one of claims 5 to 6, wherein the histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated is the same as the histological type of the organoid or enteroid from which the epithelial structure is isolated, and / or the histological type of the one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated is the same as the histological type of the organoid or enteroid from which the epithelial structure is isolated, and each of the one or more organoids and the organoid or enteroid comprises only one of the esophageal, gastric, liver, intestinal, or colonic histological types, and the resulting composite organoid is a homogeneous organoid comprising one histological type.

10. The method according to any one of claims 1 to 9, wherein the one or more organoids are derived from pluripotent stem cells (PSCs) from a first subject, and the organoids or enteroids are derived from PSCs from a second subject or isolated from gastrointestinal tissue from a second subject.

11. The method according to claim 10, wherein the first subject and the second subject are mammals.

12. The method according to any one of claims 10 to 11, wherein the first object and the second object are the same individual.

13. The method according to any one of claims 1 to 12, wherein the composite organoid is implanted in a recipient.

14. The method according to claim 13, wherein the composite organoid exhibits greater engraftment and growth in the recipient subject compared to an equivalent non-composite organoid or enteroid.

15. The method according to any one of claims 1 to 14, wherein 1) one or more organoids from which the single dissociated intestinal mesenchymal cell is isolated, or 2) the organoid or enteroid, or both, from which the epithelial structure is isolated, are manipulated to contain one or more exogenous nucleic acids or proteins, and / or include gene mutations associated with a disease state or a model of a disease state.

16. It is a composite organoid, The mesenchyme containing intestinal mesenchymal cells isolated as single dissociated cells from the first intestinal organoid, A composite organoid comprising epithelium containing epithelial cells isolated as an epithelial structure from a second organoid or enteroid.

17. The composite organoid according to claim 16, wherein the ratio of mesenchymal cells to epithelial cells in the composite organoid is greater than the ratio in the second organoid or enteroid.

18. The composite organoid according to claim 16 or 17, wherein the first organoid and the second organoid or enteroid each include a tissue type selected from the esophageal, gastric, liver, intestinal, or colon tissue type, or any combination thereof.

19. The composite organoid according to claim 18, wherein the tissue type of the first organoid and the tissue type of the second organoid or enteroid are the same.

20. The tissue type of the first organoid from which the mesenchymal cells are isolated is different from the tissue type of the second organoid or enteroid from which the epithelial cells are isolated, and the repatterning of the epithelial cells by the mesenchymal cells does not occur, and as a result the epithelium of the composite organoid maintains the tissue type of the second organoid or enteroid from which the epithelial cells are isolated, and / or The tissue type of the first organoid from which the mesenchymal cells are isolated is different from the tissue type of the second organoid or enteroid from which the epithelial cells are isolated, and the second organoid or enteroid is the second organoid, and the second organoid is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days old, or 14-30, 15-30, 18-30, 15-20, or 15-25 days old, and / or The composite organoid according to claim 19, wherein the tissue type of the first organoid from which the mesenchymal cells are isolated is different from the tissue type of the second organoid or enteroid from which the epithelial cells are isolated, and the second organoid or enteroid is a second enteroid, and the second enteroid is derived from adult tissue.

21. The composite organoid according to claim 20, wherein the second organoid or enteroid is a second organoid, and the tissue type of the first organoid from which the mesenchymal cells are isolated is different from the tissue type of the second organoid from which the epithelial cells are isolated, and repatterning of the epithelial cells by the mesenchymal cells occurs, and as a result the epithelium of the composite organoid exhibits the characteristics of the tissue type of the first organoid from which the mesenchymal cells are isolated.

22. The mesenchyme includes a first histological type, and the first histological type is an intestinal histological type. The epithelium comprises a second tissue type selected from the esophageal, gastric, liver, intestinal, or colonic tissue type, or any combination thereof. The composite organoid according to claim 16, wherein the first tissue type and the second tissue type have at least one difference between the tissue types.

23. A composite organoid according to any one of claims 16 to 22, comprising one or more exogenous nucleic acids or proteins, and / or having or being manipulated to include a gene mutation associated with a disease state or a model of a disease state.

24. A composite organoid produced by the method described in any one of claims 1 to 15.

25. A composite organoid according to any one of claims 16 to 24, for use in treating a gastrointestinal disorder in a patient requiring treatment.

26. A method for screening candidate therapeutic agents, comprising: contacting an organoid described in any one of claims 16 to 25 with the candidate therapeutic agent; and determining the effect of the candidate therapeutic agent on the organoid.

27. The method according to claim 26, wherein the organoid is genetically modified to exhibit a disease or a model thereof, and / or the mesenchyme and / or epithelium of the organoid is genetically modified to exhibit a disease or a model thereof.

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