Shaped organoid compositions and methods for making the same

Abstract

To provide methods for producing organoid compositions manipulated to form shaped or elongated morphologies that more closely resemble native organ structures.SOLUTION: A method for producing a shaped gastrointestinal organoid comprises a lumen, the method comprising: placing a plurality of spheroids in a collection channel having an enclosed predetermined shape, in which the enclosed predetermined shape is defined by sidewalls on all sides; and culturing the plurality of spheroids within the collection channel to differentiate the plurality of spheroids into the shaped gastrointestinal organoid having the predetermined shape, in which the shaped gastrointestinal organoid comprises condensed mesenchyme and a lumen.SELECTED DRAWING: Figure 7A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 855,557, filed May 31, 2019, U.S. Provisional Patent Application No. 62 / 909,868, filed October 3, 2019, and U.S. Provisional Patent Application No. 62 / 958,367, filed January 8, 2020, each of which is expressly incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under U01DK103117 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Aspects of the present disclosure generally relate to organoid compositions and methods for producing said organoid compositions. The organoids disclosed herein have a shaped or elongated structure that more closely resembles in vivo organ tissue. [Background technology]

[0004] Existing methods for providing organoids, such as intestinal organoids, are limited in their ability to form structures suitable for transplantation and subsequent functional performance. Specifically, current methods for obtaining organoids derived from pluripotent stem cells, particularly induced pluripotent stem cells, result in organoids with spherical structures that do not naturally elongate when transplanted and therefore cannot provide structures with an organization similar to that of native organoids. Furthermore, the presence of axial forces can affect organoid tissue development. Existing gastrointestinal organoids contain a lumen, and the limited shape and size available using existing methods limit their usefulness for clinical implementation. Therefore, there is an ongoing need for in vitro-grown organoid tissues derived from patients, such as human patients, with improved suitability for transplantation and improved function after transplantation. Summary of the Invention

[0005] Some aspects of the present disclosure generally relate to a method for generating shaped gastrointestinal organoids. In some embodiments, the gastrointestinal organoids comprise a lumen. In some embodiments, the method comprises: placing a plurality of spheroids into a collection channel comprising a predetermined shape; and culturing the plurality of spheroids in the collection channel to differentiate the plurality of spheroids into shaped gastrointestinal organoids having a predetermined shape. In some embodiments, the shaped gastrointestinal organoids comprise condensed mesenchyme and a lumen. In some embodiments, the collection channel is non-spherical, and the shaped gastrointestinal organoids are non-spherical gastrointestinal organoids. In some embodiments, the collection channel has an elongated shape, and the shaped gastrointestinal organoids are elongated gastrointestinal organoids. In some embodiments, the elongated gastrointestinal organoids comprise an elongated length and diameter. In some embodiments, the elongate length is 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 millimeters, about the aforementioned lengths, at least the aforementioned lengths, at least about the aforementioned lengths, less than or equal to the aforementioned lengths, or any length within a range defined by any two of the aforementioned lengths, e.g., 1-50 mm, 10-40 mm, 20-30 mm, 1-30 mm, or 20-50 mm.In some embodiments, the diameter is 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm. The diameter may be about, at least, at least about, equal to or less than the aforementioned diameter, or equal to or less than the aforementioned diameter, or any diameter within a range defined by any two of the aforementioned diameters, e.g., 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm. In some embodiments, the ratio of elongated length to diameter is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 500000, 60000, 70000, 80000, 900 The luminal ratio may be 0, 100,000, 200,000, 300,000, 400,000, or 500,000, about the aforementioned ratio, at least the aforementioned ratio, at least about the aforementioned ratio, less than or equal to the aforementioned ratio, or any ratio within a range defined by any two of the aforementioned ratios, e.g., 1-500,000, 100-500,000, 1,000-10,000, 1-500,000, or 1,000-500,000. In some embodiments, the lumen is not continuous throughout the elongated length of the shaped gastrointestinal organoid. In some embodiments, the shaped gastrointestinal organoid is a shaped human gastrointestinal organoid.In some embodiments, the plurality of spheroids are cultured in the collection channel 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the plurality of spheroids are fused with the mesenchyme of the plurality of spheroids. In some embodiments, the formed gastrointestinal organoids undergo spontaneous innervation. In some embodiments, the formed gastrointestinal organoids further comprise enteric neurons or enteric neural progenitor cells. In some embodiments, the formed gastrointestinal organoids further comprise one or more myenteric plexuses, including cells that express the neuronal marker PGP9.5. In some embodiments, the formed gastrointestinal organoids have neural activity. In some embodiments, the method further comprises inducing mechanical strain on the formed gastrointestinal organoids, wherein the mechanical strain promotes the spontaneous innervation of the formed gastrointestinal organoids or reduces the maturation time of the formed gastrointestinal organoids, or both. In some embodiments, the mechanical strain is uniaxial tensile strain. In some embodiments, the formed gastrointestinal organoids further comprise polarized columnar epithelium surrounded by mesenchyme, wherein the mesenchyme comprises a smooth muscle-like layer. In some embodiments, the formed gastrointestinal organoids further comprise epithelium patterned into crypt-like proliferative zones, or villi-like structures, or both. In some embodiments, formed gastrointestinal organoid further comprises laminated longitudinal and circular muscle.In some embodiments, formed gastrointestinal organoid further comprises smooth muscle or intestinal subepithelial myofibroblast or both markers thereof.In some embodiments, formed gastrointestinal organoid further comprises one or more of enterocyte, enteroendocrine cell, goblet cell, Paneth cell or any combination thereof.In some embodiments, the formed gastrointestinal organoid further comprises cells that express one or more of villin, Muc2, DEFA5, CHGA, or OLFM4, or any combination thereof.In some embodiments, the formed gastrointestinal organoid is derived from induced pluripotent stem cells that are reprogrammed from PBMC cells, biopsy tissue samples, or somatic cells transduced with Sendai virus.In some embodiments, the formed gastrointestinal organoid is vascularized in vitro.In some embodiments, the formed gastrointestinal organoid is vascularized when engrafted into an individual.In some embodiments, the plurality of spheroids is a plurality of hindgut middle spheroids, and the formed gastrointestinal organoid is a formed intestinal organoid.In some embodiments, the plurality of spheroids is a plurality of hindgut spheroids, and the formed gastrointestinal organoid is a formed colon organoid.In some embodiments, the plurality of spheroids is a plurality of anterior foregut spheroids, and the formed gastrointestinal organoid is an esophageal organoid. In some embodiments, the plurality of spheroids are a plurality of posterior foregut spheroids, and the formed gastrointestinal organoids are gastric organoids.In some embodiments, the method comprises: culturing induced pluripotent stem cells under conditions sufficient to differentiate the induced pluripotent stem cells into definitive endoderm; culturing definitive endoderm under conditions sufficient to differentiate the definitive endoderm into a plurality of spheroids; and collecting the plurality of spheroids before placing the plurality of spheroids in a collection channel.In some embodiments, the collecting step comprises contacting the plurality of spheroids with a binding material that can be bound to the plurality of spheroids.In some embodiments, the binding material is selected from one or more of wire, string, and fiber.In some embodiments, the plurality of spheroids are in contact with a scaffold strand.

[0006] Some aspects of the present disclosure generally relate to a method for treating an individual with impaired gastrointestinal function.In some embodiments, the method comprises transplanting gastrointestinal organoids into an individual.In some embodiments, the gastrointestinal organoid is any one of the formed gastrointestinal organoids described herein.In some embodiments, the gastrointestinal organoid is autologous or allogeneic to the individual.In some embodiments, the gastrointestinal organoid is prepared from induced pluripotent stem cells obtained from the individual.In some embodiments, the individual is in need of gastrointestinal transplantation.In some embodiments, the gastrointestinal function is intestinal function, and the gastrointestinal organoid is intestinal organoid.In some embodiments, the gastrointestinal function is colon function, and the gastrointestinal organoid is colon organoid.In some embodiments, the gastrointestinal function is esophageal function, and the gastrointestinal organoid is esophageal organoid.In some embodiments, the gastrointestinal function is stomach function, and the gastrointestinal organoid is gastric organoid.

[0007] Some aspects of the present disclosure generally relate to a formation tray for culturing one or more shaped gastrointestinal organoids. In some embodiments, the formation tray includes one or more collection channels configured to receive one or more spheroids therein. In some embodiments, the one or more collection channels have a predetermined shape and are configured to collect one or more spheroids together so that the one or more spheroids collect into the predetermined shape, and the one or more spheroids differentiate into one or more shaped gastrointestinal organoids having the predetermined shape. In some embodiments, the one or more collection channels are made of a biocompatible material configured to inhibit attachment of one or more spheroids thereto. In some embodiments, the one or more collection channels further include one or more spheroids disposed therein. In some embodiments, the one or more collection channels further include cell culture medium or extracellular matrix, or both, therein. In some embodiments, the one or more collection channels further include one or more gastrointestinal organoids disposed therein. In some embodiments, the one or more gastrointestinal organoids are any one or more shaped gastrointestinal organoids described herein.

[0008] Some aspects of the present disclosure generally relate to a kit for culturing gastrointestinal organoids.In some embodiments, the kit comprises a formation tray comprising one or more collection channels.In some embodiments, the formation tray is any one of the formation trays described herein.In some embodiments, the kit comprises a plurality of spheroids configured to be received in one or more collection channels.In some embodiments, the kit comprises a cell culture medium configured to be received in one or more collection channels.

[0009] The embodiments of the invention provided herein are described by the following numbered alternatives:

[0010] 1. A method for obtaining elongated human intestinal organoids comprising a lumen, the method comprising: (a) culturing a source of induced pluripotent stem cells under conditions sufficient to form definitive endoderm; (b) culturing the definitive endoderm until multiple spheroids are formed; (c) collecting the plurality of spheroids; (d) placing the plurality of spheroids into a collection channel; (e) forming human intestinal organoids comprising condensed mesenchyme and a lumen from the plurality of spheroids in the collection channel; The collection channel has at least one region that is at least partially tubular in structure.

[0011] 2. The method of alternative 1, wherein said collecting comprises contacting said spheroids with a binding material capable of binding to said spheroids.

[0012] 3. The method of alternative 2, wherein said bonding material is selected from one or more of wire, string, and fiber.

[0013] 4. The method of any preceding alternative, wherein said collection channel has an elongated shape.

[0014] 5. The method of any preceding alternative, wherein said collection channel has a length of at least 1 cm, or at least 2 cm, or at least 3 cm, or at least 4 cm, or at least 5 cm, or from about 1 cm to about 100 cm.

[0015] 6. The method of any preceding alternative, wherein said method is carried out in a device having scaffold strands.

[0016] 7. The method of any preceding alternative, wherein said plurality of spheroids resides in said collection channel for a period of 1 to 20 days, or 2 to 18 days, or 3 to 17 days, or 4 to 16 days, or 5 to 15 days, or 6 to 14 days.

[0017] 8. The method of any preceding alternative, wherein said spheroids are fused with mesenchyme of said spheroids.

[0018] 9. The method of Alternative 1, wherein the elongated intestinal organoids are transplanted into the host on about day 14, or from about day 13 to about day 15, or from about day 12 to about day 16, or from about day 11 to about day 17, preferably wherein the elongated intestinal organoids are transplanted adjacent to the intestine of the host.

[0019] 10. The method of any preceding alternative, wherein said intestinal organoids form a blood supply in vitro.

[0020] 11. The method of any preceding alternative, wherein the intestinal organoids form a blood supply after engraftment into an individual.

[0021] 12. The method of alternative 1, further comprising transplanting the elongated intestinal organoids into a host, wherein the host is selected from an immunodeficient mammal and an individual in need of the transplantation step.

[0022] 13. The method of any preceding alternative, wherein said spheroids are midgut / hindgut spheroids.

[0023] 14. A tray for culturing intestinal organoids,

[0024] a base formed from a biocompatible material configured to inhibit attachment of a plurality of spheroids thereto;

[0025] a collection channel extending through the base and configured to receive a plurality of spheroids therein, the collection channel being elongated and configured to collect the plurality of spheroids together such that the plurality of spheroids define a predetermined shape for culturing the plurality of spheroids into intestinal organoids having the predetermined shape.

[0026] 15. The tray of alternative 14, further comprising culture medium disposed within the collection channel.

[0027] 16. The tray of alternative 15, further comprising a plurality of spheroids disposed within a collection channel.

[0028] 17. A kit for culturing intestinal organoids, comprising: a collection channel; and a medium configured to be received in the collection channel.

[0029] 18. The kit of alternative 17, further comprising a plurality of spheroids configured to be received in the collection channel along with culture medium.

[0030] 19. A method for treating an individual with impaired intestinal function, comprising transplanting into said individual an organoid derived from induced pluripotent stem cells, said organoid comprising mesenchymal and endodermal tissue.

[0031] 20. The method of alternative 19, wherein the organoid further comprises neural tissue.

[0032] 21. The method of alternative 19, wherein said induced pluripotent stem cells are derived from said individual.

[0033] 22. The method of alternative 19, wherein the individual is in need of an intestinal transplant.

[0034] 23. The method of any of alternatives 19-22, wherein said organoids are derived from induced pluripotent stem cells generated from somatic cells transduced with Sendai virus.

[0035] 24. Any of alternatives 19-23, wherein the organoid comprises polarized columnar epithelium surrounded by mesenchyme containing a smooth muscle-like layer.

[0036] 25. Any of alternatives 19-24, wherein the organoid comprises epithelium patterned into crypt-like proliferative zones and villi-like structures.

[0037] 26. Any of alternatives 19-25, wherein said organoids comprise stacked longitudinal and circular muscles.

[0038] 27. Any of alternatives 19-26, wherein the organoids comprise markers of smooth muscle and intestinal subepithelial myofibroblasts.

[0039] 28. The method of any of alternatives 19-27, wherein said organoids comprise enterocytes, goblet, Paneth, and enteroendocrine cells or secretory, endocrine, and absorptive cell types.

[0040] 29. Any of alternatives 19-28, wherein the organoid has neural activity.

[0041] 30. The method of any of alternatives 19-29, further comprising applying tension to the organoids to shorten maturation time. [Brief explanation of the drawings]

[0042] In addition to the features described above, additional features and modifications will be readily apparent from the following drawings and description of exemplary embodiments, it being understood that these drawings illustrate embodiments and are not intended to limit the scope.

[0043] [Figure 1A] 1 illustrates one embodiment of the clinical progression from intestinal failure to intestinal transplantation. [Figure 1B] Illustrates the number of individuals on the US transplant waiting list from 1991 to 2017. [Figure 2A] FIG. 1 illustrates one embodiment of a perspective view of an exemplary formation tray having multiple collection channels for collecting multiple spheroids. [Figure 2B]2B illustrates one embodiment of a cross-sectional view of the exemplary forming tray of FIG. 2A taken along section line 2-2 of FIG. 2A. [Figure 2C] 3 illustrates one embodiment of a cross-sectional view of the exemplary forming tray of FIG. 2A taken along section line 3-3 of FIG. 2A. [Figure 3A] FIG. 1 shows one embodiment of a schematic perspective view of a biocompatible container for culturing a plurality of pluripotent stem cells. [Figure 3B-3D] 3A shows a schematic perspective view embodiment of the biocompatible container of FIG. 3A but showing (B) a plurality of pluripotent stem cells cultured into definitive endoderm, (C) a plurality of pluripotent stem cells cultured into a plurality of spheroids from a definitive endoderm intermediate, or (D) a plurality of pluripotent stem cells cultured into a plurality of spheroids arranged according to a predetermined arrangement guided by attraction to the threads of the scaffold. [Figure 4] FIG. 2C shows one embodiment of an enlarged top view of the formation tray from FIG. 2A with multiple spheroids in multiple collection channels cultured on day 1 (d1), day 3 (d3), or day 5 (d5). [Figure 5] 1 shows one embodiment of an elongated intestinal organoid (46) transplanted into a host organism (44). [Figure 6A] Shown are embodiments of light micrographs of unformed HIOs in culture. Also shown are embodiments of hematoxylin and eosin stained sections of unformed HIOs at day 14 (panel A) and day 28 (panel B). Scale bar = 0.5 mm. [Figure 6B] One embodiment shows hematoxylin and eosin-stained sections of a tHIO taken at 2, 4, and 8 weeks post-implantation (top) and a historical section of human fetal intestine (bottom). The development of epithelial structures in the tHIO proceeds in a manner similar to that of native tissue. GA: gestational age. Human data reproduced from Grand et al. (1976). [Figure 7A] 1 illustrates one embodiment of a methodology for generating elongated HIOs and subsequently implanting them into a host organism. [Figure 7B]Shown are one embodiment of hematoxylin and eosin-stained sections of elongated HIO (g-HIO) structures formed in vitro at day 6 (panel A), day 14 (panel B), and day 28 (panel C). Scale bar = 1 mm. Note: The structure in panel B is not full length. [Figure 8A] This figure shows one embodiment of successful generation of human PSC-derived tubular intestinal organoids with elongated HIO formation and a continuous epithelium. Panel A shows images of the ABS mold and PDMS scaffolding tray. Panel B shows scanning electron micrographs of the cross section (top) and edge (bottom) of the PDMS scaffolding tray. Panel C shows in vitro images of spheroids within the grooves at 1, 3, and 5 days of culture. Panel D shows a surgical image of the organoid structure at day 14 upon transplantation. Panel E shows a gross image of the engrafted organoid structure at day 14, 6 weeks after transplantation. The dashed line indicates the dissection plane. Panel F shows a hematoxylin and eosin-stained section of the transplant from Panel E. Panel G shows a tile scan of the hematoxylin and eosin-stained section of the transplant. Areas of adjacent mouse tissue are labeled. A continuous epithelium throughout the tissue is observed. [Figure 8B] 1 shows one embodiment of the transplantation of elongated intestinal organoids and the resulting vascularization. Also shown is a histology of the elongated intestinal organoids after successful engraftment. [Figure 8C] Figure 1 shows one embodiment of images of a g-HIO implanted at the time of harvest. The top panel shows a g-HIO 14 days after implantation into the mesentery of an immunodeficient rat. The bottom panel shows an unformed HIO 28 days after 8 weeks of implantation into the mesentery of an immunodeficient rat. HIOs created using the conventional protocol did not engraft in immunodeficient rats when implanted on day 14. [Figure 9A-9B] 1 shows an embodiment of an HIO that grows significantly upon transplantation in vivo. The transplanted HIO (tHIO) is significantly larger than the in vitro HIO at the time of harvest. [Figure 9C]The transplanted HIOs resemble the human intestine. The tHIOs contain major intestinal cell lineages, including mesenchymal, enterocyte (VIL1), enteroendocrine (CHGA), goblet (MUC2), and Paneth cells (DEFA5). Furthermore, they express positive expression of a marker of stem cell activity (OLFM4). [Figure 9D]

[0033] Figure 1 shows one embodiment of an anastomosis from organoids to intestine in a mesenteric transplantation model. 50% of mice survived to day 21 at the time of harvest. [Figure 9E] This figure shows one example of spontaneous myenteric plexus development that occurs after transplantation of a g-tHIO (tHIO) created in a groove. Histology of the g-tHIO reveals a robust network of myenteric plexus throughout the harvested tissue (Panel A, left). At higher magnification, bundle structures are visible (Panel A, right). Immunofluorescent staining for the pan-neuronal marker (PGP9.5) and the human-specific marker (KU80). Colocalization of these proteins indicates the human origin of the neuronal components (Panel B). Immunohistochemical staining for the pan-neuronal marker PGP9.5 in the enteric nervous system (tHIO+ENS), g-tHIO, and tHIO combined with neural crest cells to form the adult human small intestine (Panel C). Quantification of the myenteric plexus (PGP9.5+ cell bundles) from Panel C (Panel D). The size of the plexus in the g-tHIO is significantly larger than that in the tHIO+ENS using a conventional differentiation protocol. All scale bars = 100 μm. [Figure 10A] Figure 1 shows one embodiment of in vitro transcriptome isolation of g-HIOs and unformed HIOs. Principal component analysis of spheroids, day 28 unformed HIOs, and day 28 g-HIOs (Panel A). Heatmap of day 28 unformed HIOs and g-HIOs (Panel B). Venn diagram of differentially expressed genes between day 28 unformed HIOs and g-HIOs (Panel C). List of the top 10 biological processes enriched in g-HIOs related to neuronal development (Panel D). [Figure 10B]Figure 1 shows an embodiment of a transcriptome profile of g-HIOs developing in vitro. Principal component analysis of spheroids, g-HIOs at day 6, g-HIOs at day 14, and g-HIOs at day 28 (Panel A). Heatmap of spheroid and g-HIO samples (Panel B). [Figure 10C] 1 shows one embodiment of transcriptionally enriched biological processes during in vitro development of g-HIOs. Enriched biological processes are tabulated at days 0, 6, 14, and 28 during in vitro development of g-HIOs. DETAILED DESCRIPTION OF THE INVENTION

[0044] Intestinal failure (IF) is usually the result of loss of intestine due to surgical resection and / or congenital intestinal defects, resulting in altered intestinal absorption and digestion. A smaller subgroup suffers from motility problems that result in functional loss of the intestine's ability to absorb fluids and nutrients. Chronic intestinal failure occurs when the body cannot maintain its energy and nutritional needs by absorbing food and nutrients through the intestinal tract, thus requiring long-term parenteral nutrition (PN). Intestinal failure affects approximately 3-50 people per million, affecting approximately 15,000 people in the United States. Chronic intestinal failure has been granted rare disease status under the number ORPHA:294422 (Classification: Disorders).

[0045] While lifesaving, long-term PN can lead to its own set of serious complications. In the neonatal population, PN-dependent intestinal failure can be associated with multiple complications, including recurrent bloodstream infections, repeated hospitalizations, and subsequent poor growth, including metabolic bone problems. All of these place a significant burden on patients, families, and healthcare systems. Life-threatening complications in long-term PN patients, such as inaccessible veins due to thrombosis, repeated catheter-associated sepsis, and cholestatic liver disease, can ultimately lead to the need for life-saving intestinal transplantation. Figure 1A illustrates the clinical progression from intestinal failure to intestinal transplantation.

[0046] Intestinal transplantation has evolved into an established treatment for the management of patients with irreversible intestinal failure. Intestinal transplants can be performed in various forms, including isolated intestinal transplants, modified multiorgan transplants, and complete multiorgan transplants. Although the number of patients undergoing intestinal transplants is much smaller than that of other organ transplants, the number of procedures increased fivefold from 2000 to 2009. Over the past few years, the number of intestinal transplants has remained stable, with 47 pediatric transplants performed in 2017, and is trending downward toward a rate of 100–120 transplants per year. This trend is due to improved interdisciplinary care provided to patients with intestinal failure, which is likely to increase in the population over the next decade. New and improved treatments are needed for this patient population.

[0047] Although surgical techniques continue to improve, destructive alloimmunization in intestinal and multiorgan transplants remains a significant obstacle, limiting both the pool of potential patients who could benefit from transplantation and post-transplant graft maintenance. The Pittsburgh Protocol, in which T cells are first completely depleted immediately prior to transplantation, followed by continued T cell suppression and steroid maintenance, is considered the standard of care. While this protocol has reduced the rate of graft rejection, it remains significant. Donor-specific antibodies (DSA) continue to be a problem in approximately 30% of patients early after transplantation. The presence of DSA more than doubles the risk of chronic rejection.

[0048] While 1- and 5-year survival rates exceed 70%, 10- and 15-year survival rates are only 42% and 35%, respectively. This indicates that while short-term outcomes are favorable, long-term outcomes continue to be disappointing. In 2017, 90.4% of intestine-only transplants were primary transplants, and 9.6% were repeat transplants. In patients receiving combined liver and intestine transplants, 26.3% of these were repeat transplants. Intestinal repeat transplants are currently the fourth most common indication for intestinal transplantation.

[0049] Pediatric patients undergoing retransplantation tend to be younger than the primary transplant cohort as a whole. In one center's experience, the mean time from primary transplant to retransplantation was 421 days. They found a survival bias in all patients who underwent early (<90 days) retransplantation compared with patients who underwent later retransplantation (survival rates of 80% and 50%, respectively). In the pediatric subpopulation, the 3-year survival rate for retransplantation was 27%, regardless of timing, and graft survival was similar. In a second center, of 23 patients (both adults and children) who underwent retransplantation, 15 patients died at a median time of 12 months after retransplantation (survival rate of 35%). A third center in Spain reported a 5-year retransplant pediatric survival rate of 35% in 13 patients.

[0050] A rigorous characterization of one retransplant cohort (n = 23) revealed that recurrent severe rejection was common (35%), even after retransplantation. The incidence of graft rejection was significantly higher in retransplants, where patients had experienced rejection in their primary transplant, resulting in graft loss and a 33% mortality rate. Retransplanted patients were generally severely immunocompromised, with myelosuppression observed in 35% of retransplanted patients compared with 4% of primary transplants. Furthermore, these retransplant non-survivors (60%) had significantly lower absolute lymphocyte and platelet counts one month before death compared with similar time points in survivors.

[0051] Organ donations continue to experience a significant shortage relative to the need. As of July 2019, the United States had over 113,000 men, women, and children on the national transplant waiting list (Figure 1B). Every 10 minutes, one new person is added to the waiting list, and 20 people die while waiting for a transplant. Over the past 27 years, this shortage has grown from 6,953 donors / 23,198 people waiting in 1991 to 17,554 donors / 113,759 people waiting in 2018.

[0052] This increasing shortage of human organ donors has driven research scientists to consider other options, such as xenotransplantation or generating essential human transplantable organs. This approach not only presents complex scientific challenges, but also legal and ethical issues. One potential option is the use of in vitro expanded epithelial biopsies obtained from the patient's own intestine (called enteroids). Because these structures contain only epithelium, they cannot replace the majority of intestinal structures, which primarily contain mesenchymal and neural tissue. Furthermore, likely due to the lack of mesenchyme, these structures are not easily transplanted.

[0053] In contrast, organoids derived from the induced pluripotent stem cells described herein can contain both endodermal and mesodermal tissues, are easily transplantable, and contribute to the regeneration of the entire graft. The ability to generate patient-specific organoids may circumvent several scientific and ethical concerns, along with the prevention of allogeneic immune responses, which may ultimately be a solution for individuals to extend the life expectancy of their grafts and avoid being placed back on transplant waiting lists.

[0054] Currently, organoids are being utilized to study human diseases both in vitro and in vivo. As disclosed herein, there is strong evidence supporting the possibility that these iPSC-derived tissues may be fully functional in vivo and thus may be useful for rescuing organ transplants. It has been shown that pluripotent stem cells (PSCs) can be directed to differentiate into multiple organ systems in vitro, including intestinal tissue, by stepwise modulating the combinatorial activity of several signaling pathways, effectively recapitulating fetal organ development in vitro without the need for fetal tissue.

[0055] Disclosed herein is the gastrointestinal organoid derived from iPSC.In some embodiments, gastrointestinal organoid is esophageal organoid, stomach organoid, intestinal organoid or colonic organoid.In some embodiments, gastrointestinal organoid is intestinal organoid.These organoids are generated from somatic cells transduced with Sendai virus, which induces PSC to form all tissues of the body.By manipulating the factors that control embryonic organogenesis, an in vitro method has been developed that leads PSC to embryonic germ layer-restricted organoid, and then to specific cell types such as hepatocytes, nerves, muscle cells and intestinal tissues stepwise differentiation.Methods for generating organoids such as intestinal organoids are described in U.S. Patent No. 9,719,068 and 10,174,289, and PCT publication WO2016 / 061464 and WO2018 / 106628, each of which is expressly incorporated herein by reference in its entirety.

[0056] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless context dictates otherwise. The illustrative embodiments set forth in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0057] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood when read in light of the immediate disclosure by one of ordinary skill in the art to which this disclosure belongs. For purposes of this disclosure, the following terms are described below.

[0058] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0059] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 10% from a reference quantity, level, value, number, frequency, or percentage.

[0060] Throughout this specification, unless the context requires otherwise, the words "comprise," "includes," and "comprising" shall be understood to mean the inclusion of the stated steps or elements or groups of steps or elements, and not the exclusion of any others. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that other elements may not be present. "Consisting essentially of" means including the elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or behavior specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or behavior of the listed elements.

[0061] As used herein, the terms "individual," "subject," or "patient" have their plain and ordinary meaning as understood in light of the present specification, and refer to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird (e.g., chicken), as well as other vertebrates or invertebrates. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, monkeys (chimpanzees, apes, monkeys) and primates, including humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, and guinea pigs.

[0062] The terms "effective amount" or "effective dose" as used herein have their plain and ordinary meaning as understood in light of the specification and refer to that amount of the recited composition or compound that produces an observable effect. The actual dosage level of the active ingredient in the active composition of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound effective to achieve the desired response for a particular subject and / or application. The selected dosage level depends on various factors, including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0063] For clarity of disclosure, spatial terms such as "upper," "lower," "longitudinal," "lateral," "transverse," "inward," "outward," and the like are used herein or with reference to the drawings, where it is understood that such terms are used for illustrative purposes only and are not intended to be limiting or absolute. In that regard, it will be understood that the instruments as disclosed herein can be used in a variety of orientations and positions that are not limited to those shown and described herein.

[0064] The dimensions and values disclosed herein should not be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range that includes that value. For example, a dimension disclosed as "20 mm" is intended to mean "approximately 20 mm."

[0065] As used herein, the terms "function" and "functional" have their plain and ordinary meaning as understood in light of this specification and refer to biological, enzymatic, or therapeutic function.

[0066] The term "inhibit," as used herein, has its plain and ordinary meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, about the aforementioned reduction, at least the aforementioned reduction, at least about the aforementioned reduction, or less than the aforementioned reduction, or less than the aforementioned reduction, or an amount within a range defined by any two of the aforementioned values. The term "delay," as used herein, has its plain and ordinary meaning as understood in light of the specification and refers to a delay, postponement, or deferral of a biological event to a time later than would otherwise be expected. The delay is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, about the aforementioned delay, at least the aforementioned delay, at least about the aforementioned delay, less than or equal to the aforementioned delay, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay do not necessarily indicate 100% inhibition or delay; partial inhibition or delay may be achieved.

[0067] As used herein, the term "isolated" has its plain and ordinary meaning as understood in light of the specification and refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced, and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities 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, at least, at least about, less than, or equal to (or a range including and / or spanning) the aforementioned values, of other components with which they were originally associated. In some embodiments, an isolated agent is 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or equivalent to 100% pure, is about, is at least, is at least about, is less than, or is less than (or ranges including and / or spanning) the aforementioned values. As used herein, an "isolated" material can be "pure" (e.g., substantially free from other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0068] As used herein, "in vivo" is given its plain and ordinary meaning as understood in light of the present specification and refers to the performance of methods within living organisms, usually animals, mammals, including humans, and plants, rather than tissue extracts or dead organisms.

[0069] As used herein, "ex vivo" is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little change in natural conditions.

[0070] As used herein, "in vitro" is given its plain and ordinary meaning as understood in light of the specification and refers to the performance of methods outside biological conditions, e.g., in a petri dish or test tube.

[0071] As used herein, the terms "nucleic acid" or "nucleic acid molecule" have their plain and ordinary meaning as understood in light of the present specification and refer to polynucleotides, oligonucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as they naturally occur in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have alterations in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotide" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The one or more nucleic acids may be contained in a nucleic acid vector or construct (e.g., a plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC) that can be used to amplify and / or express one or more nucleic acids in various biological systems). Typically, the vector or construct also includes elements including, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a target sequence, a peptide purification tag, or an accessory gene, or any combination thereof.

[0072] A nucleic acid or nucleic acid molecule can contain one or more sequences encoding different peptides, polypeptides, or proteins, which can be adjacent within the same nucleic acid or nucleic acid molecule or joined by extra nucleic acid between them, such as linkers, repeats, or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about, at least, at least about, up to, or about, or up to, or any other length within the range defined by any two of the lengths. The term "downstream" of a nucleic acid, as used herein, has its plain and ordinary meaning as understood in light of the specification, and refers to the sequence on the strand containing the coding sequence (sense strand) that is after the 3' end of the preceding sequence, if the nucleic acid is double-stranded. The term "upstream" of a nucleic acid, as used herein, has its plain and ordinary meaning as understood in light of the specification, and refers to the sequence on the strand containing the coding sequence (sense strand) that is before the 5' end of the following sequence, if the nucleic acid is double-stranded.The term "grouped," as used herein, with respect to nucleic acids, has its plain and ordinary meaning as understood in light of the present specification and refers to two or more sequences that occur in close proximity, either directly or with extra nucleic acid between them, such as linkers, repeats, or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about said length, at least said length, at least about said length, up to or equal to said length, or any length within the range defined by any two of the above lengths, but generally without any intervening sequence encoding a functional or catalytic polypeptide, protein, or protein domain.

[0073] The nucleic acid described herein comprises nucleobases.The primary, normal, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil.Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobase, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl base, dye-labeled base, fluorescent base or biotin-labeled base.

[0074] As used herein, the terms "peptide," "polypeptide," and "protein" have their plain and ordinary meaning as understood in light of the present specification and refer to polymers composed of amino acids linked by peptide bonds. The many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymatic, structural, transport, defensive, hormonal, or signal transduction functions. Peptides, polypeptides, and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates; chemical synthesis is also available. By manipulating nucleic acid templates, protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of peptides, polypeptides, and multiple peptides, polypeptides, or proteins can be performed. These fusions of multiple peptides, polypeptides, or proteins can be joined adjacently within the same molecule, or with extra amino acids between them, such as linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about the aforementioned lengths, at least the aforementioned lengths, at least about the aforementioned lengths, or up to or equal to the aforementioned lengths, or any other length within the range defined by any two of the aforementioned lengths. As used herein, the term "downstream" of a polypeptide has its plain and ordinary meaning as understood in light of the specification, and refers to sequences following the C-terminus of the preceding sequence. The term "upstream" of a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification, and refers to sequences that precede the N-terminus of the subsequent sequence.

[0075] As used herein, the term "purity" of any given substance, compound, or material has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimal points therebetween. 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, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious pathogens. 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-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0076] The term "yield" of any given substance, compound, or material, as used herein, has its plain and ordinary meaning as understood in light of this specification and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, including all decimal points therebetween, about the aforementioned yield, at least the aforementioned yield, at least about the aforementioned yield, or less than the aforementioned yield, or less than the aforementioned yield. Yield may be affected by reaction or process efficiency, undesired side reactions, decomposition, quality of input substances, compounds, or materials, or loss of desired substances, compounds, or materials at any stage of production.

[0077] As used herein, the term "% w / w" or "% weight / weight" has its plain and ordinary meaning as understood in light of the present specification and refers to the percentage expressed as the weight of a component or agent relative to the total weight of the composition, multiplied by 100. As used herein, the term "% v / v" or "% vol / vol" has its plain and ordinary meaning as understood in light of the present specification and refers to the percentage expressed as the liquid volume of a compound, substance, component, or agent relative to the total liquid volume of the composition, multiplied by 100.

[0078] stem cells As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) are stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of building complete, viable organisms. 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.

[0079] As used herein, the term "embryonic stem cells (ESCs), commonly abbreviated as ES cells, has its plain and ordinary meaning as understood in light of the present specification and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of the present invention, the term "ESCs" is sometimes used broadly to encompass embryonic germ cells as well.

[0080] As used herein, the term "pluripotent stem cells (PSCs)" has its plain and ordinary meaning as understood in light of this specification and encompasses any cell that can differentiate into almost any cell type of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs may be the progeny of inner cell mass cells of a preimplantation blastocyst or may be obtained by the induction of non-pluripotent stem cells, e.g., 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 human, rodent, porcine, and bovine.

[0081] As used herein, the term "induced pluripotent stem cells (iPSCs)" has its plain and ordinary meaning as understood in light of the specification, and is commonly abbreviated as iPSCs. 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. hiPSCs refer to human iPSCs. In several methods known in the art, iPSCs can be derived by transfecting specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes may increase 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 embodiments, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four essential genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In another embodiment, a lentiviral system is used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whose expression is induced in iPS cells include, but are not limited to, Oct-3 / 4 (POU5F1).Certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-Catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof.

[0082] As used herein, the term "progenitor cell" has its plain and ordinary meaning as understood in light of the specification and encompasses any cell that can be used in the methods described herein, through which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent stem cells (induced or non-induced), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be derived from embryos, infants, children, or adults. In some embodiments, progenitor cells can be somatic cells that have been subjected to treatment to confer pluripotency via genetic manipulation or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0083] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some aspects, pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the early embryo, the blastocyst. Methods for deriving embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described 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.

[0084] Additional stem cells that can be used in embodiments of the present invention include, but are not limited to, those provided by or described in databases hosted by 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, Calif.), Cellartis AB (Göteborg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (13), TE04 (14), TE06 (16), UC01 (HSF1), UC06 (HSF6), WA01 (H1), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0085] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" describes the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or change the fate of the original cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0086] In some embodiments, adenovirus can be used to deliver the four necessary genes, resulting in iPSCs that are virtually identical to embryonic stem cells. Because adenovirus does not combine its own genes with the target host, the risk of tumor formation is eliminated. In some embodiments, non-viral techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system at all, albeit with very low efficiency. In other embodiments, direct protein delivery is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, mouse iPSCs can be generated using a similar methodology. Repeated treatment of cells with specific proteins delivered to the cells via polyarginine anchors was 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.

[0087] As used herein, the term "Sendai virus" has its plain and ordinary meaning as understood in light of the specification and refers to an enveloped, negative-sense, single-stranded RNA virus of the Paramyxoviridae family, also known as Japanese parainfluenza virus of mice type 1 or hemagglutinating virus (HVJ). While typically only causing disease in rodents, the virus can infect a wide range of mammalian cells, including human cells, via sialic acid receptors. Sendai virus can be used as a viral vector for delivering transgenes to cells in vitro and in vivo. In some embodiments, Sendai virus has been engineered to contain expression vectors for stem cell reprogramming factors to reprogram somatic cells into induced pluripotent stem cells. In some embodiments, stem cell reprogramming factors may include any stem cell reprogramming factor disclosed herein or known in the art, including, but not limited to, Oct3 / 4, Sox2, Klf4, and L-Myc, or any combination thereof. In some embodiments, these stem cell reprogramming factors are of human origin. In some embodiments, the Sendai virus vector comprises an expression vector for one or more (e.g., at least 1, 2, 3, 4, 5) of Oct3 / 4, Sox2, Klf4, L-Myc, or another stem cell reprogramming factor. In some embodiments, the Sendai virus vector comprises an expression vector for Klf4, Oct3 / 4, and Sox2 (KOS). In some embodiments, the Sendai virus vector comprises an expression vector for L-Myc. In some embodiments, the Sendai virus vector comprises an expression vector for Klf4. In some embodiments, one or more Sendai virus vectors are combined in different ratios to optimize cell reprogramming. In some embodiments, contacting somatic cells with one or more Sendai virus vectors successfully reprograms somatic cells into induced pluripotent stem cells.As an RNA virus, Sendai virus does not need to integrate its viral payload into the host genome or access the nucleus (as do DNA viruses). This differs from lentiviruses and adenoviruses. However, it is contemplated that other viral vectors, such as lentiviruses, adenoviruses, and adeno-associated viruses, can be used for the transduction purposes described herein where Sendai virus is used, such as for reprogramming somatic cells into stem cells.

[0088] The term "feeder cells," as used herein, has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying them on the cell surface. Feeder cells are generally adherent cells and can be growth-arrested. For example, feeder cells can be growth-arrested by irradiation (e.g., gamma rays), mitomycin-C treatment, electric pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily need to be growth-arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are murine 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 SNL76 / 7 cells, human fibroblasts, human forehead fibroblasts, human skin fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used instead of or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the expansion of target stem cells.

[0089] Some embodiments described herein relate to pharmaceutical compositions comprising, consisting essentially of, or consisting of an effective amount of a cell composition described herein and a pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0090] As used herein, "pharmaceutically acceptable" has its plain and ordinary meaning as understood in light of the specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed to the cells or mammals at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" has its plain and ordinary meaning as understood in light of the specification and is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are approved by a regulatory agency of the federal government, state government, or other regulatory body, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, including humans and non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous solutions of dextrose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers may also include one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt formation inhibitors such as sodium; non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, as desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation should be compatible with the method of administration.

[0091] Cryoprotectants are cell composition additives that improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cryopreservation medium that contains other components, such as nutrients (e.g., albumin, serum, bovine serum, fetal calf serum [FCS]), to enhance the post-thaw survival of cells. In these cryopreservation media, at least one cryoprotectant may be found at a concentration 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%, at about the aforementioned concentrations, at least the aforementioned concentrations, at least about the aforementioned concentrations, up to or equal to the aforementioned concentrations, or up to or equal to the aforementioned concentrations, or any percentage within a range defined by any two of the aforementioned numbers.

[0092] Additional excipients with 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 be residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth media components or any combination thereof. The amount of excipient may be found in the composition as 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, about the aforementioned amount, at least the aforementioned amount, at least about the aforementioned amount, up to or equal to the aforementioned amount, or up to or equal to the aforementioned amount, or any percentage within a range defined by any two of the aforementioned numbers.

[0093] The term "pharmaceutically acceptable salts," as understood in light of this specification, has its plain and ordinary meaning and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including, but not limited to, analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts can also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, classes of such organic bases may include, but are not limited to, mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, including mono-, di-, and triethanolamine; amino acids, including glycine, arginine, and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; and trihydroxymethylaminoethane.

[0094] Appropriate formulations vary depending on the selected route of administration. Techniques for the formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques for administering compounds exist in the art, including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intraarterial, intravenous), intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0095] As used herein, "carrier" has its plain and ordinary meaning as understood in light of this specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or bodily organs.

[0096] As used herein, the term "diluent" has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient 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 can also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline, which mimics the composition of human blood.

[0097] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments. The present invention also includes embodiments in which subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is completely or partially excluded.

[0098] Intestinal development In anatomy, the intestine is the segment of the digestive tract that extends from the stomach to the anus, and in humans and other mammals, it consists of two segments: the small intestine and the large intestine. In humans, the small intestine is further subdivided into the duodenum, jejunum, and ileum, and the large intestine is subdivided into the cecum and colon. The structure of the intestinal system is described herein using the human organ as an example. It will be understood by those skilled in the art that the methods and systems described herein are applicable to the intestinal systems of all mammals.

[0099] The intestinal tract can be broadly divided into two distinct parts: the small intestine and the large intestine. Grayish-purple in color and about 35 millimeters (1.5 inches) in diameter, the small intestine is the first and longest, averaging 6–7 meters (20–23 feet) in length in an adult male. The short, relatively stubby large intestine is dark reddish in color and averages about 1.5 meters (5 feet) in length.

[0100] The lumen is the cavity through which digested food passes and through which nutrients are absorbed. Both intestines share a general structure with the whole intestine, which is made up of several layers.

[0101] Radially extending from the inside of the lumen outward, the order progresses from the mucosa (epithelium and muscularis mucosa), submucosa, muscularis externa (consisting of an inner circular layer and an outer longitudinal layer), and finally the serosa. Along the entire length of the intestinal epithelium are goblet cells. These secrete mucus, which lubricates food passage and protects the intestine from digestive enzymes. Crypts are invaginations of the mucosa, and villi are finger-like projections that increase the overall surface area of the intestine while containing lacteals, which connect to the lymphatic system and help remove lipids and tissue fluids from the blood supply. During development, buckling and invagination of the epithelium occur, resulting in ridges that later resolve into crypt-villus structures. Microvilli are present in the epithelium of the villi, further increasing the surface area through which absorption can occur. The muscularis mucosa is a layer of smooth muscle that aids in the ongoing peristaltic and catastrophic action along the intestine. The submucosa contains nerves (e.g., Meissner's plexus), blood vessels, and collagen-laden elastic fibers, which stretch as volume increases but maintain the shape of the intestine. The muscularis externa contains longitudinal and smooth muscles, which again aid in ongoing peristalsis and the movement of digested material out and along the intestine. Between the two layers of muscularis lies the Auerbach's plexus. The serosa is made up of loose connective tissue and is coated with mucus to prevent damage from friction caused by the intestine rubbing against other tissues. Holding all of this in place is the mesentery, which suspends the intestine within the abdominal cavity and prevents it from being disturbed when a person is physically active.

[0102] PSC differentiation In some embodiments, PSCs, such as ESCs and iPSCs, undergo stepwise directed differentiation, first to definitive endoderm (DE), then to posterior / hindgut epithelium (e.g., hindgut spheroids), and then to intestinal tissue. In some embodiments, PSCs, such as ESCs and iPSCs, undergo non-stepwise directed differentiation, in which molecules (e.g., growth factors, ligands) to promote DE formation and molecules for subsequent tissue formation are simultaneously added.

[0103] The definitive endoderm gives rise to the gut tube. The anterior DE forms the foregut and associated organs, including the esophagus, lungs, stomach, liver, and pancreas. The posterior DE forms the midgut and hindgut, the small and large intestines, and parts of the urogenital system. Studies using mouse, chick, and frog embryos suggest that establishing an anterior-posterior pattern of the DE at the gastrula stage is a prerequisite for subsequent foregut and hindgut development. Wnt and FGF signaling pathways are important for promoting the posterior endoderm / hindgut or anterior endoderm / foregut fates. In the hindgut, a simple cuboidal epithelium first develops into a pseudostratified columnar epithelium, which then develops into a polarized columnar epithelium and a villi containing a proliferative zone at the base of the villi, corresponding to the presumptive progenitor region.

[0104] A robust and efficient process for directing the differentiation of DE into intestinal tissue in vitro has been previously described in U.S. Patent No. 9,719,068. In some embodiments, directed differentiation is achieved by selectively activating specific signaling pathways in iPSCs and / or DE cells. In some embodiments, the signaling pathways are those active in intestinal development, including, but not limited to, the Wnt signaling pathway, the Wnt / APC signaling pathway, the FGF signaling pathway, the TGF-β signaling pathway, the BMP signaling pathway, the Notch signaling pathway, the Hedgehog signaling pathway, the LKB signaling pathway, and the Par polarity signaling pathway.

[0105] Any method for generating definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) is applicable to the methods described herein. In some embodiments, the pluripotent cells are derived from a morula. In some embodiments, the pluripotent stem cells are stem cells. Stem cells used in these methods include, but are not limited to, embryonic stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or the gonadal ridges of an embryo. Embryonic stem cells or germ cells can be derived from various animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to generate definitive endoderm. In some embodiments, human embryonic germ cells are used to generate definitive endoderm. In some embodiments, iPSC cells are used to generate definitive endoderm. In some embodiments, iPSC cells (hiPSCs) are used to generate definitive endoderm.

[0106] 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 that is 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, about the aforementioned periods, at least the aforementioned periods, at least about the aforementioned periods, less than or equal to the aforementioned periods, or less than or equal to the aforementioned periods, or any period within a range defined by any two of the aforementioned periods, e.g., 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, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, multiple small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0107] 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 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 15 The cells are treated with a concentration of, for example, 1000 ng / mL, about, at least, at least about, equal to or less than the aforementioned concentrations, equal to or less than the aforementioned concentrations, or any concentration within a range defined by any two of the aforementioned concentrations, e.g., 10 ng / mL to 15,000 ng / mL, 100 ng / mL to 5,000 ng / mL, 500 ng / mL to 2,000 ng / mL, 10 ng / mL to 2,000 ng / mL, or 1,000 ng / mL to 15,000 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 treatment. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors is varied over the course of treatment. In some embodiments, more than one small molecule compound, activator, inhibitor, or growth factor is added. In these cases, the concentrations of the multiple small molecule compounds, activators, inhibitors, or growth factors may vary.

[0108] In some embodiments, ESCs, germ cells, or iPSCs are cultured in a growth medium that supports stem cell proliferation. In some embodiments, ESCs, germ cells, or iPSCs are cultured in a stem cell growth medium. In some embodiments, the stem cell growth medium is RPMI 1640, DMEM, DMEM / F12, erythroid expansion medium, Minigut medium, StemPro 34 SFM (serum-free medium), StemPro hESC SFM, 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 comprises FBS at a concentration 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%, about, at least, at least about, equal to or less than, or equal to or less than, or any percentage within a range defined by any two of the concentrations, e.g., 0%-20%, 0.2%-10%, 2%-5%, 0%-5%, or 2%-20%. In some embodiments, the stem cell growth medium is xeno-free. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0109] In some embodiments, a population of cells enriched for definitive endoderm cells is used. In some embodiments, the definitive endoderm cells are isolated or substantially purified. In some embodiments, the isolated or substantially purified definitive endoderm cells express one or more (e.g., at least 1, 3) SOX17, FOXA2, or CXRC4 markers to a greater extent than one or more (e.g., at least 1, 3, 5) of one or more (e.g., at least 1, 3) OCT4, AFP, TM, SPARC, or SOX7 markers.

[0110] Methods for enriching a cell population with definitive endoderm are also contemplated. In some embodiments, definitive endoderm cells can be isolated or substantially purified from a mixed cell population by contacting the cells with a reagent that binds to a molecule present on the surface of the definitive endoderm cells but not on the surface of other cells, and then separating the cells that bind to the reagent. In some embodiments, the cellular component present on the surface of the definitive endoderm cells is CXCR4.

[0111] Some embodiments involve a CXCR4 antibody, an SDF-1 protein or ligand, or another protein or ligand of CXCR4, and allow for obtaining definitive endoderm cells in enriched, isolated, or substantially purified form. For example, a CXCR4 antibody, an SDF-1 protein or ligand, or another protein or ligand of CXCR4, can be used as a reagent in methods such as affinity-based or magnetic-based separation to enrich, isolate, or substantially purify a preparation of definitive endoderm cells that bind to the reagent.

[0112] In some embodiments, the definitive endoderm cells and hESCs are treated with one or more growth factors. Such growth factors may include growth factors from the TGF-beta superfamily. In some embodiments, the one or more growth factors may include the Nodal / Activin and / or BMP subgroups of the TGF-beta superfamily of growth factors. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, Wnt3a, or any combination of these growth factors.

[0113] In some embodiments, activin-induced definitive endoderm (DE) can further undergo FGF / Wnt-induced posterior gut endoderm patterning, hindgut specification and morphogenesis, and finally, a foregut culture system promoting intestinal growth, morphogenesis, and cytodifferentiation into functional intestinal cell types, including mesenchyme, enterocytes, goblet, Paneth, and enteroendocrine cells. In some embodiments, human PSCs have been efficiently directed to differentiate in vitro into intestinal epithelium, including secretory, endocrine, and absorptive cell types. It will be understood that molecules such as growth factors can be added at any stage of development to promote the formation of specific types of intestinal tissue.

[0114] In some embodiments, soluble FGFs and Wnt ligands are used to mimic early hindgut specification in culture, converting DE generated from iPSCs or ESCs through directed differentiation into hindgut epithelium that efficiently gives rise to all major intestinal cell types. In humans, induction of DE differentiation is achieved by selectively activating specific signaling pathways that are important for intestinal development.

[0115] In vitro human intestinal development occurs at stages similar to fetal intestinal development: endoderm formation, posterior endoderm patterning, hindgut morphogenesis, fetal intestinal development, epithelial morphogenesis, formation of presumptive progenitor domains, and differentiation into functional intestinal cell types. For example, in humans, genes encoding Wnt signaling 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.

[0116] It will be understood by those skilled in the art that altering the concentration, expression, or function of one or more Wnt signaling proteins in combination with altering the concentration, expression, or function of one or more FGF proteins can result in directed differentiation according to the present invention. In some embodiments, cellular components associated with the Wnt and / or FGF signaling pathway, such as natural inhibitors, antagonists, activators, or agonists of the pathway, can be used to inhibit or activate the Wnt and / or FGF signaling pathway. In some embodiments, siRNA and / or shRNA targeting cellular components associated with the Wnt and / or FGF signaling pathway are used to inhibit or activate these pathways.

[0117] Fibroblast growth factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGFs are heparin-binding proteins, and their interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. FGFs play important roles in the proliferation 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 fibroblast growth factor receptors (FGFRs). FGF1 is also known as acidic fibroblast growth factor (FGF), and FGF2 is also known as basic fibroblast growth factor (bFGF). Members FGF11, FGF12, FGF13, and FGF14, also known as FGF homologous factors 1–4 (FHF1–FHF4), have been shown to have distinct functional differences compared to FGFs. Although these factors share striking sequence similarity, they do not bind to FGFRs and are involved in intracellular processes unrelated to FGFs. This group is also known as "iFGF." Members FGF15 through FGF23 are newer and less well characterized. FGF15 is the mouse ortholog of human FGF19 (hence, there is no human FGF15). Human FGF20 was identified based on its homology to Xenopus FGF-20 (XFGF-20). In contrast to the local activity of other FGFs, FGF15 / FGF19, FGF21, and FGF23 have more systemic effects.

[0118] 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 FGF used is 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.

[0119] Differentiation of PSCs into DE cultures and subsequently into various intermediate mature intestinal cell types can be determined by the presence of stage-specific cellular markers. In some embodiments, the expression of representative cellular components is used to determine DE formation. Representative cellular 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 FOXA2. , 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. In some embodiments, the absence of cellular components such as the foregut marker Pdx1 and albumin can be used to reveal directed hindgut formation. In some embodiments, one or more (e.g., at least one or three) intestinal transcription factors CDX2, KLF5, or SOX9 can be used to represent intestinal development. In some embodiments, one or more of GATA4 or GATA6 protein expression can be used to represent intestinal development.

[0120] In some embodiments, morphological changes can be used to represent the progression of directed differentiation.In some embodiments, spheroid (for example, hindgut midsection, hindgut, anterior foregut or posterior foregut spheroid) is subjected to three-dimensional culture conditions for maturation.In some embodiments, gastrointestinal organoid can be 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56, The spheroids mature in 57, 58, 59, or 60 days, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, up to the aforementioned number of days, or up to about the aforementioned number of days, or any number of days within a range defined by any two of the aforementioned numbers, e.g., 6-60 days, 20-50 days, 30-40 days, 6-50 days, or 30-60 days. In some embodiments, a highly complex epithelium surrounded by mesenchymal cells can be observed following spheroid formation. In some embodiments, gastrointestinal organoids, polarized columnar epithelium, goblet cells, or smooth muscle cells are 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, It can be observed for 4, 55, 56, 57, 58, 59, or 60 days, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, not more than the aforementioned number of days, not more than the aforementioned number of days, or any number of days within a range defined by any two of the aforementioned numbers, e.g., 6 to 60 days, 20 to 50 days, 30 to 40 days, 6 to 50 days, or 30 to 60 days.

[0121] In some embodiments, pluripotent stem cells are converted to intestinal cell types by a "one-step" process, for example, by directly treating pluripotent stem cells with one or more molecules capable of differentiating pluripotent stem cells in DE culture (e.g., activin A) in combination with additional molecules capable of promoting directed differentiation in DE culture (e.g., Wnt3a and FGF4).

[0122] 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 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.

[0123] 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 at an MOI 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, about, at least, at least about, equal to or less than, or equal to or less than, or any MOI within a range defined by any two of the MOIs, e.g., 0-5.0, 1.0-4.0, 2.0-3.0, 0-3.0, or 1.0-5.0. In some embodiments, after transduction, the PBMCs express stem cell reprogramming factors. In some embodiments, after transduction, the PBMCs are reprogrammed into iPSCs. In some embodiments, the iPSCs are grown on a feeder cell substrate. In some embodiments, the iPSCs are grown on a MEF feeder cell substrate. In some embodiments, iPSCs are grown on irradiated MEF feeder cell substrates. In some embodiments, iPSCs are grown on 0.1% gelatin. In some embodiments, iPSCs are grown in RPMI 1640, DMEM, DMEM / F12, Erythroid Expansion Medium, Minigut Medium, StemPro 34 SFM (serum-free medium), StemPro hESC SFM, mTeSR 1, or mTeSR Plus medium.

[0124] In some embodiments, the reprogrammed iPSCs are expanded in cell culture. In some embodiments, the iPSCs are expanded in Matrigel. In some embodiments, the iPSCs are expanded in cell culture medium containing a ROCK inhibitor (e.g., Y-27632). In some embodiments, the iPSCs are expanded to 80-95% confluence. In some embodiments, the iPSCs are differentiated into definitive endoderm cells. In some embodiments, the iPSCs are differentiated into definitive endoderm cells by contacting the iPSCs with activin A. In some embodiments, the iPSCs are further contacted with BMP4. In some embodiments, the iPSCs are contacted with a concentration of BMP4 that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, about the aforementioned concentrations, at least the aforementioned concentrations, at least about the aforementioned concentrations, or at or below the aforementioned concentrations.

[0125] In some embodiments, definitive endoderm cells are differentiated into mid-hindgut spheroids. In some embodiments, definitive endoderm cells are differentiated into mid-hindgut spheroids by contacting the definitive endoderm cells with one or more (e.g., at least one or two) GSK3 inhibitors or FGF4. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the FGF4 is recombinant FGF4. In some embodiments, definitive endoderm cells are differentiated into mid-hindgut spheroids without contacting the definitive endoderm cells with one or more (e.g., at least one or two) GSK3 inhibitors or FGF4. In some embodiments, definitive endoderm cells are differentiated into mid-hindgut spheroids without contacting the definitive endoderm with CHIR99021, FGF4, or both. In some embodiments, definitive endoderm cells are differentiated into mid-hindgut spheroids by contacting the definitive endoderm cells with epidermal growth factor (EGF).

[0126] In some embodiments, the mid-hindgut spheroids are embedded in a basement membrane or a basement membrane mimic. In some embodiments, the mid-hindgut spheroids are embedded in Matrigel. In some embodiments, the mid-hindgut spheroids are cultured in a basal intestinal medium. In some embodiments, the mid-hindgut spheroids are cultured in a basal intestinal medium to differentiate the mid-hindgut spheroids into intestinal organoids. In some embodiments, the basal intestinal medium comprises one or more of: advanced DMEM-F12, N2 supplement, vitamin A-free B27 supplement, HEPES, L-glutamine, penicillin-streptomycin, or epidermal growth factor (EGF), or any combination thereof. In some embodiments, the basal intestinal medium comprises EGF. In some embodiments, the mid-hindgut spheroids are filtered through pores. In some embodiments, the mid-hindgut spheroids are filtered through a pore size of 70 μm. In some embodiments, the mid-hindgut spheroids are separated into spheroids smaller than 70 μm and spheroids larger than 70 μm, hi some embodiments, spheroids larger than 70 μm are used in the methods described herein.

[0127] In some embodiments, definitive endoderm cells are differentiated into spheroids. In some embodiments, definitive endoderm cells are differentiated into spheroids by contacting the definitive endoderm cells with one or more (e.g., at least 1, 2, 3, 4) GSK3 inhibitors, FGF4, BMP inhibitors, or retinoic acid (RA). In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the FGF4 is recombinant FGF4. In some embodiments, the BMP inhibitor is noggin. In some embodiments, definitive endoderm cells are differentiated into spheroids without contacting the definitive endoderm cells with one or more (e.g., at least 1, 2, 3, 4) GSK3 inhibitors, FGF4, BMP inhibitors, or RA, or any combination thereof. In some embodiments, definitive endoderm cells are differentiated into spheroids without contacting the definitive endoderm with CHIR99021, FGF4, noggin, or RA, or any combination thereof. In some embodiments, definitive endoderm cells are differentiated into spheroids by contacting the definitive endoderm cells with epidermal growth factor (EGF).

[0128] In some embodiments, the spheroids are embedded in a basement membrane or a basement membrane mimic. In some embodiments, the spheroids are embedded in Matrigel. In some embodiments, the spheroids are cultured in a growth medium to differentiate the spheroids into organoids. In some embodiments, the spheroids are filtered through pores. In some embodiments, the spheroids are filtered through pores with a pore size of 70 μm. In some embodiments, the spheroids are divided into spheroids smaller than 70 μm and spheroids larger than 70 μm. In some embodiments, spheroids larger than 70 μm are used in the methods described herein.

[0129] Unformed organoids In some embodiments, gastrointestinal organoids are esophageal organoids, gastric organoids, fundus gastric organoids, antral gastric organoids, small intestinal (intestinal) organoids, or large intestinal (colonic) organoids. In some embodiments, gastrointestinal organoids are intestinal organoids. In some embodiments, gastrointestinal organoids are human intestinal organoids (HIOs). In some embodiments, gastrointestinal organoids are not formed by any of the methods disclosed herein. In some embodiments, gastrointestinal organoids comprise a generally spherical three-dimensional structure comprising polarized columnar epithelium. In some embodiments, the polarized columnar epithelium is surrounded by mesenchyme. In some embodiments, the mesenchyme comprises a smooth muscle-like layer. In some embodiments, the epithelium comprises crypt-like proliferative zones and villus-like structures. In some embodiments, the mesenchyme comprises laminated longitudinal and circular muscles. In some embodiments, gastrointestinal organoids comprise a lamina propria that contains all the major functional cell types of the gastrointestinal organ. In some embodiments, the generally spherical gastrointestinal organoids comprise layered mesenchyme.

[0130] How to create the same organoids In some embodiments, the gastrointestinal organoids are formed gastrointestinal organoids. In some embodiments, the gastrointestinal organoids are formed esophageal organoids, formed stomach organoids, formed fundus organoids, formed antral stomach organoids, formed small intestinal (intestinal) organoids, or formed large intestinal (colonic) organoids, or any combination thereof. In some embodiments, the gastrointestinal organoids are intestinal organoids. In some embodiments, the formed gastrointestinal organoids are HIOs. In some embodiments, the formed gastrointestinal organoids comprise a generally tubular three-dimensional structure comprising polarized columnar epithelium. In some embodiments, the polarized columnar epithelium is surrounded by mesenchyme. In some embodiments, the mesenchyme comprises a smooth muscle-like layer. In some embodiments, the epithelium comprises crypt-like proliferative zones and villus-like structures. In some embodiments, the mesenchyme comprises laminated longitudinal and circular muscles. In some embodiments, the formed gastrointestinal organoids comprise a lamina propria that contains all the major functional cell types of the gastrointestinal organ. In some embodiments, the shaped gastrointestinal organoids comprise stratified mesenchyme.

[0131] In some embodiments, the shaped gastrointestinal organoids are elongated gastrointestinal organoids. In some embodiments, the gastrointestinal organoids are formed into elongated structures. In some embodiments, the gastrointestinal organoids are formed into elongated structures using one of the embodiments of the forming tray described herein. In some embodiments, the shaped gastrointestinal organoids have a straight or essentially straight shape. In some embodiments, the shaped gastrointestinal organoids have a straight or essentially straight shape in at least one dimension. In some embodiments, the shaped gastrointestinal organoids have a straight or essentially straight shape in all dimensions. In some embodiments, the shaped gastrointestinal organoids have a rectangular parallelepiped, cubic, cylindrical, conical, or pyramidal shape. In some embodiments, the shaped gastrointestinal organoids have a curved or essentially curved shape. In some embodiments, the shaped gastrointestinal organoids have a curved or essentially curved shape in at least one dimension. In some embodiments, the shaped gastrointestinal organoids have a curved or essentially curved shape in all dimensions. In some embodiments, the formed gastrointestinal organoid has a spherical shape.In some embodiments, the formed gastrointestinal organoid has a non-spherical shape.In some embodiments, the formed gastrointestinal organoid has a shape that has at least one curved surface, but is otherwise straight.In some embodiments, the formed gastrointestinal organoid has a curved cube, a curved cube, a curved cylinder, a curved cone, a curved pyramid, a parabolic shape, a parabolic shape, a hyperbolic shape, a diploid shape, an elliptical shape, a spiral shape, a helix, a sine wave, a sinusoid, a serpentine shape, a square wave, a wave with a triangular wave, a sawtooth wave, a spindle shape, a dendritic shape, a branched shape, or a radial shape, or any combination thereof.In some embodiments, the spheroids (for example, hindgut midsection spheroids) prepared as disclosed herein are seeded in the groove of the collection channel of the forming tray.In some embodiments, the number of spheroids seeded into a collection channel is 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 10,000, or about, at least, at least about, up to, or about, up to, or any number of spheroids within a range defined by any two of the preceding numbers, e.g., 100-10,000 spheroids, 2,000-8,000 spheroids, 3,000-4,000 spheroids, 100-4,000 spheroids, or 3,000-10,000 spheroids per collection channel. In some embodiments, the spheroids have a density of mm. 3 The density may be 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids per mm, at about the aforementioned densities, at least the aforementioned densities, at least about the aforementioned densities, or at or below the aforementioned densities, or any density within a range defined by any two of the aforementioned densities, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids per mm 3 100-2000 spheroids per mm 3 500-1500 spheroids per mm 3 100-1000 spheroids per mm 3Spheroids are seeded into the collection channel at 1,000-2,000 spheroids per spheroid. In some embodiments, the spheroids in the collection channel of the formation tray, the Tissue Train culture plates, or both, are cultured in Minigut medium containing one or more of advanced DMEM-F12, glutamine, HEPES, penicillin, streptomycin, N2 supplement, B27 supplement, or EGF, or any combination thereof. In some embodiments, the Minigut medium contains EGF. In some embodiments, the EGF is recombinant EGF. In some embodiments, the spheroids in the collection channel of the formation tray, the Tissue Train culture plates, or both, are cultured in Matrigel. In some embodiments, the spheroids in the collection channel of the formation tray, the Tissue Train culture plates, or both, are cultured in a 50% mixture of Matrigel and Minigut medium. In some embodiments, the spheroids are grown in the collection channel for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, or less than the aforementioned number of days, or less than the aforementioned number of days, or a range between any two of the aforementioned values, e.g., 1-10 days, 3-7 days, 1-5 days, 4-10 days, 6-9 days, or 7-10 days. The type of spheroid selected is determined by the desired organoid, e.g., mid-hindgut spheroids for the preparation of intestinal organoids, hindgut spheroids for the preparation of colon organoids, anterior foregut spheroids for the preparation of esophageal organoids, or posterior foregut spheroids for the preparation of gastric organoids. In some embodiments, the spheroids are mid-hindgut spheroids. In some embodiments, the spheroids are hindgut spheroids. In some embodiments, the spheroids are foregut spheroids. In some embodiments, the spheroids are anterior foregut spheroids. In some embodiments, the spheroids are posterior foregut spheroids.

[0132] In some embodiments, the formed gastrointestinal organoid is an elongated gastrointestinal organoid.In some embodiments, the elongated gastrointestinal organoid comprises an elongated length, width, depth or diameter, or any combination thereof. In some embodiments, the elongate length is 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 millimeters, about the aforementioned lengths, at least the aforementioned lengths, at least about the aforementioned lengths, less than or equal to the aforementioned lengths, or any length within a range defined by any two of the aforementioned lengths, e.g., 1-50 mm, 10-40 mm, 20-30 mm, 1-30 mm, or 20-50 mm. In some embodiments, the width is 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 30 00 μm, about the aforementioned width, at least the aforementioned width, at least about the aforementioned width, equal to or less than the aforementioned width, or equal to or less than the aforementioned width, or any width within a range defined by any two of the aforementioned widths, e.g., 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm.In some embodiments, the depth is 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm. m, about the aforementioned depth, at least the aforementioned depth, at least about the aforementioned depth, less than or equal to the aforementioned depth, or less than or equal to about the aforementioned depth, or any depth within a range defined by any two of the aforementioned depths, for example, 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm. In some embodiments, the diameter is 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm. The diameter may be about, at least, at least about, equal to or less than the aforementioned diameter, or equal to or less than the aforementioned diameter, or any diameter within a range defined by any two of the aforementioned diameters, e.g., 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm.In some embodiments, the ratio of the elongate length to one or more (e.g., 1, 2, 3) of the width, depth, or diameter, or any combination thereof, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 500000 , 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, or 500,000, about the aforementioned ratio, at least the aforementioned ratio, at least about the aforementioned ratio, less than or equal to the aforementioned ratio, or any ratio within a range defined by any two of the aforementioned ratios, for example, 1 to 500,000, 100 to 500,000, 1,000 to 10,000, 1 to 500,000, or 1,000 to 500,000. In some embodiments, the elongated gastrointestinal organoids are 100 μm. 3 , 200 μm 3 , 300 μm 3 , 400 μm 3 , 500 μm 3 , 600 μm 3 , 700 μm 3 , 800 μm 3 , 900 μm 3 , 1000 μm 3 , 10000μm 3 , 100,000 μm 3 , 1,000,000 μm 3 , or 0.01 mm 3 , 0.1mm 3 , 1mm 3 , 2mm 3 , 3mm 3 , 4mm 3 , 5mm 3 , 6mm 3 , 7mm 3 , 8mm 3 , 9mm 3 , 10mm 3 , 100mm 3 , 1000mm 3, 1500mm 3 , or 2000mm 3 is, is about the aforementioned volume, is at least the aforementioned volume, is at least about the aforementioned volume, is less than or equal to the aforementioned volume, or is less than or equal to about the aforementioned volume, or any volume within a range defined by any two of the aforementioned volumes, e.g., 100 μm 3 ~2000mm 3 , 1000 μm 3 ~1000mm 3 , 0.1mm 3 ~5mm 3 , 100 μm 3 ~1mm 3 , or 1 mm 3 ~2000mm 3 is. In some embodiments, the elongated gastrointestinal spheroids are comprised of or formed from a number of spheroids, where the number of spheroids is, about, at least, at least about, up to, or equal to 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 10000 spheroids, or any number within a range defined by any two of the foregoing, e.g., 100-10000 spheroids, 2000-8000 spheroids, 3000-4000 spheroids, 100-4000 spheroids, or 3000-10000 spheroids. In some embodiments, the elongated gastrointestinal spheroids have a density of less than 1000 mm 3 The density of the spheroids may be 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids per mm, at about the aforementioned densities, at least the aforementioned densities, at least about the aforementioned densities, or at or below the aforementioned densities, or any density within a range defined by any two of the foregoing, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids per mm. 3100-2000 spheroids per mm 3 500-1500 spheroids per mm 3 100-1000 spheroids per mm 3 The spheroids are composed of or formed from collected spheroids, with 1,000 to 2,000 spheroids per spheroid. As described above, the type of spheroid selected is determined by the desired organoid, for example, mid-hindgut spheroids for preparing intestinal organoids, hindgut spheroids for preparing colon organoids, anterior foregut spheroids for preparing esophageal organoids, or posterior foregut spheroids for preparing gastric organoids. In some embodiments, the spheroids are mid-hindgut spheroids. In some embodiments, the spheroids are hindgut spheroids. In some embodiments, the spheroids are foregut spheroids. In some embodiments, the spheroids are anterior foregut spheroids. In some embodiments, the spheroids are posterior foregut spheroids.

[0133] In some embodiments, spheroids are subjected to tension while they are formed in the collection channel. In some embodiments, spheroids are subjected to tension after they are formed in the collection channel. In some embodiments, spheroids formed in the collection channel are grown in a Tissue Train culture plate, as described in more detail herein. In some embodiments, the Tissue Train culture plate includes nylon mesh tabs and a deformable rubber membrane. In some embodiments, spheroids in a molded form are aligned between and secured to the nylon mesh tabs. In some embodiments, the deformable rubber membrane applies a mechanical load to the formed spheroids. In some embodiments, the deformable rubber membrane applies uniaxial strain to the formed spheroids. In some embodiments, the deformable rubber membrane applies a uniaxial strain that induces an elongation of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, about, at least, at least about, less than, or equal to about, or any percentage within a range defined by any two of the percentages, e.g., 0%-20%, 5%-15%, 8%-12%, 0%-10%, or 10%-20%. In some embodiments, the uniaxial strain reinforces the elongated structure of the spheroids. In some embodiments, spheroids experiencing uniaxial strain differentiate into elongated gastrointestinal organoids. The type of spheroid that is selected is determined by the desired organoid, for example, the hindgut middle spheroid for preparing intestinal organoid, the hindgut spheroid for preparing colon organoid, the anterior foregut spheroid for preparing esophageal organoid, or the posterior foregut spheroid for preparing gastric organoid.In some embodiments, spheroid is the hindgut middle spheroid.In some embodiments, spheroid is the hindgut spheroid.In some embodiments, spheroid is the foregut spheroid.In some embodiments, the spheroids are anterior foregut spheroids. In some embodiments, the spheroids are posterior foregut spheroids.

[0134] In some embodiments, the gastrointestinal organoid described herein is esophageal organoid, gastric organoid, fundus stomach organoid, antral stomach organoid, intestinal organoid, or colonic organoid, or any combination thereof.In some embodiments, the gastrointestinal organoid described herein is intestinal organoid.In some embodiments, the gastrointestinal organoid described herein is HIO.In some embodiments, intestinal organoid is produced according to the method described herein.In some embodiments, gastrointestinal organoid is produced according to the method described herein.In some embodiments, unformed gastrointestinal organoid is produced according to the method known in the art.In some embodiments, spheroid (for example, hindgut mid, hindgut, anterior foregut or posterior foregut spheroid) and unformed gastrointestinal organoid (for example, esophagus, stomach, fundus stomach, antral stomach, intestine or colonic organoid) and the method for producing them are described. The method for producing the same is described in United States Patent (USP) Nos. 9,719,068 and 10,174,289, and PCT publications WO2016 / 061464, WO2017 / 192997, WO2018 / 106628, WO2019 / 074793, each of which is expressly incorporated herein by reference for the purpose of producing each unformed gastrointestinal organoid.In some embodiments, the gastrointestinal organoid described herein or the unformed gastrointestinal organoid described in reference publication is prepared as formed gastrointestinal organoid using one or more forming trays as described herein. In some embodiments, spheroids described herein or in referenced publications (e.g., mid-hindgut, hindgut, anterior foregut, or posterior foregut spheroids) are used to prepare shaped gastrointestinal organoids by culturing the spheroids in one or more collection channels of a formation tray described herein to differentiate the spheroids into shaped gastrointestinal organoids (e.g., esophageal, stomach, fundus stomach, antral stomach, intestinal, or colonic organoids). In some embodiments, culturing the spheroids in the collection channels is carried out under the conditions disclosed in the referenced publications for the particular organoids of interest.In some embodiments, the spheroids described herein or in the reference publications are used to prepare shaped intestinal organoids.In some embodiments, the spheroids described herein or in the reference publications are used to prepare shaped HIOs.In some embodiments, one or more forming trays described herein are used to form shaped gastrointestinal organoids (e.g., esophageal, stomach, fundus, antral stomach, intestinal, or colonic organoids) comprising one or more features described herein.In some embodiments, one or more forming trays described herein are used to form shaped intestinal organoids.In some embodiments, one or more forming trays described herein are used to form shaped HIOs.

[0135] Forming Tray Embodiments Disclosed herein are embodiments of a formation tray used to prepare a molded organoid structure from a plurality of spheroids. In some embodiments, the molded organoid structure is intended for use, for example, in investigating gastrointestinal function or for transplantation into a host organism (e.g., human, mouse, rat, dog, cat, or other mammal). In some embodiments, the molded organoid structure is an elongated organoid structure. In some embodiments, the spheroid is a mid-hindgut spheroid, and the elongated organoid is an intestinal organoid, e.g., an elongated HIO. In some embodiments, the formation tray (10) has a structure designed for a predetermined shape configured to more closely complement the desired organ to be used. More specifically, the formation tray (10) has one or more (e.g., at least 1, 3, 5, 10) collection channels (12) configured to receive spheroids and collect the spheroids according to a collective arrangement that defines the predetermined shape. Continued culturing of spheroids within one or more collection channels (12) for a predetermined formation time effectively positions the spheroids relative to one another in a cast state configured to maintain the spheroids in a predetermined shape upon removal from one or more collection channels (12) for further culture and / or transplantation. In some embodiments, the predetermined shape is a non-spherical predetermined shape, such as an elongated column. In some embodiments, the predetermined shape of the elongated column of one or more collection channels (12) defines a cast state for maintaining the arrangement of spheroids in the predetermined shape of the elongated column. In some embodiments, the term "cast state" as used herein has its plain and ordinary meaning in light of this specification and refers to spheroids that are fixed relative to one another to maintain a predetermined shape while allowing movement such that the predetermined shape remains flexible, including, but not limited to, elastic flexibility or malleable flexibility. In some embodiments, the cast state and predetermined shape are not intended to limit the arrangement of the spheroids to a rigid, fixed state, and it is understood that the predetermined shape is sufficiently maintained to complement a desired organ function while allowing manipulation and structural connection to the desired organ by a surgeon during transplantation.

[0136] 2A-C show one embodiment of a forming tray (10) including a plurality of collection channels (12). Each of the plurality of collection channels (12) in this embodiment has an elongated length (14), a width (16), and a depth (18). In some embodiments, the elongated length (14) is 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 millimeters. , at least the aforementioned length, at least about the aforementioned length, not more than the aforementioned length, not more than about the aforementioned length, or any length within a range defined by any two of the aforementioned lengths, e.g., 1 to 50 mm, 10 to 40 mm, 20 to 30 mm, 1 to 30 mm, or 20 to 50 mm, and defined by opposing longitudinal sidewalls (20) of the forming tray (10). In some embodiments, width (16) is 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm, or is about the aforementioned width, or is at least The lateral sidewalls (22) extend laterally, perpendicular to the longitudinal direction, and are defined by opposing lateral sidewalls (22) that are at least as wide as, at least about as wide as, equal to or less than, or equal to or less than, or any width within the range defined by any two of the widths defined above, e.g., 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm.In some embodiments, the depth (18) is 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm, or about the aforementioned. The depth (18) extends transversely, perpendicular to the longitudinal and lateral directions, to a depth, at least the aforementioned depth, at least about the aforementioned depth, less than or equal to the aforementioned depth, or less than or equal to about the aforementioned depth, or any depth within a range defined by any two of the aforementioned depths, e.g., 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm. In some embodiments, the depth (18) is defined between a channel opening (24) in the top surface (26) of the forming tray (10) and a floor (28) of the forming tray (10). Thus, in some embodiments, each of the plurality of collection channels (12) is defined between a respective longitudinal sidewall (20), lateral sidewall (22), channel opening (24), and floor (28).In some embodiments, each of the plurality of collection channels (12) has a radius of curvature of 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm. or 3000 μm, about the aforementioned radius, at least the aforementioned radius, at least about the aforementioned radius, equal to or less than the aforementioned radius, or equal to or less than the aforementioned radius, or any radius within a range defined by any two of the aforementioned radii, for example, 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm. and a radius of about 0.2 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2500 μm, or 3000 μm. In some embodiments, each of the plurality of collection channels (12) has a generally cylindrical shape extending therebetween with another radius of curvature that is at least, at least about, equal to or less than, or equal to or less than the aforementioned radius, or any radius within the range defined by any two of the aforementioned radii, e.g., 0.2 μm to 3000 μm, 200 μm to 1500 μm, 500 μm to 1000 μm, 0.2 μm to 1000 μm, or 500 μm to 3000 μm. In some embodiments, each of the plurality of collection channels (12) has a radius of curvature of 100 μm. 3 , 200 μm 3 , 300 μm 3 , 400 μm 3 , 500 μm 3 , 600 μm 3 , 700 μm 3 , 800 μm 3 , 900 μm 3 , 1000 μm 3, 10000μm 3 , 100,000 μm 3 , 1,000,000 μm 3 , or 0.01 mm 3 , 0.1mm 3 , 1mm 3 , 2mm 3 , 3mm 3 , 4mm 3 , 5mm 3 , 6mm 3 , 7mm 3 , 8mm 3 , 9mm 3 , 10mm 3 , 100mm 3 , 1000mm 3 , 1500mm 3 or 2000 mm3, about the aforementioned volume, at least the aforementioned volume, at least about the aforementioned volume, less than or equal to the aforementioned volume, or less than or equal to the aforementioned volume, or any volume within a range defined by any two of the aforementioned volumes, e.g., 100 μm 3 ~2000mm 3 , 1000 μm 3 ~1000mm 3 , 0.1mm 3 ~5mm 3 , 100 μm 3 ~1mm 3 , or 1 mm 3 ~2000mm 3has a volume. In some embodiments, the forming tray (10) has a plurality of collection channels (12) or one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) collection channels (12) having the same or approximately the same length (14), the same or approximately the same width (16), and the same or approximately the same depth (18) dimensions. In some embodiments, the collection channels of the plurality of collection channels (12) or one or more collection channels (12) do not necessarily have the same or approximately the same length (14), the same or approximately the same width (16), or the same or approximately the same depth (18) dimensions, or any combination thereof. In some embodiments, the forming tray (10) has a plurality of collection channels (12) or one or more collection channels (12) that are parallel or approximately parallel to one another. In some embodiments, the collection channels of the plurality of collection channels (12) or one or more collection channels (12) do not necessarily have to be parallel or approximately parallel to one another. In some embodiments, the forming tray (10) includes a lid configured to cover one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the plurality of collection channels (12) or one or more collection channels (12). In some embodiments, the one or more collection channels (12) are formed without channel openings (24) such that they are encapsulated rather than open at the top surface (26). In some embodiments, the one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) encapsulated collection channels (12) comprise a tube or hose structure. In some embodiments, the forming tray (10) is not intended to be unnecessarily limited to the specific number, arrangement, or size of collection channels (12) shown in the embodiment of FIGS. 2A-C or described herein.

[0137] In some embodiments, one or more collection channels (12) are configured to gather spheroids together toward a collection arrangement that defines a predetermined shape. In some embodiments, one or more collection channels (12) taper together from a relatively wide channel opening (24) toward a relatively narrow floor (28). Figures 2A-C show an embodiment of a formation tray (10) in which opposing longitudinal sidewalls (20) taper toward each other from the channel opening (24) to the floor (28), while opposing lateral sidewalls (22) similarly taper toward each other from the channel opening (24) to the floor (28). In some embodiments, gravity forces spheroids in one or more collection channels (12) downward in the transverse direction, while recoil forces exerted on the spheroids by the longitudinal and lateral sidewalls (20, 22) orient the spheroids upward and inward relative to each other, effectively gathering the spheroids into a predetermined shape.

[0138] In some embodiments, the one or more collection channels (12) are not limited by the embodiments shown in FIGS. 2A-C. In some embodiments, the one or more collection channels have a straight or essentially straight shape. In some embodiments, the one or more collection channels have a straight or essentially straight shape in at least one dimension. In some embodiments, the one or more collection channels have a straight or essentially straight shape in all dimensions. In some embodiments, the one or more collection channels have a rectangular, cubic, cylindrical, conical, or pyramidal shape. In some embodiments, the one or more collection channels have a curved or essentially curved shape. In some embodiments, the one or more collection channels have a curved or essentially curved shape in at least one dimension. In some embodiments, the one or more collection channels have a curved or essentially curved shape in all dimensions. In some embodiments, the one or more collection channels have a spherical shape. In some embodiments, the one or more collection channels have a non-spherical shape. In some embodiments, the one or more collection channels have a shape that has at least one curved surface but is otherwise straight. In some embodiments, one or more collection channels have the shape of a curved cube, a curved cube, a curved cylinder, a curved cone, a curved pyramid, a parabolic shape, a parabolic shape, a hyperbolic shape, a diploid shape, an elliptical shape, a spiral shape, a helix shape, a sine wave shape, a sinusoidal wave shape, a serpentine wave shape, a square wave shape, a wave shape with a triangular wave shape, a sawtooth wave shape, a spindle shape, a dendritic shape, a branched shape or a radial shape, or any combination thereof.In some embodiments, the formed gastrointestinal organoid is suitably formed with any one of the shapes of one or more collection channels described herein and elsewhere.

[0139] In some embodiments, each of the one or more collection channels (12) contains a number of spheroids and liquid medium appropriate for the volume of the one or more collection channels (12). In some embodiments, each of the one or more collection channels (12) contains a number of spheroids that is 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 10,000, about, at least, at least about, up to, or up to, or any number within a range defined by any two of the foregoing, e.g., 100-10,000 spheroids, 2,000-8,000 spheroids, 3,000-4,000 spheroids, 100-4,000 spheroids, or 3,000-10,000 spheroids per collection channel (12).In some embodiments, each of the one or more collection channels (12) containing a number of spheroids contains 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 10000 spheroids, about the aforementioned number, at least the aforementioned number, at least about the aforementioned number, up to the aforementioned number, or up to the aforementioned number, or any number of spheroids within a range defined by any two of the foregoing, e.g., 100-10000 spheroids, 2000-8000 spheroids, 3000-4000 spheroids, The spheroids may comprise 100 to 4000 spheroids, or 3000 to 10000 spheroids, and have a length (14) that is about, at least, at least about, less than, or equal to about 10, 15, or 20 mm; a width (16) that is about, at least, at least about, less than, or equal to about 0.5 mm; and a depth (18) that is about, at least, at least about, less than, or equal to about 0.5 mm. In some embodiments, the spheroids are 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids / mm. 3 is about the aforementioned density, is at least the aforementioned density, is at least about the aforementioned density, is equal to or less than the aforementioned density, or is equal to or less than the aforementioned density, or any density within a range defined by any two of the foregoing, e.g., mm 3 100-2000 spheroids per mm 3 500-1500 spheroids per mm 3 100-1000 spheroids per mm 3Collect at a predetermined density, which is 1000-2000 spheroids per well.

[0140] In the embodiment shown in Figures 2A-C, the longitudinal sidewalls (20), lateral sidewalls (22), and floors (28) of one or more collection channels (12) are arcuate, each having a radius of curvature such that they are tubular and continuous with one another. In some embodiments, one or more (e.g., at least 1, 3, 5, 10) of the longitudinal sidewalls (20), lateral sidewalls (22), or floors (28) intersect such that they are not continuous. In some embodiments, the sidewalls (20, 22) of one or more (e.g., at least 1, 3, 5, 10) and floors (28) of one or more (e.g., at least 1, 3, 5, 10) of the one or more collection channels (12) are not intended to be unnecessarily limited to the smooth, continuous surfaces shown in the embodiment of Figures 2A-C or described herein. In some embodiments, the shape and dimensions of the one or more (e.g., at least 1, 3, 5, 10) collection channels (12) are configured for effective growth of spheroids, such as gastrointestinal spheroids, as described herein. In some embodiments, the shape and dimensions of the one or more (e.g., at least 1, 3, 5, 10) collection channels (12) are configured for effective growth of spheroids that are not gastrointestinal spheroids. In some embodiments, the one or more collection channels (12) are not intended to be unnecessarily limited to the particular shapes and / or dimensions shown in the illustrated embodiments or described herein.

[0141] In some embodiments, the formation tray (10) has a single, unitary structure. In some embodiments, the formation tray (10) is fabricated from a biocompatible material. In some embodiments, the formation tray (10) is fabricated from a biocompatible material that inhibits attachment of spheroids to the formation tray (10) within one or more collection channels (12) while allowing the development of spheroids into gastrointestinal organoid structures. In some embodiments, the formation tray (10) is formed from multiple components, and the surfaces within at least one or more collection channels (12) are fabricated from a biocompatible material. In some embodiments, the biocompatible material comprises, consists essentially of, or consists of stainless steel, titanium, polymeric organosilicone compounds, polydimethylsiloxane (PDMS), glass, plastic, PVC, PE, PP, PMMA, PS, PTFE, nylon, polyurethane, PET, PES, hyaluronan, chitosan, sugar, ceramic, alumina, zirconia, bioglass, hydroxyapatite, or any combination thereof, or other biocompatible materials known in the art. In some embodiments, the formation tray (10) is sterile, resistant to adhesion by tissues and / or cells, includes a hydrophobic surface, includes features that improve the formation and subsequent removal and / or use of the disclosed tissues, or any combination thereof. In some embodiments, the formation tray (10) includes one or more (e.g., at least 1, 3, 5, 10) small molecule compounds, activators, inhibitors, growth factors, nucleic acids, DNA, RNA, peptides, polypeptides, or proteins, or any combination thereof that promote growth and / or differentiation.

[0142] 3A-D show one embodiment of a plurality of pre-arranged spheroids (30) for culturing in a formation tray (10). In some embodiments, a plurality of iPSCs (32) are cultured in a biocompatible container (34) under conditions that differentiate the plurality of iPSCs into a plurality of definitive definitive endoderm cells (36), such as those described herein or otherwise known in the art. In some embodiments, a plurality of definitive endoderm cells (36) are cultured in a biocompatible container (34) under conditions that differentiate the plurality of definitive endoderm cells into a plurality of spheroids (38), such as those described herein or otherwise known in the art. In some embodiments, the spheroids (38) are hindgut spheroids. In some embodiments, the spheroids (38) are foregut spheroids. In some embodiments, the spheroids (38) are anterior foregut spheroids. In some embodiments, the spheroids (38) are posterior foregut spheroids. In some embodiments, the spheroid (38) is a mid-hindgut spheroid. In some embodiments, the spheroid (38) is not a mid-hindgut spheroid. In some embodiments, as the plurality of spheroids (38) is formed, a scaffold strand (40) is introduced in proximity to the plurality of spheroids (38). In some embodiments, the scaffold strand (40) is permanently or semi-permanently positioned or housed in or near the biocompatible container (34) so that the spheroids can contact the scaffold strand (40) as they form. In some embodiments, the scaffold strand (40) is formed from a biocompatible material configured to attract and contact the developing spheroids (38), and then the developing spheroids (38) are pressed into the plurality of pre-positioned spheroids (30). In some embodiments, the scaffold strands (40) have a shape complementary to the collection channel (12) so that the plurality of pre-arranged spheroids (30) can be more efficiently collected within and removed from the biocompatible container (34). In some embodiments, the scaffold strands (40) are generally linear and fibrous.In some embodiments, the scaffold strands (40) are strings, fibers, wires, cables, or other structures configured to attract and position the spheroids (38). In some embodiments, the scaffold strands (40) are constructed from a suitable metallic or non-metallic biocompatible material configured to attract the spheroids while allowing the spheroids to develop into organoids.

[0143] In some embodiments, once a sufficient number of pre-arranged spheroids (30) have been seeded, the scaffold strands (40) to which the pre-arranged spheroids (30) are attached are removed, and the pre-arranged spheroids (30) are transferred to one or more collection channels (12) (Figure 4). In some embodiments, the scaffold strands (40) can then be discarded, leaving the pre-arranged spheroids (30) in their predetermined shape. In some embodiments, linear pre-arranged spheroids (30) simplify placement into a complementary shaped collection channel (12), although such spheroids (38) can be cultured and removed from the biocompatible container (34) without the use of the scaffold strands (40). Figure 4 shows one embodiment of pre-arranged spheroids (30) cultured in one or more collection channels (12) on days 1 (d1), 3 (d3), and 5 (d5). In some embodiments, the pre-arranged spheroids (30) are cultured in one or more collection channels (12) for a predetermined formation time as described herein (e.g., see above), such that fusion occurs between the mesenchyme of the pre-arranged spheroids (30), a blood supply forms, innervation occurs, or the spheroids adopt a predetermined shape (e.g., an elongated column for the elongated gastrointestinal organoids described herein), or any combination thereof.

[0144] In some embodiments, the pre-positioned spheroids are transferred to a Tissue Train culture plate (Flexcell International Corp, Burlington, NC). In some embodiments, the Tissue Train culture plate includes nylon mesh tabs and a deformable rubber membrane disposed between the nylon mesh tabs. In some embodiments, the pre-positioned spheroids are aligned on the deformable rubber membrane between the nylon mesh tabs so that the length (i.e., longest dimension) of the pre-positioned spheroids is located on and between the nylon mesh tabs. In some embodiments, the nylon mesh tabs serve as anchors to hold the ends of the pre-positioned spheroids. In some embodiments, the Tissue Train culture plate includes a vacuum chamber below the deformable rubber membrane, and application of vacuum to the Tissue Train culture plate stretches the deformable rubber membrane toward the vacuum chamber. In some embodiments, while the vacuum is applied to the Tissue Train culture plate, the pre-positioned spheroids are positioned on the deformable rubber membrane between the nylon mesh tabs. Then, in some embodiments, the vacuum is released, causing the deformable rubber membrane to return to its unstretched state. In some embodiments, the return to the unstretched state imparts a strain to the pre-positioned spheroids located on top of the deformable rubber membrane. In some embodiments, the strain is a uniaxial strain. In some embodiments, the strain is a uniaxial strain directed outward toward the nylon mesh tab.In some embodiments, the uniaxial strain imparts to the pre-positioned spheroids an elongation of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, about the aforementioned elongation, at least the aforementioned elongation, at least about the aforementioned elongation, or less than or equal to the aforementioned elongation, or any percentage within a range defined by any two of the aforementioned percentages, e.g., 0%-20%, 5%-15%, 8%-12%, 0%-10%, or 10%-20%. In some embodiments, the uniaxial strain imparted by the deformable rubber membrane maintains the pre-positioned spheroids in an elongated shape. In some embodiments, the pre-positioned spheroids are subjected to a Tissue In the Train culture plate, the cells are cultured under strain 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, about, at least, at least about, up to, or about, or any number of days within a range defined by any two of the preceding numbers, e.g., 1 to 50 days, 10 to 40 days, 20 to 30 days, 1 to 30 days, or 20 to 50 days.

[0145] In some embodiments, the size and dimensions of the one or more collection channels (12) and / or the size and dimensions of the shaped gastrointestinal organoids are appropriately configured for a mouse or other organism that is approximately the size of a mouse. In some embodiments, the size and dimensions of the one or more collection channels (12) and / or the size and dimensions of the shaped gastrointestinal organoids are appropriately configured for a human. In some embodiments, the elongated length (14) of the collection channel (12) or the length of the formed gastrointestinal organoids is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460 , 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, or 600 cm, about the aforementioned lengths, at least the aforementioned lengths, at least about the aforementioned lengths, less than or equal to the aforementioned lengths, or less than or equal to the aforementioned lengths, or any length within a range defined by any two of the aforementioned lengths, e.g., 1 to 600 cm, 100 to 500 cm, 200 to 300 cm, 1 to 300 cm, or 200 to 600 cm. In some embodiments, the width (16) of the collection channel (12) or the width of the formed gastrointestinal organoids extends laterally across 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 cm, about said width, at least said width, at least about said width, no more than said width, no more than about said width, or any width within a range defined by any two of the aforementioned widths, e.g., 1-30 cm, 5-25 cm, 10-20 cm, 1-20 cm, or 10-30 cm.In some embodiments, the depth (18) of the collection channel (12) or the depth of the formed gastrointestinal organoids is 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 cm, or about the aforementioned depth, or at least the aforementioned depth, or at least about the aforementioned depth, or less than the aforementioned depth, or any depth within a range defined by any two of the aforementioned depths, e.g., 1-30 cm, 5-25 cm, 10-20 cm, 1-20 cm, or 10-30 cm, extending laterally. In some embodiments, the diameter of the formed gastrointestinal organoids is 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 cm, about said diameter, at least said diameter, at least about said diameter, less than or equal to said diameter, or less than or equal to said diameter, or any diameter within a range defined by any two of the foregoing diameters, e.g., 1-30 cm, 5-25 cm, 10-20 cm, 1-20 cm, or 10-30 cm. In some embodiments, the collection channel (12) or shaped gastrointestinal organoids are 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000, or 10,000,000 cm. 3 is, is about the aforementioned volume, is at least the aforementioned volume, is at least about the aforementioned volume, is less than or equal to the aforementioned volume, or is less than or equal to about the aforementioned volume, or any volume within a range defined by any two of the aforementioned volumes, e.g., 1 to 10,000,000 cm 3 , 500~1,000,000cm 3 , 10,000~100,000cm 3 , 1~100000cm 3 , or 10,000 to 10,000,000 cm 3In some embodiments, the collection channel (12) contains a number of spheroids and liquid medium appropriate for the volume of the collection channel to form molded gastrointestinal organoids of a size and dimensions appropriate for humans. In some embodiments, the collection channel (12) has a volume of 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 Or 10 20 spheroids, about the aforementioned number, at least the aforementioned number, at least about the aforementioned number, up to the aforementioned number, or up to about the aforementioned number, or any number of spheroids within a range defined by any two of the foregoing, e.g., 10 2 ~10 20 , 10 5 ~10 15 , 10 8 ~10 12 , 10 2 ~10 10 , or 10 10 ~10 20 The number of spheroids that are spheroids.In some embodiments, the formed gastrointestinal organoid is formed from several spheroids that are suitable for forming the formed gastrointestinal organoid with the size and dimension suitable for human.In some embodiments, the formed gastrointestinal organoid is formed from 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11, 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , or 10 20 spheroids, about the aforementioned number, at least the aforementioned number, at least about the aforementioned number, up to the aforementioned number, or up to about the aforementioned number, or any number of spheroids within a range defined by any two of the foregoing, e.g., 10 2 ~10 20 , 10 5 ~10 15 , 10 8 ~10 12 , 10 2 ~10 10 , or 10 10 ~10 20 In some embodiments, the gastrointestinal organoid that is suitable for human beings is formed esophageal organoid, formed stomach organoid, formed fundus stomach organoid, formed antrum stomach organoid, formed small intestine (intestine) organoid or formed large intestine (colon) organoid.In some embodiments, the gastrointestinal organoid that is suitable for human beings is formed intestinal organoid.In some embodiments, the gastrointestinal organoid that is suitable for human beings is formed HIO.

[0146] As disclosed herein in some embodiments, spheroids are grown in vitro under specific conditions to yield formed gastrointestinal organoids derived from PSCs. In some embodiments, the resulting formed gastrointestinal organoids serve as clinically useful tissues that can be used to study or treat a variety of different diseases, including, but not limited to, short bowel, intestinal failure, necrotizing enterocolitis (NEC), injury, ulcers, celiac disease, Crohn's disease, pathogenic infections, cancer, intestinal obstruction, irritable bowel syndrome, or any combination thereof. In some embodiments, the resulting formed gastrointestinal organoids are used to study esophageal, stomach, intestinal, or colonic function, including, but not limited to, drug screening, neurological function, microbiota interactions, or transplantation, or any combination thereof. In some embodiments, the resulting formed gastrointestinal organoids are used to study intestinal function. In some embodiments, the formed gastrointestinal organoids comprise a functional lumen. In some embodiments, the formed gastrointestinal organoids have the ability to further differentiate upon transplantation. In some embodiments, the formed gastrointestinal organoids are grown to the fetal stage in vitro and further differentiate upon transplantation. In some embodiments, formed gastrointestinal organoid is elongated gastrointestinal organoid.In some embodiments, formed gastrointestinal organoid is elongated intestinal organoid.In some embodiments, formed gastrointestinal organoid is elongated HIO.In some embodiments, formed gastrointestinal organoid is prepared by using any one of the forming trays described herein and according to any one of the methods described herein.

[0147] Disclosed herein is a method for generating shaped gastrointestinal organoids. In some embodiments, the shaped gastrointestinal organoids comprise a lumen. In some embodiments, the shaped gastrointestinal organoids are elongated gastrointestinal organoids described herein. In some embodiments, the method comprises: placing a plurality of spheroids into a collection channel comprising a predetermined shape; culturing the plurality of spheroids in the collection channel to differentiate the plurality of spheroids into shaped gastrointestinal organoids having a predetermined shape. In some embodiments, the shaped gastrointestinal organoids comprise mesenchyme and a lumen. In some embodiments, the mesenchyme is condensed mesenchyme. In some embodiments, the shaped gastrointestinal organoids are subject to spontaneous innervation. In some embodiments, the plurality of spheroids comprises 2500, 3000, 3500, 4000, 4500, or 5000 spheroids, about, at least, at least about, up to, or about, or any number of spheroids within a range defined by any two of the preceding numbers, e.g., 2500-5000 spheroids, 3000-4000 spheroids, 2500-4000 spheroids, or 3000-5000 spheroids. In some embodiments, the predetermined shape comprises a length. In some embodiments, the length is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 millimeters, about the aforementioned lengths, at least the aforementioned lengths, at least about the aforementioned lengths, less than or equal to the aforementioned lengths, or any length within a range defined by any two of the aforementioned lengths, e.g., 10-25 mm, 15-20 mm, 10-20 mm, or 15-25 mm. In some embodiments, the length is an elongated length. In some embodiments, the predetermined shape includes a diameter.In some embodiments, the diameter is 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm, about the aforementioned diameter, at least the aforementioned diameter, at least about the aforementioned diameter, less than or equal to the aforementioned diameter, or any diameter within a range defined by any two of the aforementioned diameters, e.g., 300 μm to 1000 μm, 500 μm to 800 μm, 300 μm to 600 μm, or 500 μm to 1000 μm. In some embodiments, the length to diameter ratio is 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, about the aforementioned ratio, at least the aforementioned ratio, at least about the aforementioned ratio, less than or equal to the aforementioned ratio, or any ratio within a range defined by any two of the aforementioned ratios, e.g., 10 to 100, 30 to 80, 40 to 60, 10 to 50, or 50 to 100. In some embodiments, the volume of the gastrointestinal organoid is less than 0.1 mm. 3 , 0.5mm 3 , 1mm 3 , 2mm 3 , 3mm 3 , 4mm 3 , 5mm 3 , 6mm 3 , 7mm 3 , 8mm 3 , 9mm 3 , 10mm 3 , 11mm 3 , 12mm 3 , 13mm 3 , 14mm 3 , 15mm 3 , 16mm 3 , 17mm 3 , 18mm 3 , 19mm 3 , 20mm 3 , 21mm 3 , 22mm 3 , 23mm 3 , 24mm 3 , or 25mm 3is, is about the aforementioned volume, is at least the aforementioned volume, is at least about the aforementioned volume, is less than or equal to the aforementioned volume, or is less than or equal to about the aforementioned volume, or any volume within a range defined by any two of the aforementioned volumes, e.g., 0.1 mm 3 ~25mm 3 , 10mm 3 ~25mm 3 , or 10mm 3 ~20mm 3 In some embodiments, the elongated gastrointestinal spheroids have a density of mm 3 The density of the spheroids may be 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids per mm, at about the aforementioned densities, at least the aforementioned densities, at least about the aforementioned densities, or at or below the aforementioned densities, or any density within a range defined by any two of the foregoing, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 spheroids per mm. 3 100-2000 spheroids per mm 3 500-1500 spheroids per mm 3 100-1000 spheroids per mm 3 The organoids are composed of or formed from collected spheroids, with 1,000 to 2,000 spheroids per spheroid. In some embodiments, the collection channel is non-spherical, and the formed gastrointestinal organoids are non-spherical gastrointestinal organoids. In some embodiments, the collection channel has an elongated shape, and the formed gastrointestinal organoids are elongated gastrointestinal organoids. In some embodiments, the lumen is not continuous throughout the length of the formed gastrointestinal organoid. In some embodiments, the formed gastrointestinal organoids are formed human gastrointestinal organoids. In some embodiments, the formed gastrointestinal organoids are derived from PBMC cells, biopsy tissue samples, or induced pluripotent stem cells reprogrammed from somatic cells transduced with Sendai virus.

[0148] In some embodiments, the plurality of spheroids are cultured in the collection channel 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, or 30 days, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, or less than the aforementioned number of days, or less than the aforementioned number of days. In some embodiments, the plurality of spheroids are cultured in growth medium. In some embodiments, the growth medium is advanced DMEM-F12. In some embodiments, the growth medium is Minigut medium. In some embodiments, the growth medium is supplemented with EGF. In some embodiments, the growth medium is not supplemented with CHIR99021 or FGF4, or both. In some embodiments, the plurality of spheroids comprise mesenchyme. In some embodiments, the multiple spheroids fuse together at the mesenchyme of the multiple spheroids.

[0149] In some embodiments, the method described herein further comprises inducing mechanical strain in the shaped gastrointestinal organoid.In some embodiments, the mechanical strain promotes the spontaneous innervation of the shaped gastrointestinal organoid.In some embodiments, the mechanical strain reduces the maturation time of the shaped gastrointestinal organoid.In some embodiments, the mechanical strain is uniaxial tensile strain.

[0150] In some embodiments, the formed gastrointestinal organoids further comprise enteric neurons or enteric neural progenitor cells, or both. In some embodiments, the formed gastrointestinal organoids comprise one or more myenteric plexuses. In some embodiments, the one or more myenteric plexuses comprise cells expressing the neuronal marker PGP9.5. In some embodiments, the formed gastrointestinal organoids have neural activity. In some embodiments, the formed gastrointestinal organoids comprise polarized columnar epithelium surrounded by mesenchyme. In some embodiments, the mesenchyme comprises a smooth muscle-like layer. In some embodiments, the formed gastrointestinal organoids comprise epithelium patterned into crypt-like proliferative zones, or villus-like structures, or both. In some embodiments, the formed gastrointestinal organoids comprise stacked longitudinal and circular muscles. In some embodiments, the formed gastrointestinal organoids comprise markers of smooth muscle or intestinal subepithelial myofibroblasts, or both. In some embodiments, the formed gastrointestinal organoids comprise one or more of enterocytes, enteroendocrine cells, goblet cells, Paneth cells, or any combination thereof. In some embodiments, formed gastrointestinal organoid comprises the cell that expresses one or more of villin, Muc2, DEFA5, CHGA or OLFM4, or any combination thereof.In some embodiments, formed gastrointestinal organoid is vascularized in vitro.In some embodiments, formed gastrointestinal organoid is vascularized when implanted in individual.

[0151] Described herein are embodiments of a formation tray. In some embodiments, the formation tray is used to culture one or more gastrointestinal organoids. In some embodiments, the formation tray is used to culture one or more molded gastrointestinal organoids. In some embodiments, the formation tray is used to culture one or more elongated gastrointestinal organoids. In some embodiments, the formation tray includes one or more collection channels configured to receive one or more spheroids therein. In some embodiments, the one or more collection channels have an elongated shape. In some embodiments, the one or more collection channels have a non-spherical shape. In some embodiments, the one or more collection channels are configured to collect one or more spheroids together such that the one or more spheroids define a predetermined shape. In some embodiments, the one or more spheroids differentiate into one or more gastrointestinal organoids having a predetermined shape. In some embodiments, the one or more spheroids differentiate into one or more elongated gastrointestinal organoids having a predetermined shape. In some embodiments, the one or more collection channels are made of a biocompatible material configured to inhibit attachment of one or more spheroids thereto. In some embodiments, one or more collection channels comprise one or more spheroids arranged therein.In some embodiments, one or more collection channels comprise cell culture medium or extracellular matrix or both therein.In some embodiments, one or more collection channels further comprise one or more gastrointestinal organoids arranged therein.In some embodiments, one or more gastrointestinal organoids are one or more molded gastrointestinal organoids produced by any one of the methods described herein.

[0152] Described herein are embodiments of kits. In some embodiments, the kit is used for culturing gastrointestinal organoids. In some embodiments, the kit includes a formation tray comprising one or more collection channels. In some embodiments, the formation tray is any one of the formation trays described herein. In some embodiments, the kit includes a plurality of spheroids configured to be received in one or more collection channels of the formation tray. In some embodiments, the kit includes a cell culture medium configured to be received in one or more collection channels of the formation tray.

[0153] Transplantation and Treatment Methods In some embodiments, after a predetermined period of time for forming and generating the shaped gastrointestinal organoids described herein, the shaped gastrointestinal organoids are transplanted into a host organism, for example, as a therapeutic or experimental model described herein.In some embodiments, the shaped gastrointestinal organoids are elongated gastrointestinal organoids.In one embodiment, after a predetermined period of time for forming and generating the shaped gastrointestinal organoids (46), the shaped gastrointestinal organoids (46) are transplanted into a host organism (44), as shown in Figure 5. In some embodiments, transplantation occurs after culturing the organoids for several days, which may be 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, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, up to the aforementioned number of days, or up to about the aforementioned number of days, or any number of days within a range defined by any two of the aforementioned numbers, e.g., 1 to 50 days, 10 to 40 days, 20 to 30 days, 1 to 30 days, or 20 to 50 days. In some embodiments, transplantation occurs after culturing the organoids for several days, such as 11, 12, 13, 14, 15, 16, or 17 days, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, or less than the aforementioned number of days, or less than the aforementioned number of days. In some embodiments, the formed gastrointestinal organoids are mature enough to be studied several days before gastrointestinal organoids prepared by transplantation and / or other methods known in the art reach the same or similar state of maturity, that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, about the aforementioned number of days, at least the aforementioned number of days, at least about the aforementioned number of days, not more than the aforementioned number of days, or not more than the aforementioned number of days, or any number of days within a range defined by any two of the aforementioned numbers, e.g., 1-20 days, 5-15 days, 10-15 days, 1-15 days, or 10-20 days.In some embodiments, the host organism is a mammal. In some embodiments, the host organism is an immunodeficient mammal. In some embodiments, the host organism is an immunodeficient mouse. In some embodiments, the host organism is a monkey, dog, hamster, or rat. In some embodiments, the host organism is an immunodeficient monkey, dog, hamster, or rat. In some embodiments, the host organism is a human. In some embodiments, the host organism is an immunodeficient human. In some embodiments, the host organism is an immunocompetent human. In some embodiments, the host organism is an immunocompetent human treated with an immunosuppressant. In some embodiments, the host organism is an immunocompetent human, and the shaped gastrointestinal organoids are autologous to the host organism. In some embodiments, the host organism is an immunocompetent human, and the shaped gastrointestinal organoids are allogeneic to the host organism. In some embodiments, the host organism is a mammal in need of a gastrointestinal organ transplant. In some embodiments, the host organism is a human in need of a gastrointestinal organ transplant. In some embodiments, the gastrointestinal organoids are not intended to be unnecessarily limited to the formed gastrointestinal organoids shown as (46) or described herein.

[0154] In some embodiments, gastrointestinal organoid is generally spherical gastrointestinal organoid, molded gastrointestinal organoid or elongated gastrointestinal organoid as described herein.In some embodiments, gastrointestinal organoid is transplanted adjacent to the intestine of animal.In some embodiments, gastrointestinal organoid is transplanted onto the mesenteric vasculature of animal.In some embodiments, gastrointestinal organoid is fixed with adhesive.In some embodiments, adhesive is cyanoacrylate adhesive.In some embodiments, gastrointestinal organoid is connected to the gastrointestinal tract of animal through anastomosis from organoid to intestine.In some embodiments, anastomosis is left-right anastomosis or end-to-end anastomosis. In some embodiments, the gastrointestinal organoids grow in the animal for a number of days that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, gastrointestinal organoids grow larger than in vitro gastrointestinal organoids prepared at the same time. In some embodiments, gastrointestinal organoids show integration with host organism tissue.

[0155] In some embodiments, gastrointestinal organoid comprises gastrointestinal cell lineage.In some embodiments, gastrointestinal organoid comprises one or more of mesenchymal cells, mucus cells, parietal cells, chief cells, gastrin cells, alveolar cells, enterocytes, enteroendocrine cells, goblet cells, microfold cells, cup cells, tuft cells or Paneth cells, or any combination thereof.In some embodiments, gastrointestinal organoid comprises the cell that expresses one or more (for example, 1, 3, 5) VILLIN, MUC2, DEFA5, CHGA or OLFM4, or any combination thereof.In some embodiments, gastrointestinal organoid spontaneously develops gastrointestinal cell lineage.

[0156] In some embodiments, intestinal organoid comprises gastrointestinal cell lineage.In some embodiments, gastrointestinal organoid comprises one or more of mesenchyme, enterocyte, enteroendocrine cell, goblet cell or Paneth cell, or any combination thereof.In some embodiments, intestinal organoid comprises the cell that expresses one or more (for example, 1, 3, 5) VILLIN, MUC2, DEFA5, CHGA or OLFM4, or any combination thereof.In some embodiments, intestinal organoid spontaneously develops intestinal cell lineage.

[0157] In some embodiments, gastrointestinal organoids comprise neural structures. In some embodiments, gastrointestinal organoids comprise cells that express neural cell markers. In some embodiments, gastrointestinal organoids comprise cells that express PGP9.5. In some embodiments, gastrointestinal organoids comprising neural cell structures or cells that express neural cell markers have not been combined with any neural cell lineage cells, such as neural crest cells, during their formation. In some embodiments, gastrointestinal organoids spontaneously develop neural structures. In some embodiments, gastrointestinal organoids spontaneously become innervated. In some embodiments, gastrointestinal organoids spontaneously become innervated without experiencing mechanical strain. In some embodiments, gastrointestinal organoids comprise one or more myenteric plexuses. In some embodiments, gastrointestinal organoids spontaneously develop one or more myenteric plexuses. In some embodiments, the myenteric plexus size of the gastrointestinal organoids is greater than the myenteric plexus size of gastrointestinal organoids combined with neural crest cells according to previous methods (e.g., methods found in PCT Publication WO2016 / 06146). In some embodiments, the gastrointestinal organoids comprise one or more myenteric plexuses at a percentage of total cell density that is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total cell density, at about the aforementioned density, at least the aforementioned density, at least about the aforementioned density, or less than the aforementioned density, or less than the aforementioned density, or any percentage within a range defined by any two of the aforementioned percentages, e.g., 1% to 20%, 5% to 15%, 8% to 12%, 1% to 15%, or 10% to 20%.

[0158] In some embodiments, gastrointestinal organoid comprises blood vessel or endothelial structure.In some embodiments, gastrointestinal organoid comprises cells that express blood vessel or endothelial marker.In some embodiments, gastrointestinal organ is not combined with any endothelial lineage cells.In some embodiments, gastrointestinal organoid spontaneously develops blood vessel or endothelial structure.In some embodiments, gastrointestinal organoid spontaneously becomes vascularized.In some embodiments, blood vessel or endothelial structure is derived from host organism.

[0159] In some embodiments, gastrointestinal organoid comprises lumen.In some embodiments, gastrointestinal organoid comprises lumen that occupies the total volume percentage of gastrointestinal organoid.In some embodiments, lumen occupies 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 gastrointestinal organoid. %, about the aforementioned volume, at least the aforementioned volume, at least about the aforementioned volume, less than the aforementioned volume, or less than about the aforementioned volume, or any percentage within a range defined by any two of the aforementioned percentages, e.g., 1%-40%, 10%-30%, 15%-20%, 1%-20%, or 10%-40% of the total volume of the gastrointestinal organoids.

[0160] In some embodiments, gastrointestinal organoids show gene upregulation compared with the organoids produced by traditional methods.In some embodiments, gastrointestinal organoids show gene upregulation compared with the organoids produced by traditional methods, about the above-mentioned number of genes, at least the above-mentioned number of genes, at least about the above-mentioned number of genes, less than the above-mentioned number of genes, or less than the above-mentioned number of genes, or any number of genes within the range defined by any two of the above-mentioned number of genes, for example, 100-800 genes, 200-600 genes, 300-500 genes, 100-400 genes or 400-800 genes. In some embodiments, the gastrointestinal organoids have about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 genes, compared to organoids produced by conventional methods. The upregulated genes may be at least, at least about, up to, or about the aforementioned number of genes, or any number of genes within a range defined by any two of the aforementioned numbers, e.g., downregulation of 100-2000 genes, 400-1500 genes, 700-1000 genes, 100-1000 genes, or 1000-2000 genes. In some embodiments, the upregulated genes are involved in one or more (e.g., at least 1, 3, 5, 10) of neuronal differentiation, neurogenesis, neuronal generation, neuronal projection development, regulation of multicellular organism development, neuronal development, neuronal projection morphogenesis, cell adhesion, axon development, or biological adhesion, or any combination thereof.In some embodiments, the upregulated genes are involved in one or more (e.g., at least 1, 3, 5, 10) of pattern specification processes, regional formation, anterior / posterior pattern specification, anatomical structure formation involved in morphogenesis, animal organ morphogenesis, embryonic development, tube morphogenesis, epithelial development, epithelial tube morphogenesis, embryonic morphogenesis, circulatory system development, positive regulation of multicellular organism processes, regulation of multicellular organism development, tube development, vasculature development, regulation of cell differentiation, blood vessel development, positive regulation of developmental processes, gut development, extracellular matrix organization, extracellular structure organization, inflammatory response, biological adhesion, cell adhesion, response to wounding, regulation of cell proliferation, defense response, regulation of cell migration, regulation of motility, neuronal differentiation, neuronal generation, neurogenesis, neuronal projection development, neuronal development, regulation of multicellular organism development, cell adhesion, biological adhesion, neuronal projection morphogenesis, or cell projection organization, or any combination thereof.

[0161] Described herein is a method for treating an individual with impaired gastrointestinal function, or for improving or inhibiting harmful gastrointestinal disorders in an individual in need thereof.In some embodiments, the method comprises transplanting or engrafting gastrointestinal organoids into an individual.In some embodiments, the gastrointestinal organoid is the gastrointestinal organoid of any one of the methods described herein.In some embodiments, the gastrointestinal organoid is esophageal organoid, gastric organoid, fundus gastric organoid, antral gastric organoid, small intestine (intestinal) organoid, or large intestine (colon) organoid.In some embodiments, the gastrointestinal organoid is intestinal organoid.In some embodiments, the gastrointestinal organoid is HIO.In some embodiments, the gastrointestinal organoid is the formed gastrointestinal organoid of any one of the methods described herein.In some embodiments, the gastrointestinal organoid is the formed or elongated gastrointestinal organoid of any one of the methods described herein.In some embodiments, the gastrointestinal organoid is autologous or allogeneic to the individual. In some embodiments, gastrointestinal organoid is prepared from the induced pluripotent cells that are derived from an individual or obtained.In some embodiments, the individual needs gastrointestinal transplantation.In some embodiments, gastrointestinal organoid is transplanted or engrafted as a whole gastrointestinal organoid.In some embodiments, the transplantation site is gastrointestinal tissue. [Example]

[0162] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will appreciate that many other embodiments are also within the scope of the present invention, as described herein above and in the claims.

[0163] Example 1 Generation of iPSCs from human somatic tissue (biopsy or blood) Human somatic cells were collected and utilized for iPSC generation. Either the peripheral blood mononuclear cell (PBMC) fraction from fresh whole blood by Ficoll centrifugation or thawed cryopreserved PBMCs were used as starting material. PBMCs were cultured at 1-5 x 10 cells per well of a 6-well dish in 2 mL of erythrocyte expansion medium (EEM). 6 The cells were plated and incubated at 37°C, 5% CO2 for 24 hours. On day 2, 2 mL of the suspension was transferred to a new 6-well dish to select for non-adherent cells. The non-adherent cells were incubated at 37°C, 5% CO2 for 5 days. For the next 5 days, 1 mL of fresh EEM was added to each cell-containing well every other day. On day 7, 2 mL of 0.1% gelatin was added to a new 6-well plate per donor and placed in a 37°C, 5% CO2 incubator overnight. On day 8, approximately 187,500 irradiated mouse embryonic fibroblasts (MEFs) were plated onto a gelatin-coated 6-well plate (one well per PBMC donor).

[0164] On day 9, PBMCs were transduced with Sendai virus. For each sample to be transduced, 3 × 10 5 The cells were transferred to a 14 mL round-bottom tube in a volume of 500 μL or less of EEM. Sendai virus master mix containing human Klf4, Oct3 / 4, Sox2 (KOS), human L-Myc (hL-Myc), and human Klf4 (hKlf4) transgenes (CTS CytoTune 2.1, Invitrogen) was added at a MOI of (MOI × cell number) / (viral titer × 10 -3The virus was prepared at a ratio of 1000 μL (mL / μL). The MOI for KOS was 2.5, the MOI for hL-Myc was 2.5, and the MOI for hKlf4 was 1.5. The MOI (multiplicity of infection) refers to the number of infectious units (CIU) per cell, and the viral titer varies between virus preparations. The master mix was warmed to 37°C. 1 mL of Sendai virus master mix was added to each sample in a round-bottom tube. The samples were centrifuged at 1000 × g for 30 minutes at room temperature. After centrifugation, 1 mL of warm EEM was added to each tube, the cells were gently resuspended, and the entire volume was plated into the wells of a 12-well plate. The plate containing the samples was placed in a 37°C, 5% CO2 incubator.

[0165] On day 10, harvest the cells and medium from the 12-well plate and transfer them to a 15 mL conical tube. Rinse the wells with 1 mL of fresh EEM to ensure all cells are collected. Centrifuge the tubes at 200 x g for 5 minutes at room temperature to remove the Sendai virus from the cells. After centrifugation, discard the supernatant into 15% bleach disinfectant to inactivate the virus. Resuspend the cell pellet in 0.5 mL of EEM and plate it into a well of a 24-well plate. Incubate the cells at 37°C, 5% CO2 for 48 hours. Prepare a gelatin-coated 6-well dish for each sample transduced according to the process above.

[0166] On day 11, the gelatin was aspirated from the gelatin-coated plates, and approximately 187,500 irradiated MEFs were immediately plated onto the gelatin-coated plates in MEF medium. The MEFs were incubated at 37°C, 5% CO for 24 hours.

[0167] On day 12, remove the MEF medium and rinse the MEF plates twice with 2 mL of PBS per well. Add 2.5 mL of StemPro 34 SFM medium to each MEF well. A viable cell count is performed on the transduced samples to determine the total number of PBMCs. Four concentrations (cells / well): 5 x 10 3 , 1×10 5 , 2.5×105 , 5×10 5 PBMCs are replated onto the MEF-coated wells at 20°C. Cells are incubated at 37°C, 5% CO2 for 24 hours.

[0168] On days 13 and 15, a 50% medium change is performed by removing approximately 50% of the medium from the wells and adding an equal volume of fresh StemPro 34 SFM medium.

[0169] On day 16, a 50% medium change is performed by removing approximately 50% of the medium from the wells and adding an equal volume of StemPro hESC medium plus bFGF (2 μg / mL).

[0170] Perform a complete medium change using fresh hESC medium + bFGF (2 µg / mL) daily and monitor cell colony formation from days 17 to 40 (usually around days 21 to 28). Once the cells have reached the desired state, they can be frozen in 1 mL of cryopreservation medium in 1.5 mL cryovials at approximately 1-2 million cells per vial for future use.

[0171] Example 2 Generation of Unformed Human Intestinal Organoids (HIO) Human PSCs, either hESCs or hiPSCs, are cultured in feeder-free conditions in 6-well Nunclon Delta surface-treated tissue culture dishes (Nunc) coated with hESC-qualified Matrigel (Corning) and maintained in mTeSR1 medium (StemCell Technologies). hPSCs are first passaged with either Dispase (Thermo Fisher) for clump passaging or Accutase (Thermo Fisher) for single-cell passaging, then replated at high or low confluence in mTeSR1 medium onto hESC-qualified Matrigel-coated Nunclon 24-well plates. mTeSR1 medium for hPSCs undergoing single-cell passaging is supplemented with 10 μM Y-27632 dihydrochloride (a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, Tocris) on the first day only. hPSCs passaged at low confluence receive mTeSR1 medium on day 2 to allow the monolayer to reach 80-95% confluence, while hPSCs passaged at high confluence are expected to be 80-95% confluent already after day 1.

[0172] Differentiate cells into definitive endoderm by treating them with 100 ng / mL activin A (Cell Guidance Systems) for 3 days in RPMI 1640 medium (Invitrogen) supplemented with 1x NEAA (Invitrogen) and increasing concentrations of defined FBS (dFBS, Hyclone) at 0%, 0.2%, and 2.0% on days 1, 2, and 3 of activin A treatment, respectively. Additionally, a low concentration of BMP4 (1–15 ng / mL BMP4 (R&D Systems)) may or may not be supplemented on the first day of Activin A treatment. Following this, the DE monolayer is treated with mid-hindgut spheroid induction medium for 4 days. Mid-hindgut spheroid induction medium contains 3 µM CHIR99021 (glycogen synthase kinase 3 (GSK3) inhibitor, Stemgent) and 500 ng / mL FGF4 (R&D Systems) in RPMI 1640 supplemented with 1x NEAA and 2.0% dFBS.

[0173] Four days after mid-hindgut spheroid induction, floating spheroids are collected and embedded in 3D basement membrane Matrigel "domes / bubbles," then maintained in basal intestinal medium. Basal intestinal medium contains Advanced DMEM-F12 (Invitrogen), 1x N2 supplement (Invitrogen), 1x B27 supplement without vitamin A (Thermo Fisher), 15 mM HEPES (Life Technologies), 2 mM L-glutamine (Life Technologies), and 100 units / mL (1x) penicillin-streptomycin (Life Technologies) supplemented with 100 ng / mL epidermal growth factor (EGF, R&D Systems). Medium is changed every 3–4 days for approximately 2 weeks, or whenever the medium turns yellow due to pH, whichever occurs first. HIOs are then replated in fresh Matrigel with fewer organoids per Matrigel dome to allow for continued expansion. The same basic intestinal medium treatment schedule is usually maintained for an additional 2 weeks, allowing for long-term culture.

[0174] The resulting HIOs are three-dimensional structures (Figure 6A) containing polarized columnar epithelium surrounded by mesenchyme containing a smooth muscle-like layer. The epithelium is patterned into crypt-like proliferative zones and villus-like structures, while the mesenchyme is patterned into laminated longitudinal and circular muscle, and a lamina propria with all the major functional cell types of the intestine. Furthermore, organoids cultured using this method contain stratified mesenchyme and express markers of smooth muscle and intestinal subepithelial myofibroblasts, which are essential for the ability of these tissues to engraft into the intestine, and also resemble fetal intestinal morphology (Figure 6B). HIO mesenchymal differentiation precedes epithelial differentiation, indicating that they create and comprehend their own niche.

[0175] Example 3: Generation of molded elongated HIOs The in vitro containment protocol using the collection channel embodiments described herein generates continuous cylindrical organoid structures suitable for transplantation into immunodeficient animal models, etc. When compared to the HIO generation protocol of Example 2, a reduction of approximately 14 days in the time required in vitro before successful engraftment is observed.

[0176] Figure 7A illustrates the methodology for forming cylindrical intestinal organoid structures. hPSCs were cultured, induced into definitive endoderm, and differentiated into intestinal spheroids as described herein. Once spheroids were collected, they were filtered through a 70 µm pore size, retaining spheroids larger than 70 µm and discarding smaller spheroids. This size cutoff appears to be associated with a high ability to form HIOs, but this may vary for other tissue types. Retained spheroids were resuspended in 2 mL of Minigut medium, and a 50 µL sample was taken to quantify the number of spheroids under a microscope. Based on the quantification, the total number of spheroids was estimated. Approximately 3,000–4,000 spheroids were seeded per groove of the collection channel in 50% Matrigel diluted in Minigut medium. The number of spheroids used depends on the shape of the groove; the spheroids must be tightly packed in the groove. The parameters disclosed herein are for a groove having a hemispherical cross section with a diameter of 0.5 mm and a length of 15 mm.

[0177] The collection channels containing the spheroids were incubated at 37°C for 30–45 minutes. Five mL of Minigut medium supplemented with 100 ng / mL EGF was added to each collection channel. The medium was replaced with fresh medium on day 4 of culture. On day 6 of culture, the organoid structures were carefully removed from the grooves using Dumont #4 forceps. The organoid structures were placed into the wells of a Tissue Train culture plate, aligning the structures between the nylon mesh tabs. 200–400 μL of growth factor-reduced (GFR) Matrigel (Corning) was added to the plate, covering both the nylon mesh tabs and the organoid structures between them. The plate was incubated at 37°C for 90 minutes. Six mL of Minigut medium supplemented with 100 ng / mL EGF was added to each well. The medium was replaced with fresh medium twice a week until day 14.

[0178] Figure 7B shows the in vitro growth progression of elongated intestinal organoids forming in collecting channel grooves (g-HIO) by immunohistochemistry. Hematoxylin and eosin stained sections of g-HIO structures at days 6, 14, and 28.

[0179] Materials: mTeSR1 medium (StemCell Technologies); Advanced DMEM-F12 (Invitrogen), RPMI 1640 (Invitrogen), hESC-qualified Matrigel (Corning), defined FBS (Hyclone), L-glutamine (100x) (Invitrogen), penicillin-streptomycin (100x) (Invitrogen), 50x B27 supplement (Invitrogen), HEPES buffer (Invitrogen), dispase (Invitrogen), activin A (R&D Systems), FGF4 (R&D Systems), CHIR99021 (R&D Systems), polydimethylsiloxane (PDMS) tissue culture collection channel scaffold with appropriately sized grooves, GFR Matrigel, phenol red-free (Corning), Minigut medium: 2 mM glutamine, 10 mM Advanced DMEM-F12 medium supplemented with HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 1x N2 supplement, 1x B27 supplement, human recombinant EGF (R&D Systems), Dumont #4 forceps (Fine Science Tools), and Tissue Train culture plates with nylon mesh anchors (FlexCell International).

[0180] It is envisioned that alternative collection channel scaffolds can be constructed using a wide range of materials, such as metal, glass, plastic (e.g., acrylonitrile butadiene styrene (ABS), PLA, PP, PC, PS, PET, nylon, PE, polyurethane, PVC, PVDC, PTFE, polyester, PMMA, PEEK, PEI), using machining processes or 3D printers. In some embodiments, the scaffold is first fabricated and used to prepare a mold so that similarly shaped scaffolds can be fabricated from biologically inert or generally biologically inert materials such as PDMS or other silicones.

[0181] Example 4. Implantation of HIO Immunocompromised mice were maintained on an antibiotic chow diet (275 ppm sulfamethoxazole and 1365 ppm trimethoprim). Food and water were provided ad libitum before and after surgery. Mice were anesthetized with 2% inhaled isoflurane, and the abdominal wall was aseptically prepared with isopropyl alcohol and povidone-iodine. A 1-2 cm midline incision was made to access the abdominal cavity. The cecum was identified and gently removed, followed by the colon and small intestine. The mesentery was then spread, along with the distal ileum and ascending colon. A drop of octyl / butyl cyanoacrylate glue was applied over the area where the branching mesenteric vessels are located, one to two arcades from the ileocecal area. HIO was then applied to the glue and allowed to dry in place for a minimum of 3 minutes. The organoid structure was positioned adjacent to the intestine on top of the mesenteric vasculature. The intestine was then returned to the abdominal cavity, and the mice were given an intraperitoneal flush of piperacillin / tazobactam (100 mg / kg). The skin was closed in two layers, and the mice were injected subcutaneously with Buprenex (0.05 mg / kg). Mice were followed for 10 weeks for survival, at which point they were humanely euthanized. Organoid grafts were excised and processed for histology. Successful engraftment of organoids and integration of the human PSC-derived tissue with adjacent mouse host tissue were observed (Figure 8A-B). Whole-molded g-HIOs demonstrated successful engraftment and vascularization when transplanted into immunodeficient rats (Figure 8C). Transplantation of unmolded HIOs prepared according to previous methods (e.g., in WO2016 / 061464) failed to engraft in immunodeficient rats.

[0182] The engraftment rate and size of the HIO were measured. The overall survival rate was 85% (n=17 / 20), with 82% (n=14 / 17) successfully engrafting the HIO into the host mesentery. The transplanted organoids (tHIO) were approximately 46 times larger than time-matched in vitro HIOs (Figure 9A-B). Similar to the human intestine, histological analysis of this confirmed native-emerging mesenchyme with subepithelial elements and a muscle layer, as well as continued expansion of the epithelium with the presence of major cell lineages, including mesenchyme, enterocytes, enteroendocrine cells, goblet cells, and Paneth cells (Figure 9C).

[0183] If necessary, mice underwent organoid-to-intestine anastomosis. In a second surgery after the initial organoid transplantation, the organoids and adjacent small intestine were identified and removed from the abdominal cavity. Alternating left-right anastomosis was performed using 9-0 nylon in an interrupted fashion. After completion of the anastomosis, the anastomosis was checked for gross leaks and the intestine was returned to the abdominal cavity, taking care to avoid twisting. Fifty percent (n = 6) of mice survived up to 21 days at the time of harvest (Figure 9D).

[0184] After further testing, elongated intestinal organoids transplanted into NSG mice after culturing in the collecting channel (g-tHIO, g-tHIO) spontaneously developed neuronal structures upon engraftment (Figure 9E, using anti-PGP9.5 antibody as a pan-neuronal marker). A robust network of the myenteric plexus, the major collection of neurons in the enteric nervous system, is observed throughout the transplanted g-tHIO. It has previously been shown that intestinal organoids can be innervated by mechanically aggregating mid-hindgut spheroids with PSC-derived neural crest cells (NCCs) (see WO2016 / 061464). It is demonstrated herein that intestinal organoids generated from PSCs and prepared according to the protocol provided herein develop enteric nervous system structures without the addition of separate NCCs. Furthermore, the size of the nerve plexus in transplanted g-tHIOs is consistently larger than that seen in previous spheroid / NCC aggregate organoids.

[0185] Materials: Mice: Female or male immunodeficient NSG (NOD-scid IL2Rgamma) mice nullMice were housed in a microisolator system in a barrier facility. Mice were 6 to 14 weeks old. Other immunodeficient mouse models or other immunodeficient animal models, such as immunodeficient monkeys, dogs, hamsters, or rats, are contemplated. Diet: Modified solid diet (Picolab Rodent Diet 20, LabDiet) supplemented with 275 ppm sulfamethoxazole and 1365 ppm trimethoprim (LabDiet). For alternating left-right anastomosis surgery, a liquid diet (Jevity 1 Cal) was used. 0.3 mg / mL of 275 ppm sulfamethoxazole and 1365 ppm trimethoprim (Bactrim) were diluted in sterile water and given ad libitum after alternating left-right anastomosis. Antibiotics: 100 mg / kg of piperacillin and tazobactam were diluted in sterile saline and used for any surgery (ZOSYN, Pfizer). Fine Science Tools: Suture tying forceps, ring forceps, dissecting scissors, Bishop-Harmon forceps, Halsey needle holder, sterile tray. Isoflurane and anesthesia system. Sterile 7-0 non-absorbable silk suture (PERMA-HAND), sterile 4-0 coated absorbable suture (VICRYL RAPIDE), sterile 9-0 non-absorbable nylon suture with tapered needle (ETHILON), and octyl / butyl cyanoacrylate local tissue adhesive (GLUture).

[0186] Immunohistochemistry: Transplanted HIOs were harvested and fixed overnight in 4% paraformaldehyde (PFA), then processed and embedded in paraffin. Five-micrometer-thick sections of the tissue were prepared and deparaffinized for heat-induced epitope retrieval and staining. Both primary and secondary antibody incubations were performed overnight at 4°C in 1% bovine serum albumin in phosphate-buffered saline (PBS). The following primary antibodies and their respective dilutions were used: goat anti-villin (1:100), mouse anti-HuMuc2 (1:1250), mouse anti-DEFA5 (1:500), mouse anti-CHGA (1:500), and rabbit anti-OLFM4 (1:400). The following secondary antibodies were used: horse anti-goat biotin (1:1000), horse anti-mouse biotin (1:1000), and goat anti-rabbit biotin (1:1000). A peroxidase-based detection system was used, followed by Nuclear Fast Red (NUC) as a counterstain (Vector Labs, Polysciences, Inc.).

[0187] Example 5. Transcriptome profiling of forming intestinal organoids We evaluated the gene expression profiles of mid-hindgut spheroids, unformed intestinal organoids, and formed elongated intestinal organoids. Day 28 cultured g-HIOs showed 499 up-regulated genes and 1,546 down-regulated genes compared to day 28 cultured unformed HIOs (Figure 10A), indicating that the process of organoid formation in the collection channel significantly alters the biological activity of the constituent cells. Genes related to neuronal differentiation, neuronal development, neurogenesis, neuronal generation, neuronal projection development, regulation of multicellular organism development, neuronal development, neuronal projection morphogenesis, cell adhesion, axon development, or biological adhesion support the observation of spontaneous neurogenesis in g-HIOs and the development of organoids into a mature, organ-like state.

[0188] We also evaluated the gene expression profiles of g-HIOs grown in the collection channel at different culture times (day 0 [spheroids], day 6, day 14, and day 28) (Figure 10B). Each growth stage showed enrichment of genes related to different biological processes (Figure 10C). Day 0 spheroids were associated with pattern specification processes, region formation, anterior / posterior pattern specification, anatomical structure formation involved in morphogenesis, animal organ morphogenesis, embryonic development, tube morphogenesis, epithelial development, epithelial tube morphogenesis, and embryonic morphogenesis. Day 6 g-HIOs were associated with circulatory system development, positive regulation of multicellular organism processes, regulation of multicellular organism development, tube development, vasculature development, regulation of cell differentiation, blood vessel development, positive regulation of developmental processes, and gastrointestinal development. Day 14 g-HIOs were associated with extracellular matrix organization, extracellular structure organization, inflammatory response, biological adhesion, cell adhesion, response to wounding, regulation of cell proliferation, defense response, regulation of cell migration, and regulation of motility. The g-HIOs at day 28 were associated with neuronal differentiation, neuronal generation, neurogenesis, neuronal projection development, neuronal development, regulation of multicellular organism development, cell adhesion, biological adhesion, neuronal projection morphogenesis, and cell projection organization. These data suggest that significant developmental and morphological changes occur during organoid culture, similar to the in vivo development of intestinal tissue.

[0189] In at least some of the above-described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, except where such substitution is technically not feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0190] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein.

[0191] In general, terms used herein, and particularly in the appended claims (e.g., the appended claims), are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). Where a specific number of introduced claims is intended, such intention will be explicitly set forth in the claim; in the absence of such a setting, it will be further understood by those skilled in the art that no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as meaning that introducing a claim recitation with the indefinite article "a" or "an" limits a particular claim that includes such introduced claim recitation to embodiments that include only one such recitation. The same applies to the use of definite articles to introduce claim enumerations, even when the same claim contains the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). Furthermore, even when a specific number of enumerations in an introduced claim is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare enumeration of "two enumerations" without other modifiers means at least two enumerations, or two or more enumerations). Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one skilled in the art would understand the convention (e.g., "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 A, B, C together, etc.).Where a convention similar to "at least one of A, B, or C, etc." is used, generally, such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually all disjunctive words and / or phrases presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0192] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0193] As will be understood by those skilled in the art, for all purposes, including in terms of providing a written description, all ranges disclosed herein also encompass all possible subranges and combinations of subranges. A stated range can be readily recognized as sufficiently descriptive so that the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range described herein can be easily broken down into a lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the recited numbers and can subsequently be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, ranges include individual members. Thus, for example, a group having 1 to 3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4, or 5 items, etc.

[0194] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0195] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over such conflicting material.

[0196] References Selvaggi,G and Khan F.A.Overview of intestinal and multivisceral transplantation.UpToDate.2019 Nov.Available on the world wide web at uptodate.com / contents / overview-of-intestinal-and-multivisceral-transplantation / print Abu-Elmagd et al.Current status of intestinal and multivisceral transplantation.Gastroentrerol Rep(Oxf).2017 Feb;5(1):20-28 Ekser B,Kubal CA,Fridell JA,Mangus RS.Comparable outcomes in intestinal retransplantation:Single-center cohort study.Clin Transplant.2018;32:e13290. Venick RS,Wozniak LJ,Ngo K,et al.Unique technical and patient characteristics of retransplantation:a detailed single center analysis of intestinal transplantation.International Small Bowel Symposium 2013;Abstract 5.203(Available on the world wide web at www.tts.org / component / tts / ?view=presentation&id=13190) Kubal,CA,Pennington K,Fridell J,Ekser B,Mihaylov P and Mangus R.Challenges with intestinal and multivisceral re-transplantation:importance of timing of re-transplantation and optimal immunosuppression.Ann Transplant,2018;23:98-104. Hernandez F,Andres AM,Encinas JL,et al.Refining indications for intestinal retransplantation.International Small Bowel Symposium 2013;Abstract 12.241(Available on the world wide web at www.tts.org / component / tts / ?view=presentation&id=13241) Loike,JD and Pollack,R.Opinion:Develop Organoids,Not Chimeras,for Transplantation.The Scientist Magazine.2019 Aug.https: / / www.the-scientist.com / news-opinion / opinion--develop-organoids--not-chimeras--for-transplantation-66339. Wells et al.United States Patent.Patent No.US 9,719,068 B2.Aug 1,2017. McCracken et al.Generating human intestinal tissue from pluripotent stem cells in vivo.Nat Protoc.2011 Nov 10;6(12);1920-1928. A.Gurkan.Advances in small bowel transplantation.Turk J Surg.2017;33(3):135-141. McCracken et al.Generating human intestinal tissue from pluripotent stem cells in vivo.Nat Protoc.2011 Nov 10;6(12);1920-1928. Cortez AR,Poling HM,Brown NE,Singh A,Mahe MM and Helmrath MA.Transplantation of Human Intestinal Organoids into the Mouse Mesentery:A More Physiologic and Anatomic Engraftment Site.Surgery.2018 October;164(4):643-650. Capeling et al.Nonadhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids.Stem Cell Reports.2019 Feb;(12):381-394. Wiley LA,Burnight ER,DeLuca AP,Anfinson KR,Cranston CM,Kaalberg EE,Penticoff JA,Affatigato LM,Mullins RF,Stone EM and Tucker BA.cGMP production of patient-specific iPSCs and photoreceptors precursor cells to treat retinal degenerative blindness.Scientific Reports 2016.DOI:10.1038 / srep30742. Watson,CL et al.An in vivo model of human small intestine using pluripotent stem cells.Nat.Med.2014.20(11):1310-4. Workman MJ et al.Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.Nat.Med.2017.23(1):49-59. Poling,HM et al.Mechanically induced development and maturation of human intestinal organoids in vivo.Nat.Biomed.Eng.2018.2(6):429-442. Mahe MM et al.In vivo model of small intestine.Methods Mol.Biol.2017.1597:229-245. Grand,RJ et al.Development of the human gastrointestinal tract.Gastroenterology.1976.80:790-810.

Claims

1. 1. A method for generating formed gastrointestinal organoids comprising a lumen, comprising: disposing a plurality of spheroids into a collection channel having an enclosed predetermined shape, the enclosed predetermined shape being surrounded on all sides by sidewalls; Culturing the plurality of spheroids in the collection channel to differentiate the plurality of spheroids into the shaped gastrointestinal organoids having the predetermined shape; The method, wherein the formed gastrointestinal organoids comprise condensed mesenchyme and a lumen.

2. The method of claim 1 , wherein the collection channel has a groove shape and is elongated.

3. 3. The method of claim 1 or claim 2, wherein the collection channel has a length with opposing longitudinal sidewalls and a width with opposing lateral sidewalls.

4. 4. The method of any one of claims 1 to 3, wherein the collection channel has an elongated shape, and the shaped gastrointestinal organoids are elongated gastrointestinal organoids, the elongated gastrointestinal organoids having a length of 1 to 50 mm and a diameter of 0.2 μm to 3000 μm.

5. The method of any one of claims 1 to 4, wherein the elongated gastrointestinal organoids have a depth of 0.2 μm to 3000 μm.

6. 6. The method of any one of claims 1 to 5, wherein the lumen is not continuous throughout the length of the elongated gastrointestinal organoid.

7. 7. The method of any one of claims 1 to 6, wherein the shaped gastrointestinal organoids are shaped human gastrointestinal organoids.

8. The method of any one of claims 1 to 7, wherein the shaped gastrointestinal organoids further comprise enteric neurons or enteric neural progenitor cells.

9. The method of any one of claims 1 to 8, wherein the shaped gastrointestinal organoids further comprise one or more myenteric plexuses comprising cells that express the neuronal marker PGP9.

5.

10. 10. The method of any one of claims 1 to 9, wherein the shaped gastrointestinal organoids have neural activity.

11. 11. The method of any one of claims 1 to 10, further comprising inducing mechanical strain in the shaped gastrointestinal organoids, wherein the mechanical strain promotes spontaneous innervation of the shaped gastrointestinal organoids or reduces the maturation time of the shaped gastrointestinal organoids, or both.

12. The method of claim 11 , wherein the mechanical strain is a uniaxial tensile strain.

13. 13. The method of any one of claims 1 to 12, wherein the shaped gastrointestinal organoids further comprise a polarized columnar epithelium surrounded by mesenchyme, the mesenchyme comprising a smooth muscle-like layer.

14. 14. The method of any one of claims 1 to 13, wherein the shaped gastrointestinal organoids further comprise epithelium patterned into crypt-like proliferative zones or villi-like structures, or both.

15. 15. The method of any one of claims 1 to 14, wherein the shaped gastrointestinal organoids further comprise laminated longitudinal and circular muscles.

16. 16. The method of any one of claims 1 to 15, wherein the shaped gastrointestinal organoids are vascularized a) in vitro or b) upon engraftment into an individual.

17. The plurality of spheroids a) a plurality of mid-hindgut spheroids, wherein said formed gastrointestinal organoids are formed intestinal organoids; b) a plurality of hindgut spheroids, wherein said formed gastrointestinal organoids are formed colon organoids; c) a plurality of anterior foregut spheroids, wherein the shaped gastrointestinal organoids are esophageal organoids; and d) a plurality of posterior foregut spheroids, wherein the shaped gastrointestinal organoids are gastric organoids; The method of any one of claims 1 to 16, selected from the group consisting of:

18. A shaped gastrointestinal organoid produced by the method of any one of claims 1 to 17.

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