Improved methods for producing organoid compositions

Abstract

Disclosed herein is the organoid or its composition that is produced by aggregating intestinal endoderm monolayer and culturing the resulting intestinal endoderm aggregate.The example of these aggregated organoids includes but is not limited to aggregated liver organoid, aggregated stomach organoid, aggregated colon organoid and aggregated colon organoid.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 885,903, filed August 13, 2019, which is expressly incorporated by reference herein in its entirety.

[0002] Aspects of the present disclosure relate generally to organoid compositions and methods of making same, for example involving aggregation of progenitor cells in a formation plate. [Background technology]

[0003] Human pluripotent stem cells (hPSCs; including both embryonic stem cells and induced pluripotent stem cells) represent a renewable resource for generating human three-dimensional gastrointestinal tissues (e.g., human intestinal organoids; HIOs) organized into distinct epithelial and mesenchymal layers. For example, in HIOs, the epithelium contains all known intestinal epithelial cell types and exhibits several properties of functional intestinal tissue, including absorption, gut hormone synthesis, and mucosal secretion. After transplantation into experimental animal models, HIOs undergo significant proliferation and maturation to resemble the postnatal human intestine, including the mucosa, submucosa, and muscularis propria. Epithelial cells are arranged in crypt-villus structures that contain the adult stem cell activity / progenitor zone of the crypt, and the mature epithelium capable of functions such as nutrient absorption and brush border enzyme activity. As a result, organoids derived from pluripotent stem cells are a physiologically relevant and powerful tool to study intestinal development and disease, and also provide a novel platform for drug development. Moreover, given that induced pluripotent stem cells can be derived from any individual, including those with intestinal disease, it is possible to generate disease / patient-specific organoids such as HIOs for personalized medical applications. There is a persistent need for improved organoid compositions that are more similar to in vivo tissues, and for methods to generate organoid compositions that are more robust, scalable, faster, and cost-effective. The prior art documents relevant to the invention of this application are as follows (including documents cited during the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent Documents) (Patent Document 1) U.S. Patent Application Publication No. 2019 / 0093076 (Patent Document 2) U.S. Patent Application Publication No. 2018 / 0258400 (Patent Document 3) U.S. Patent Application Publication No. 2018 / 0043357 (Patent Document 4) U.S. Patent Application Publication No. 2017 / 0202885 (Non-Patent Literature) (Non-Patent Document 1) ARORA et al. "A process engineering approach to increase organoid yield," Development, 15 March 2017 (15.03.2017). Vol. 144, No. 6, Pgs. 1128-1136. entire document (Non-Patent Document 2) MILLER et al. "Generation of lung organoids from human pluripotent stem cells in vitro," Nature Protocols. 28 February 2019 (28.02.2019), Vol. 14, No. 2, Pgs. 518-540. entire document Summary of the Invention

[0004] Disclosed herein is a method for producing one or more aggregated organoids. In some embodiments, the method comprises: differentiating definitive endoderm into gut endoderm monolayer and gut spheroids; separating gut endoderm monolayer from gut spheroids; dissociating gut endoderm monolayer into single cell suspension of gut endoderm cells; aggregating the single cell suspension of gut endoderm cells into one or more gut endoderm aggregates; and culturing the one or more gut endoderm aggregates to produce one or more aggregated organoids. In some embodiments, gut endoderm monolayer is adherent, and gut spheroids are separated and suspended in growth medium. In some embodiments, definitive endoderm is differentiated from pluripotent stem cells. In some embodiments, definitive endoderm is differentiated from embryonic stem cells or induced pluripotent stem cells. In some embodiments, definitive endoderm is human definitive endoderm. In some embodiments, the separation step comprises aspirating growth medium and suspended gut spheroids from gut endoderm monolayer. In some embodiments, the dissociation step comprises enzymatically dissociating the gut endoderm monolayer. In some embodiments, the gut endoderm monolayer is enzymatically dissociated using Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express, or TrypLE Select, or any combination thereof. In some embodiments, the aggregation step comprises aggregating the single cell suspension in a hanging drop, centrifuging the single cell suspension in a "v" or "u" bottom microwell culture plate, aggregating the single cell suspension using an orbital shaker, or centrifuging the single cell suspension in a formation plate, or any combination thereof. In some embodiments, the formation plate is an Aggrewell plate.In some embodiments, each of the one or more enteric endoderm aggregates comprises about 250, about 500, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 7500, about 8000, about 8500, about 9000, about 9500, or about 10000 enteric endoderm cells, or any number of enteric endoderm cells within a range defined by any two of the aforementioned cell numbers. In some embodiments, the culturing step comprises contacting the one or more enteric endoderm aggregates with an extracellular matrix, or a mimic or derivative thereof. In some embodiments, the extracellular matrix, or a mimic or derivative thereof comprises Matrigel.

[0005] In any of the embodiments disclosed herein, the gut endoderm monolayer is a foregut endoderm monolayer, and the gut spheroid is a foregut spheroid.In some embodiments, differentiating definitive endoderm into foregut endoderm monolayer and foregut spheroid comprises contacting definitive endoderm with one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof.In some embodiments, the one or more FGF signaling pathway activators comprise FGF4, the one or more Wnt signaling pathway activators comprise CHIR99021, or the one or more BMP signaling pathway inhibitors comprise Noggin, or any combination thereof.In some embodiments, the one or more aggregated organoids are aggregated liver organoids. In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated liver organoids comprises contacting one or more enteric endoderm aggregates with one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, or any combination thereof.In some embodiments, one or more FGF signaling pathway activators comprise FGF2, or one or more BMP signaling pathway activators comprise BMP4, or both.In some embodiments, one or more aggregated organoids are aggregated gastric organoids.In some embodiments, one or more aggregated organoids are aggregated gastric organoids.In some embodiments, one or more aggregated gastric organoids are aggregated antral gastric organoids.In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated antral gastric organoids comprises contacting one or more enteric endoderm aggregates with EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, or any combination thereof. In some embodiments, the one or more BMP signaling pathway inhibitors include Noggin.

[0006] In any of the embodiments disclosed herein, the gut endoderm monolayer is a hindgut endoderm monolayer, and the gut spheroid is a hindgut spheroid.In some embodiments, differentiating definitive endoderm into hindgut endoderm monolayer and hindgut spheroid comprises contacting definitive endoderm with one or more FGF signaling pathway activators, or one or more Wnt signaling pathway activators, or both.In some embodiments, the one or more FGF signaling pathway activators comprise FGF4, or the one or more Wnt signaling pathway activators comprise CHIR99021, or both.In some embodiments, the one or more aggregated organoids are aggregated intestinal organoids.In some embodiments, culturing the one or more gut endoderm aggregates to form one or more aggregated intestinal organoids comprises contacting the one or more gut endoderm aggregates with EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof. In some embodiments, one or more Wnt signaling pathway activators comprise R-spondin, or one or more BMP signaling pathway inhibitors comprise Noggin, or both.In some embodiments, one or more aggregated organoids are aggregated colon organoids.In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated colon organoids comprises contacting one or more enteric endoderm aggregates with EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators, or any combination thereof.In some embodiments, one or more Wnt signaling pathway activators comprise R-spondin, or one or more BMP signaling pathway activators comprise BMP2, or any combination thereof.

[0007] In any of the embodiments disclosed herein, the one or more gut endoderm aggregates comprise at least 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 gut endoderm aggregates, or any number of gut endoderm aggregates within a number defined by any two of the aforesaid gut endoderm aggregate numbers. In some embodiments, each of the one or more intestinal endoderm aggregates comprises a diameter within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of the one or more intestinal endoderm aggregates, or any diameter within a range defined by any two of the aforesaid diameters, or a volume within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the one or more intestinal endoderm aggregates, or any volume within a range defined by any two of the aforesaid volumes, or both.

[0008] In any of the embodiments disclosed herein, the method further comprises transplanting one or more aggregated organoids into recipient subject.In some embodiments, the recipient subject is a mammal.In some embodiments, the recipient subject is human.

[0009] Also disclosed herein is any of one or more aggregated organoids produced by any one of the methods disclosed herein.Also disclosed herein is a plurality of intestinal endoderm aggregates.In some embodiments, the plurality of intestinal endoderm aggregates comprises at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 intestinal endoderm aggregates, or any number of intestinal endoderm aggregates within the number defined by any two of the aforementioned intestinal endoderm aggregate numbers, and each of the plurality of intestinal endoderm aggregates is within ±10%, ±9%, ±8%, ±10%, ±15%, ±20%, ±30%, ±40%, ±50%, ±60%, ±70%, ±80%, ±100%, ±25%, ±30%, ±40%, ±50%, ±60%, ±70%, ±80%, ±100%, ±150%, ±25%, ±30%, ±40%, ±50%, ±100%, ±25%, ±30%, ±40%, ±5 ...50%, ±25%, ±30%, ±40%, ±50%, ±150%, ±25%, ±30%, ±40%, ±50%, ±150%, ±250%, ±250%, ±30%, ±4 The diameter may be within ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, or any diameter within a range defined by any two of the aforementioned diameters, or a volume within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, or any volume within a range defined by any two of the aforementioned volumes, or both. In some embodiments, the multiple gut endoderm aggregates are derived from the same subject. Also disclosed herein is a formation plate. In some embodiments, the formation plate comprises a plurality of microwells and a plurality of gut endoderm aggregates according to claim 38 or 39, each of the multiple microwells comprising a single gut endoderm aggregate of the multiple gut endoderm aggregates. In some embodiments, for any one of the plurality of gut endoderm aggregates disclosed herein, or any one of the formation plates disclosed herein, the plurality of gut endoderm aggregates are produced according to any one of the methods disclosed herein.

[0010] The embodiments of the present disclosure provided herein are described by the following numbered alternatives:

[0011] 1. A method for producing an organoid composition, comprising:

[0012] dissociating the hindgut endoderm (HGE) to form a single cell population derived from the HGE;

[0013] Aggregating the HGE-derived single cell population in a formation plate;

[0014] Culturing the HGE-derived single cell population in the formation plate to form aggregates;

[0015] and culturing the aggregates with EGF, a BMP signaling pathway activator, and a Wnt signaling pathway activator until intestinal organoids are formed.

[0016] 2. The method of alternative 1, wherein said hindgut endoderm (HGE) is obtained from definitive endoderm (DE).

[0017] 3. The method according to alternative 2, wherein the DE is cultured with an FGF signaling pathway activator and a Wnt signaling pathway activator to form the hindgut endoderm (HGE).

[0018] 4. The method according to the preceding alternative, wherein said organoids are obtained from progenitor cells.

[0019] 5. The method of the preceding alternative, wherein said progenitor cells are induced pluripotent stem cells.

[0020] 6. The method of alternative 1, wherein the HGE-derived single cell population comprises hindgut endoderm cells.

[0021] 7. The method of the preceding alternative, wherein the aggregates comprise about 1000 hindgut endoderm cells, or about 2000 hindgut endoderm cells, or about 3000 hindgut endoderm cells, or about 4000 hindgut endoderm cells, or about 5000 hindgut endoderm cells.

[0022] 8. The method of the preceding alternative, wherein the aggregates are contacted with an anti-adhesion rinse solution.

[0023] 9. The method according to the preceding alternative, comprising contacting the aggregates with a three-dimensional structure, preferably Matrigel (basement membrane matrix), and further culturing the aggregates until organoids are formed.

[0024] 10. The method of the preceding alternative, wherein the formation plate is selected from a microwell culture plate, a V-bottom microwell culture plate, a hanging drop culture plate, or a plate capable of physically agglomerating a population of cells. [Brief description of the drawings]

[0025] In addition to the features described herein, additional features and modifications will be readily apparent from the following drawings and description of exemplary embodiments, it being understood that these drawings depict embodiments and are not intended to be limiting in scope.

[0026] [Figure 1] 1 shows an embodiment of a timeline of differentiation and development of hPSCs into HIOs. [Figure 2A-C] 1 shows an embodiment of the preparation of intestinal endoderm monolayers for single cell dissociation and aggregation. [Figure 3A-B] 1 shows an embodiment of a formation plate and aggregation of cells to form aggregates. [Figure 4-6] 1 illustrates an embodiment of a forming plate or a component thereof. [Figure 7A] Shown is a schematic embodiment of an existing spheroid production protocol for human intestinal organoid (HIO) generation. [Figure 7B] 13 shows an embodiment of inter-experimental variability in spheroid production using an embodiment of an existing protocol. [Figure 7C] 13 shows an embodiment of line-to-line variability in spheroid production using an embodiment of an existing protocol. [Figure 7D] 7D shows an embodiment of an exemplary image corresponding to FIG. 7C. [Figure 8] 1 shows an embodiment of uniform CDX2+ hindgut endoderm produced by an embodiment of an existing protocol. [Figure 9A] FIG. 1 shows a schematic embodiment of an aggregation-based spheroid production protocol for HIO generation. [Figure 9B] 1 shows an embodiment of successful homogenous aggregation of multiple hPSC lines. [Figure 9C] 13 shows an embodiment of an image demonstrating a greatly increased yield of uniform spheroids produced by the aggregation method. [Figure 9D] 1 illustrates an embodiment in which the yield of spheroids per well from HGE aggregation is greatly increased. [Figure 9E] 1 shows an embodiment of the different organization of epithelial and mesenchymal cells in dissociated spontaneous and aggregated spheroids. [Figure 10A] 1 shows an embodiment of an image demonstrating that detached spontaneous spheroids and aggregated spheroids are morphologically indistinguishable after growth in Matrigel. [Figure 10B] 1 shows an embodiment of an image showing the indistinguishable organization of epithelial and mesenchymal cells in dissociated spontaneous and aggregated spheroids 3 days after embedding in Matrigel. [Figure 11A] 1A-1C show embodiments of images demonstrating that the growth and morphology of aggHIOs are indistinguishable from spontaneous HIOs. [Figure 11B] 1 shows an embodiment of images showing that AggHIOs contain CDX2+ intestinal epithelium and Emilin1+ mesenchyme. [Figure 11C] FIG. 1 shows an embodiment of images demonstrating that both spontaneously dissociated HIOs and AggHIOs are patterned in the proximal small intestine. [Figure 12A] 1 shows an embodiment of images demonstrating that AggHIOs undergo robust proliferation and maturation following in vivo transplantation. [Figure 12B] 1 shows an embodiment of immunofluorescence analysis of mature small intestine markers. [Figure 13A] FIG. 1 shows an embodiment of a schematic for the generation of antral human gastric organoids (aHGOs) by aggregation. [Figure 13B]1 shows an embodiment of images demonstrating that aHGO derived from condensed foregut endoderm are morphologically indistinguishable from spontaneous aHGO. [Figure 13C] 1 shows an embodiment of images showing that expression of gastric epithelial markers cannot distinguish between spontaneous and aggregated aHGO. [Figure 14A] FIG. 1 shows an embodiment of a schematic for the generation of human colon organoids (HCO) by aggregation. [Figure 14B] 13 shows an embodiment of images demonstrating that HCOs derived from aggregated hindgut endoderm spheroids are morphologically indistinguishable from HCOs derived from spontaneous spheroids. [Figure 14C] 13 shows an embodiment of images demonstrating that expression of the colonic epithelial marker SATB2 is indistinguishable between HCOs derived from spontaneous or aggregated hindgut endoderm. [Figure 15] FIG. 1 shows an embodiment of a schematic for the generation of human liver organoids (HLO) by aggregation. [Figure 16A] 1 shows an embodiment of an image showing the population density of mesoderm (detected by T expression) and definitive endoderm (detected by FOXA2 expression) in definitive endoderm cultures differentiated from PSCs. [Figure 16B] FIG. 16B shows an embodiment of quantification of the proportions of mesoderm and definitive endoderm populations in the cultures of FIG. 16A. [Figure 16C] 1 shows an embodiment of images depicting population densities of mesenchyme (detected by FOXF1 expression) and gut endoderm (detected by FOXA2 expression) in foregut and hindgut endoderm monolayer cultures differentiated from PSC-derived definitive endoderm. [Figure 16D] FIG. 16C shows an embodiment of quantification of the proportions of mesenchymal and endodermal populations in the cultures. [Figure 16E] 1 shows an embodiment of quantification of proliferating mesoderm and endoderm comparing day 3 definitive endoderm to day 7 hindgut endoderm cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Current techniques for organoid generation rely on the stepwise in vitro differentiation of hPSCs into organ tissue lineages. For example, in the case of gastrointestinal organoids, stem cells are first differentiated into definitive endoderm (DE) cells, followed by foregut endoderm (FGE) or hindgut endoderm (HGE) intermediates (~7 days of culture), during which spontaneous morphogenesis occurs, resulting in the formation and separation of three-dimensional spheroids that resemble the embryonic gut tube. These spheroids are embedded in an extracellular matrix, or a mimic or derivative thereof (e.g., Matrigel), and cultured in a medium that promotes proliferation and organ differentiation. After ~28 days in these conditions (35 days of total culture), human organoids can be harvested and used for purposes such as studying organ function and morphology, drug screening, or engraftment into animal models for further proliferation and maturation.

[0028] However, current organoid generation methods have several limitations. In particular, there is a large variability in the efficiency of spontaneous spheroid production and separation between different hPSC lines. Even in lines known to have a strong ability to produce spontaneous spheroids, there is a large variability in spontaneous spheroid production and separation from experiment to experiment. In a given organoid generation experiment, there is also a large variability in the efficiency of spontaneous spheroid production and separation from well to well. Spontaneous spheroid formation often occurs in large "chains" of multiple attached spheroids. The size of spontaneous spheroids generated between strains can be highly variable. Thus, reliance on spontaneous morphogenesis is associated with inefficient and inconsistent spheroid generation. Furthermore, these methods are not well suited to the increased scalability required for biopharmaceutical manufacturing applications.

[0029] Provided herein is an improved method for producing organoid or its composition, which overcomes one or more limitations of existing methods.In some embodiments, the disclosed method eliminates or reduces the low efficiency of spontaneous spheroid production associated with the variability between strains, between experiments, and between wells, and provides a method for improving scalability.

[0030] The method disclosed herein takes advantage of the ability of hPSC-derived cells to self-organize upon aggregation. In the case of gastrointestinal organoids, hPSCs are differentiated into DE, then FGE or HGE are subsequently dissociated into single cells, including when spontaneous morphogenesis and spheroid formation and separation are not detected using standard methods and the cells remain attached to the cell culture plate. In some embodiments, single FGE or HGE cells are then aggregated in a formation plate, for example, overnight. In some embodiments, the formation plate is an Aggrewell plate (StemCell Technologies). In some embodiments, the aggregates are then harvested and embedded in Matrigel for growth and differentiation into organoids (e.g., intestinal organoids). Analysis of HIOs obtained by the improved aggregation method shows no significant difference compared to HIOs obtained by spontaneous spheroid generation in the same experiment. Furthermore, HIOs derived from aggregated HGE retain the ability to grow into mature human intestinal tissue after engraftment in mouse models. In some embodiments, processes for differentiating other types of organoids known in the art can be used with the methods described herein.

[0031] Disclosed herein are aggregated organoids and compositions thereof, and methods for making them, which involve forming aggregated organoids by aggregating single cell suspensions of intestinal endoderm monolayers (as opposed to intestinal spheroids) and then culturing the aggregates.The methods disclosed herein produce organoids with higher reliability and reproducibility than conventional methods known in the art, and impact the feasibility of scaling up organoid production.These aggregated organoids can be used for purposes such as drug screening or personalized medicine, and are suitable for, for example, autologous or allogeneic transplantation into subjects such as humans or other mammals, or xenotransplantation into immunodeficient animals.In some embodiments, the aggregated organoids are liver, stomach, antral stomach, fundus, intestine, or colon organoids.In some embodiments, the aggregated organoids are derived from cells isolated from patients. Methods for producing organoids can be found in U.S. Patent Nos. 9,719,068 and 10,174,289, as well as PCT Publication Nos. WO2016 / 061464, WO2017 / 192997, WO2018 / 106628, WO2018 / 200481, WO2018 / 085615, WO2018 / 085622, WO2018 / 085623, WO2018 / 226267, and WO2020 / 023245, each of which is expressly incorporated herein by reference in its entirety.

[0032] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like elements unless the context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0033] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood when read in light of this 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.

[0034] 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.

[0035] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 10% from the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0036] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of the recited steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including everything that the phrase "consisting of" follows. Thus, the phrase "consisting of" indicates that the recited elements are required or mandatory, and that no other elements may be present. "Consisting essentially of" means the inclusion of every element recited before this phrase, with other elements being limited to those that do not interfere with or contribute to the activity or function set forth in this disclosure with respect to the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they have a substantial effect on the activity or action of the recited elements.

[0037] 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 will be 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 may be used in a variety of orientations and positions that are not limited to those shown and described herein.

[0038] The terms "individual", "subject" or "patient" as used herein have their common and usual meaning as understood in the context of this specification, and refer to human or non-human mammals, such as dogs, cats, mice, rats, cows, sheep, pigs, goats, non-human primates, or birds, such as chickens, as well as other vertebrates or invertebrates. The term "mammal" is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including monkeys (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0039] The term "effective amount" or "effective dose" as used herein has its common and ordinary meaning as understood in light of the specification, and refers to that amount of the described 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 effective amount of the active composition or compound to achieve a desired response for a particular subject and / or application. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the composition, the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, the minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Contemplated herein are the determination and adjustment of the effective dose, as well as the evaluation of when and how to make such adjustments.

[0040] The terms "function" and "functional" as used herein have their plain and ordinary meaning as understood in light of this specification and refer to biological, enzymatic, or therapeutic functions.

[0041] The term "inhibit" as used herein has its general and usual meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be about, about, at least, at least about, less than, or a percentage less than about, or an amount within a range defined by any two of the aforementioned values, of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The term "delay" as used herein has its general and usual meaning as understood in light of the present specification and refers to a delay, postponement, or delay of a biological event to a later time than would otherwise be expected. The delay may be about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, about, at least about, less than, or a percentage less than about, or an amount within a range defined by any two of the preceding values. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0042] As used herein, the term "isolated" has its ordinary and usual meaning as understood in light of the specification and refers to a substance and / or entity that is (1) separated from at least some of the components with which it is associated when originally produced (in nature and / or in an experimental environment) and / or (2) separated from at least some of the components with which it is associated when produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from, about, at least, at least about, less than, or equal to 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, about, at least, at least about, less than, or less than about (or ranges including and / or spanning the aforementioned values) of other components with which they were originally associated. In some embodiments, an isolated agent is equal to, about, at least, at least about, less than, or about less than 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, an "isolated" material can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0043] As used herein, "in vivo" is given its ordinary and ordinary meaning as understood in light of the present specification and refers to the performance of methods within living organisms, usually animals, mammals, including humans, and plants, as opposed to tissue extracts or dead organisms.

[0044] As used herein, "ex vivo" is given its ordinary and usual meaning as understood in light of the specification and refers to the performance of the method outside a living body with little change in natural conditions.

[0045] As used herein, "in vitro" is given its common and ordinary meaning as understood in light of the specification, and refers to the performance of the method outside biological conditions, for example in a petri dish or test tube.

[0046] The terms "nucleic acid" or "nucleic acid molecule" as used herein have their common and usual meaning as understood in the context of this specification and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those occurring naturally in cells, fragments generated by polymerase chain reaction (PCR), and fragments generated 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 changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. 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 substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotides" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The nucleic acid(s) can be included in a nucleic acid vector or construct (e.g., a plasmid, a virus, a retrovirus, a lentivirus, a bacteriophage, a cosmid, a fosmid, a phagemid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a human artificial chromosome (HAC)) that can be used for amplifying and / or expressing the nucleic acid(s) in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a targeting sequence, a peptide purification tag, or an accessory gene, or any combination thereof.

[0047] 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 can be joined to any other sequence, e.g., by extra nucleic acid between linkers, repeats, or restriction enzyme sites, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within a range defined by any two of the aforementioned lengths, at or about, at or about, or at or about. The term "downstream" as used herein with respect to a nucleic acid has its ordinary and usual meaning as understood in the context of the specification and refers to the sequence behind the 3' end of the previous sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded. The term "upstream" as used herein with respect to a nucleic acid has its ordinary and usual meaning as understood in the context of the specification and refers to the sequence ahead of the 5' end of the subsequent sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded. The term "grouping" as used herein with respect to nucleic acids has its general and ordinary meaning as understood in the context of the present specification and refers to two or more sequences that occur directly or in close proximity to any other sequence, e.g., extra nucleic acid between linkers, repeats or restriction enzyme sites, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within a range defined by any two of the foregoing lengths, but generally not between sequences that encode functional or catalytic polypeptides, proteins, or protein domains.

[0048] The nucleic acid described herein comprises nucleobase.The primary, normal, natural or unmodified base is 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.

[0049] The terms "peptide", "polypeptide" and "protein" as used herein have their common and usual meaning as understood in the context of this specification and refer to a polymer composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides and proteins are known in the art, including, but not limited to, enzymatic, structural, transport, defensive, hormonal or signal transduction. Peptides, polypeptides and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating the nucleic acid template, peptide, polypeptide and protein mutations such as substitution, deletion, truncation, addition, duplication or fusion of two or more peptides, polypeptides and proteins can be performed. These fusions of two or more peptides, polypeptides, or proteins can be adjacent in the same molecule, or can be joined with extra amino acids between, for example, linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within the range defined by any two of the aforementioned lengths, or about, or at least about, or less than, or about, or less than. The term "downstream" as used herein with respect to a polypeptide has its ordinary and ordinary meaning as understood in light of the specification, and refers to a sequence following the C-terminus of the preceding sequence. The term "upstream" as used herein with respect to a polypeptide has its ordinary and usual meaning as understood in the context of the specification, and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0050] The term "purity" of any given substance, compound, or material as used herein has its common and ordinary meaning as understood in light of the 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 is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals 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 infectious agents. Purity can be measured using techniques including, but not limited to, electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0051] The term "yield" of any given substance, compound, or material as used herein has its common and ordinary meaning as understood in the context of the specification, and refers to the actual total amount of the substance, compound, or material relative to the expected amount present. For example, the yield of a substance, compound, or material may be, about, at least, at least about, less than, or about less than 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, including all decimal points therebetween. Yield may be affected by the efficiency of a reaction or process, undesired side reactions, decomposition, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material at any step of production.

[0052] The term "w / w%" or "weight / weight %" as used herein has its ordinary and ordinary meaning as understood in the context of this specification and refers to a percentage expressed in terms of the weight of a component or agent relative to the total weight of the composition multiplied by 100. The term "v / v%" or "volume / volume %" as used herein has its ordinary and ordinary meaning as understood in the context of this specification and refers to a percentage expressed in terms of the liquid volume of a compound, substance, component or agent relative to the total liquid volume of the composition multiplied by 100.

[0053] 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 constructing a complete, viable organism. These cells are produced from the fusion of an egg and a sperm cell. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0054] As used herein, the term "embryonic stem cells (ESCs)", also commonly abbreviated as ES cells, as used herein has its plain and ordinary meaning as understood in the context 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 this disclosure, the term "ESCs" may be used broadly to encompass embryonic germ cells.

[0055] As used herein, the term "pluripotent stem cells (PSCs)" has its plain and ordinary meaning as understood in light of the present 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 descendants of inner cell mass cells of a preimplantation blastocyst, or may be obtained by induction of non-pluripotent stem cells, e.g., adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0056] 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, refers to a type of pluripotent stem cell artificially derived from normally non-pluripotent cells, such as adult somatic cells, by inducing "forced" expression of certain genes, and hiPSCs refers to human iPSCs. In several methods known in the art, iPSCs can be derived by transfection of certain 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 can include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes also improve the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically, and are usually isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSC includes first generation iPSC, second generation iPSC in mouse, and human induced pluripotent stem cell.In some methods, retrovirus system is used to transform human fibroblasts into pluripotent stem cells with four crucial genes: Oct3 / 4, Sox2, Klf4, and c-Myc.In other methods, lentivirus system is used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4 (POU5F1), certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klfl, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, E-cadherin, or any combination thereof.

[0057] 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, the progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent stem cells (induced or non-induced), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, the progenitor cells can be from embryos, infants, children, or adults. In some embodiments, the progenitor cells can be somatic cells that are subjected to treatment to confer pluripotency via genetic engineering or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0058] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which are derived from the totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early stage embryo. Methods for deriving embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in this application, but it will be understood by those skilled in the art that the methods and systems described herein are applicable to any stem cells.

[0059] Additional stem cells that can be used in embodiments according to the present disclosure include, but are not limited to, those provided 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 (Goteborg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present disclosure include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (13), TE04 (14), TE06 (16), UC01 (HSF1), UC06 (HSF6), WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0060] 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 initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0061] In some embodiments, adenovirus can be used to deliver the four necessary genes, resulting in iPSCs that are virtually identical to embryonic stem cells. Adenovirus does not combine its own genes with the target host, thus eliminating the risk of creating tumors. In some embodiments, non-viral based techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without any viral transfection system at all, albeit with very low efficiency. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, generation of mouse iPSCs is possible using similar methodologies. Repeated treatment of cells with specific proteins delivered to the cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0062] The term "feeder cells" as used herein has its common and usual meaning as understood in light of the present specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying them on the cell surface. Feeder cells are generally adherent cells and may be growth arrested. For example, feeder cells are growth arrested by irradiation (e.g., gamma radiation), mitomycin-C treatment, electric pulses, or mild chemical fixation (e.g., formaldehyde or glutaraldehyde). However, feeder cells are not necessarily growth arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cell is mouse fibroblast, mouse embryonic fibroblast, mouse STO cell, mouse 3T3 cell, mouse SNL 76 / 7 cell, human fibroblast, human forehead fibroblast, human skin fibroblast, human adipose mesenchymal cell, human bone marrow mesenchymal cell, human amniotic mesenchymal cell, human amniotic epithelial cell, human umbilical cord mesenchymal cell, human fetal muscle cell, human fetal fibroblast, or human adult fallopian tube epithelial cell.In some embodiments, the conditioned medium prepared from the feeder cell is used instead of or in combination with the feeder cell co-culture.In some embodiments, the feeder cell is not used during the proliferation of the target stem cell.

[0063] The term "extracellular matrix" as used herein has its plain and ordinary meaning in the context of the present specification and refers to any biological or synthetic compound, substance, or composition that enhances cell adhesion and / or proliferation. Any extracellular matrix known in the art, as well as mimics or derivatives thereof, can be used in the methods disclosed herein. Some examples of extracellular matrices, or mimics or derivatives thereof, include, but are not limited to, cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, polylysine, polyornithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, basement membrane, matrigel, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof.

[0064] 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 pharma- ceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0065] As used herein, "pharmacologically 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 used. As used herein, "pharmacologically acceptable", "diluent", "excipient", and / or "carrier" have their plain and ordinary meaning as understood in light of the specification and are 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 agency for use in animals, including humans and non-human mammals such as cats and dogs, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias. 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, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, carbohydrates such as amino acids, glucose, mannose, dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol and sorbitol, salt formation counteracting agents such as sodium, non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), PLURONICS®. The compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation should be compatible with the method of administration.

[0066] Cryoprotectants are cell composition additives to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene, glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cryopreservation medium that contains other components such as nutrients (e.g., albumin, serum, bovine serum, fetal calf serum [FCS]) to increase the post-thaw survival of cells. In these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, is about, is at least about, is less than, or is about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the foregoing numbers.

[0067] Additional excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugar, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth media components or any combination thereof. The amount of excipient may be found in the composition at, about, at least, at least about, less than, or equal to 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or any percentage within a range defined by any two of the aforesaid numbers.

[0068] The term "pharmaceutically acceptable salts" has its plain and ordinary meaning as understood in light of this specification and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including, but not limited to, analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, classes of such organic bases may include, but are not limited to, mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, including mono-, di-, and triethanolamine; amino acids, including glycine, arginine, and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; and trihydroxymethylaminoethane.

[0069] Appropriate formulations vary depending on the route of administration selected. Techniques for 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.

[0070] 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 body organs.

[0071] As used herein, "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 pharma- ceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It may also be a liquid for dissolution of 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.

[0072] The disclosure herein generally uses positive language to describe many embodiments, and the disclosure also includes embodiments in which subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is entirely or partially excluded.

[0073] Differentiation of PSCs In some embodiments, PSCs, such as ESCs and iPSCs, undergo stepwise directed differentiation, first to definitive endoderm (DE), then to foregut or hindgut lineages, and to gastrointestinal tissue. In some non-limiting embodiments, PSCs may include H1 hESCs, iPSC72_3, iPSC75_1, or iPSC285_1, or any combination thereof. In some embodiments, PSCs undergo non-stepwise directed differentiation, in which molecules (e.g., growth factors, ligands) for promoting DE formation and molecules for subsequent tissue formation are added simultaneously. 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 include, but are not limited to, Wnt signaling pathway, Wnt / APC signaling pathway, FGF signaling pathway, TGF-β signaling pathway, BMP signaling pathway, Notch signaling pathway, Hedgehog signaling pathway, LKB signaling pathway, and Par polarity signaling pathway. Each of the listed signaling pathways has signaling pathway activators and signaling pathway inhibitors conventionally known in the art.

[0074] 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, while 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 development of the foregut and hindgut. Wnt and FGF signaling pathways are important for promoting posterior endoderm / hindgut or anterior endoderm / foregut fates. In the hindgut, a simple cuboidal epithelium first develops into a pseudostratified columnar epithelium, then into villi containing polarized columnar epithelium and a proliferation zone at the base of the villi that corresponds to the putative progenitor region.

[0075] Any method for producing 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 from the gonadal ridges of an embryo. Embryonic stem cells or germ cells can be derived from a variety of animal species, including, but not limited to, a variety of mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSC cells are used to produce definitive endoderm. In some embodiments, iPSC cells (hiPSCs) are used to produce definitive endoderm. In some embodiments, PSCs are first modified before differentiation into definitive endoderm. In some embodiments, PSCs are genetically modified to express exogenous nucleic acids or proteins before differentiation into definitive endoderm.

[0076] In some embodiments, the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time period that is, is about, is at least, is at least about, is less than, or is less than about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or any time period within a range defined by any two of the aforementioned times, such as 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, multiple small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, multiple small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0077] In some embodiments, the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration 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 is treated with a concentration of about, at least about, at most, or at most about 15000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, 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 changed over the course of treatment. In some embodiments, multiple small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the concentrations of multiple small molecule compounds, activators, inhibitors, or growth factors may vary.

[0078] In some embodiments, the ESCs, germ cells, or iPSCs are cultured in a growth medium that supports the proliferation of stem cells. In some embodiments, the 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, mTeSR1, mTeSR Plus, DE differentiation, hindgut endoderm differentiation, gut-based, or complete Sato medium. In some embodiments, the stem cell growth medium includes fetal bovine serum (FBS). In some embodiments, the stem cell growth medium comprises FBS at a concentration that is, is about, is at least about, is less than, or is less than 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, e.g., 0%-20%, 0.2%-10%, 2%-5%, 0%-5%, or 2%-20%. In some embodiments, the stem cell growth medium does not contain xenogeneic components. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0079] In some embodiments, use a cell population that is enriched in definitive endoderm cells.In some embodiments, definitive endoderm cells are isolated or substantially purified.In some embodiments, isolated or substantially purified definitive endoderm cells express one or more (e.g., at least one, three) SOX17, FOXA2 or CXRC4 markers more than one or more (e.g., at least one, three, five) OCT4, AFP, TM, SPARC or SOX7 markers.

[0080] 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-β superfamily. In some embodiments, the one or more growth factors include the Nodal / Activin and / or BMP subgroups of the TGF-β 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, Wnt proteins, or any combination of these growth factors. For example, Wnt proteins in humans 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.

[0081] In some embodiments, Activin-induced definitive endoderm (DE) can further undergo FGF and / or Wnt-induced anterior or posterior patterning, foregut or hindgut specification and morphogenesis, and ultimately gastrointestinal proliferation, morphogenesis, and cell differentiation into functional gastrointestinal cell types. In some embodiments, PSCs are efficiently directed to differentiate in vitro into gastrointestinal epithelium or mesenchyme, including secretory, endocrine, and absorptive cell types. It will be appreciated that molecules such as growth factors can be added at any developmental stage to promote specific types of intestinal tissue formation.

[0082] Human gastrointestinal development in vitro occurs at stages that approximate fetal intestinal development; endoderm formation, anterior or posterior endodermal patterning, foregut or hindgut morphogenesis, fetal intestinal development, epithelial morphogenesis, formation of putative progenitor domains, and differentiation into functional cell types.

[0083] 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 disclosure.In some embodiments, cellular components related to Wnt and / or FGF signaling pathways, such as natural inhibitors, antagonists, activators or agonists of the pathways, can be used to inhibit or activate Wnt and / or FGF signaling pathways.In some embodiments, siRNA and / or shRNA that target cellular components related to Wnt and / or FGF signaling pathways are used to inhibit or activate these pathways.

[0084] Fibroblast growth factors (FGFs) are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGFs are heparin-binding proteins, and interactions with cell surface-associated heparan sulfate proteoglycans have been shown to be essential for FGF signaling. FGFs play important roles in the proliferation and differentiation processes of a wide variety of cells and tissues. In humans, 22 members of the FGF family have been identified, all of which are structurally related signaling molecules. Members FGF1-FGF10 all bind to the fibroblast growth factor receptor (FGFR). FGF1 is also known as acidic fibroblast growth factor, and FGF2 is also known as basic fibroblast growth factor (bFGF). Members FGF11, FGF12, FGF13, and FGF14, also known as FGF homologous factors 1-4 (FHF1-FHF4), have been shown to have distinct functional differences compared to FGFs. Although these factors have remarkably similar sequence homology, they do not bind to FGFRs and are involved in intracellular processes independent of FGFs. This group is also known as "iFGFs". Members FGF15-FGF23 are newer and less well characterized. FGF15 is the mouse orthologue of human FGF19 (so 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.

[0085] 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, or FGF23.

[0086] Differentiation of PSCs into DE cultures and then into various intermediate mature gastrointestinal cell types can be determined by the presence of stage-specific cell markers. In some embodiments, the expression of representative 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, 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 foregut markers Pdx1 and albumin can be used to reveal directed hindgut formation. In some embodiments, one or more (e.g., at least 1, 3) 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 may be used to represent intestinal development.

[0087] In some embodiments, morphological changes can be used to represent the progression of directed differentiation. In some embodiments, gut endoderm monolayers (e.g., mid-hindgut, hindgut, foregut, anterior foregut, or posterior hindgut endoderm monolayers), or cells thereof, are subjected to three-dimensional culture conditions for maturation. In some embodiments, the gut endoderm monolayer matures into gastrointestinal organoids in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 days, about, at least, at least about, less than, or about less than 1, or any number of days within a range defined by any two of the aforementioned days, e.g., 1-40 days, 20-30 days, 30-40 days, or 1-20 days. In some embodiments, a highly complex epithelium surrounded by mesenchymal cells can be observed. In some embodiments, gastrointestinal organoids, epithelium, polarized columnar epithelium, mesenchyme, neuronal cells, or smooth muscle cells can be observed at, about, at least, at least about, less than, or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or any number of days within a range defined by any two of the foregoing days, e.g., 1-40 days, 20-30 days, 30-40 days, or 1-20 days.

[0088] In some embodiments, pluripotent stem cells are converted to gastrointestinal 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., CHIR99021 and FGF4).

[0089] In some embodiments, the pluripotent stem cells are prepared from somatic cells. In some embodiments, the pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, the pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, the PBMCs are grown on a feeder cell substrate. In some embodiments, the PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, the PBMCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, the PBMCs are grown on 0.1% gelatin.

[0090] 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 that is, is about, is at least about, is less than, or is about equal to 0, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 MOI, or any MOI within a range defined by any two of the aforementioned 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, the iPSCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, iPSCs are grown on 0.1% gelatin. In some embodiments, iPSCs are grown in RPMI 1640, DMEM, DMEM / F12, mTeSR1, mTeSR Plus, DE differentiation, hindgut endoderm differentiation, gut-based, or complete Sato medium.

[0091] In some embodiments, PSCs (e.g., ESCs or iPSCs) are cultured according to methods known in the art. In some embodiments, PSCs are expanded in extracellular matrix, or a mimic or derivative thereof. In some embodiments, PSCs are expanded in Matrigel. In some embodiments, PSCs in culture are dissociated (e.g., using dispase) and plated on a plate coated with Matrigel for expansion. In some embodiments, PSCs are expanded in cell culture medium containing a ROCK inhibitor (e.g., Y-27632). In some embodiments, PSCs are expanded to at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% confluence. In some embodiments, PSCs are differentiated into definitive endoderm cells. In some embodiments, PSCs are differentiated into definitive endoderm cells by contacting PSCs with activin A. In some embodiments, PSCs are further contacted with one or more BMP signaling pathway activators, such as BMP4. In some embodiments, the PSCs are contacted with activin A or one or more BMP signaling pathway activators at a concentration that is, is about, is at least about, is less than, or is about equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-200 ng / mL, 10-150 ng / mL, 1-100 ng / mL, or 100-200 ng / mL of each of the activin A or one or more BMP signaling pathway activators. In some embodiments, iPSCs are differentiated to definitive endoderm in RPMI 1640, DMEM, DMEM / F12, mTeSR1, mTeSR Plus, day 1 DE differentiation, day 2 DE differentiation, day 3 DE differentiation, hindgut endoderm differentiation, gut-based, or complete Sato medium.In some embodiments, the DE differentiation medium comprises one or more (e.g., at least 1, 2, 3, 4) of RPMI 1640, non-essential amino acids (NEAA), dialyzed fetal bovine serum (dFCS), or activin A, or any combination thereof. In some embodiments, the DE differentiation medium on day 1 comprises 0% or about 0% dFCS, the DE differentiation medium on day 2 comprises 0.2% or about 0.2% dFCS, and the DE differentiation medium on day 3 comprises 2% or about 2% dFCS.

[0092] Definitive endoderm differentiation Definitive endoderm represents the embryonic precursor of many major organs, including the gastrointestinal tract (e.g., esophagus, lung, thyroid, liver, pancreas, small intestine, large intestine). Methods for producing definitive endoderm cells from pluripotent stem cells (PSCs) include methods conventionally known in the art. In some embodiments, the definitive endoderm is differentiated or has been differentiated from PSCs. In some embodiments, the definitive endoderm is differentiated or has been differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the definitive endoderm or PSCs are derived from a human. In some embodiments, the definitive endoderm is human definitive endoderm.

[0093] In some embodiments, the method described herein for producing one or more aggregated organoids comprises differentiating definitive endoderm into gut endoderm monolayer and gut spheroids. In some embodiments, gut endoderm monolayer is attached to, for example, tissue culture plate or embodiment of formation plate disclosed herein, and gut spheroids are separated and suspended in the growth medium used to culture definitive endoderm, gut endoderm monolayer, and gut spheroids. As used herein, gut endoderm refers to cells derived from definitive endoderm that have undergone patterning into gastrointestinal lineage. In some embodiments, gut endoderm can include foregut endoderm, midgut endoderm, hindgut endoderm, or any combination thereof. In some embodiments, hindgut endoderm as used herein encompasses both midgut and hindgut endoderm, and refers to small intestine and large intestine organ lineage. During differentiation from definitive endoderm to gut endoderm, gut spheroids are formed spontaneously and separated from gut endoderm monolayer as floating cell masses. These intestinal spheroids display early characteristics of organoids, in particular the heterogeneity of the constituent cell populations, including both epithelial and mesenchymal cell lineages.

[0094] Disclosed herein are methods for differentiating definitive endoderm into gut endoderm monolayers and gut spheroids, however, previously known methods can also be used to produce gut endoderm monolayers. Methods can be found, for example, in U.S. Pat. Nos. 9,719,068 and 10,174,289, and PCT Publication Nos. WO2016 / 061464, WO2017 / 192997, WO2018 / 106628, WO2018 / 200481, WO2018 / 085615, WO2018 / 085622, WO2018 / 085623, WO2018 / 226267, and WO2020 / 023245, each of which is expressly incorporated by reference herein in its entirety. Methods previously described for differentiating definitive endoderm into gut spheroids can be considered synonymous with differentiating definitive endoderm into both gut endoderm monolayers and gut spheroids, as production of a gut endoderm monolayer usually results in the concomitant production of a gut endoderm monolayer.

[0095] In some embodiments, definitive endoderm is differentiated into gut endoderm monolayers and gut spheroids by contacting definitive endoderm with one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof (e.g., at least one, two, or three). In some embodiments, the one or more FGF signaling pathway activators comprise one or more FGF proteins disclosed herein or known in the art. In some embodiments, the one or more FGF signaling pathway activators comprise FGF4. In some embodiments, the one or more Wnt signaling pathway activators comprise one or more Wnt proteins disclosed herein or known in the art. In some embodiments, the one or more Wnt signaling pathway activators comprise one or more GSK3 inhibitors. In some embodiments, the one or more Wnt signaling pathway activators comprise CHIR99021. In some embodiments, the one or more BMP signaling pathway inhibitors comprise any BMP signaling pathway inhibitors disclosed herein or known in the art. In some embodiments, the one or more BMP signaling pathway inhibitors include Noggin. In some embodiments, the definitive endoderm is further contacted with retinoic acid. In some embodiments, the definitive endoderm is further contacted with EGF.In some embodiments, each of the one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, one or more BMP signaling pathway inhibitors, retinoic acid, or EGF, when provided, is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 65 The contact is at a concentration that is, about, at least, at least about, less than, or about 0, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of the one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, one or more BMP signaling pathway inhibitors, retinoic acid, or EGF, as provided, is contacted at a concentration that is, is about, is at least, is at least about, is less than, or is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, the definitive endoderm is differentiated into gut endoderm monolayers and gut spheroids by culturing the definitive endoderm for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

[0096] In some embodiments, the definitive endoderm is differentiated into foregut endoderm monolayer and foregut spheroids. In some embodiments, the gut endoderm monolayer is a foregut endoderm monolayer, and the gut spheroid is a foregut spheroid. In some embodiments, differentiating the definitive endoderm into foregut endoderm monolayer and foregut spheroids comprises contacting the definitive endoderm with one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof (e.g., at least 1, 2, or 3). In some embodiments, the one or more FGF signaling pathway activators comprise FGF4, the one or more Wnt signaling pathway activators comprise CHIR99021, or the one or more BMP signaling pathway inhibitors comprise Noggin, or any combination thereof (e.g., at least 1, 2, or 3).

[0097] In some embodiments, the definitive endoderm is differentiated into a hindgut endoderm monolayer and a hindgut spheroid. In some embodiments, the gut endoderm monolayer is a hindgut endoderm monolayer, and the gut spheroid is a hindgut spheroid. In some embodiments, differentiating the definitive endoderm into a hindgut endoderm monolayer and a hindgut spheroid comprises contacting the definitive endoderm with one or more FGF signaling pathway activators, or one or more Wnt signaling pathway activators, or both. In some embodiments, the one or more FGF signaling pathway activators comprise FGF4, or the one or more Wnt signaling pathway activators comprise CHIR99021, or both. In some embodiments, differentiating the definitive endoderm further comprises contacting the definitive endoderm with one or more BMP signaling pathway activators, such as one or more BMP proteins described herein or known in the art. In some embodiments, the definitive endoderm is differentiated into a gut endoderm monolayer and a gut spheroid in a hindgut endoderm differentiation medium. In some embodiments, the hindgut endoderm differentiation medium comprises one or more (e.g., at least 1, 2, 3, 4, 5) of RPMI 1640, NEAA, dFCS, FGF4, or CHIR99021, or any combination thereof. In some embodiments, the hindgut endoderm differentiation medium comprises 2% or about 2% dFCS, 500 ng / mL or about 500 ng / mL FGF4, or 3 μM or about 3 μM CHIR99021, or any combination thereof.

[0098] In some embodiments, the gut endoderm monolayer produced by any of the methods disclosed herein is distinguished from gut spheroids produced according to previous methods by the relative abundance of mesoderm and / or mesenchyme lineages. In some embodiments, culturing the gut endoderm monolayer results in an increase in mesoderm and / or mesenchyme. In some embodiments, the gut endoderm monolayer comprises mesoderm and / or mesenchyme that is, is about, is at least about, is less than, or is less than 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 number, or any percentage of the total cell number that is within a range defined by any two of the aforementioned percentages, for example, 1%-20%, 1%-10%, 10%-20%, or 5%-15%. In some embodiments, the gut endoderm monolayer contains more mesoderm and / or mesenchyme compared to gut spheroids at the same stage of culture. In some embodiments, the gut endoderm monolayer contains some mesoderm and / or mesenchyme, i.e., 1, 2, 3, 4, or 5 times, about, at least, at least about, less than, or about less than the number of mesoderm and / or mesenchyme found in gut spheroids, or any multiple of mesoderm and / or mesenchyme within a range defined by any two of the aforementioned multiples.

[0099] Isolation and dissociation of gut endoderm After differentiating the definitive endoderm into gut endoderm monolayer and gut spheroids, any one of the methods disclosed herein includes separating the gut endoderm monolayer from the gut spheroids, both of which are in growth medium. Any method of separating adherent cells (e.g., gut endoderm monolayer) and floating cells (e.g., gut spheroids) known in the art can be used. For example, as a non-limiting embodiment, the growth medium and suspended gut spheroids are aspirated, leaving the gut endoderm monolayer. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) washing steps can be performed to ensure that all or most of the gut spheroids are removed. In some embodiments, the growth medium can be gently agitated to resuspend the settled gut spheroids. In another non-limiting embodiment, the intestinal endoderm monolayer and intestinal spheroids are subjected to continuous flow conditions (e.g., in a flow chamber or intracellularly) of fresh growth medium or washing solution to continuously remove the detached and suspended intestinal spheroids while retaining the attached intestinal endoderm monolayer.

[0100] After separating the gut endoderm monolayer from the gut spheroid in any one of the methods disclosed herein, the method further comprises dissociating the gut endoderm monolayer into a single cell suspension of gut endoderm cells. In some embodiments, the gut endoderm cells comprise foregut endoderm cells or hindgut endoderm cells, or both. In some non-limiting embodiments, dissociating the gut endoderm monolayer comprises mechanically dissociating the gut endoderm monolayer or enzymatically dissociating the gut endoderm monolayer, or both. In some embodiments, the gut endoderm monolayer is dissociated with proteolytic enzymes and / or collagenolytic enzymes. In some embodiments, the gut endoderm monolayer is dissociated enzymatically using Accutase (StemCell Technologies), Accumax (StemCell Technologies), trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express (Thermo Fisher), TrypLE Select (Thermo Fisher), or any combination thereof. In some embodiments, the intestinal endoderm monolayer is mechanically dissociated, for example, by trituration with a pipette. In some embodiments, the single cell suspension of intestinal endoderm cells is filtered to remove undissociated cell clumps.

[0101] Gut endoderm aggregation After dissociating the gut endoderm monolayer into a single cell suspension of gut endoderm cells in any one of the methods disclosed herein, the method further comprises aggregating the single cell suspension of gut endoderm cells into one or more gut endoderm aggregates. In some embodiments, the one or more gut endoderm aggregates are, comprise, consist essentially of, or consist of one or more foregut endoderm aggregates. In some embodiments, the gut endoderm aggregates are, comprise, consist essentially of, or consist of one or more hindgut endoderm aggregates. In some non-limiting embodiments, aggregating the single cell suspension of gut endoderm cells into one or more gut endoderm aggregates comprises one or more (e.g., at least one, two, or three) of aggregating the single cell suspension in a hanging drop, centrifuging the single cell suspension in a microwell culture plate, centrifuging the single cell suspension in a "v" or "u" bottom microwell culture plate, aggregating the single cell suspension using an orbital shaker, or centrifuging the single cell suspension in a formation plate, or any combination thereof. In some embodiments, centrifugation of the single cell suspension may be replaced by allowing the single cell suspension to settle into aggregates by gravity. In some embodiments, the formation plate is one of the formation plates disclosed herein. In some embodiments, each of the one or more gut endoderm aggregates is 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or The number of cells may be, about, at least, at least about, or less than about 10,000 enteric endoderm cells, or any number of enteric endoderm cells within a range defined by any two of the aforementioned numbers, such as 50-10,000 cells, 50-4,000 cells, 1,000-10,000 cells, or 1,000-5,000 enteric endoderm cells.In some embodiments, after aggregation, one or more gut endoderm aggregates are cultured in RPMI 1640, DMEM, DMEM / F12, mTeSR1, mTeSR Plus, DE differentiation, hindgut endoderm differentiation, gut-based, or complete Sato medium. In some embodiments, the gut-based medium comprises one or more (e.g., at least 1, 2, 3, 4, 5, 6) of advanced DMEM / F12, B27 supplement, insulin, N2 supplement, HEPES buffer, penicillin / streptomycin, or L-glutamine, or any combination thereof. In some embodiments, the complete Sato medium comprises one or more (e.g., at least 1, 2, 3, 4) of gut-based medium, EGF, Noggin, or R-spondin, or any combination thereof. In some embodiments, the complete Sato medium comprises 500ng / mL or about 500ng / mL recombinant human EGF, 100ng / mL or about 100ng / mL recombinant human Noggin, or 500ng / mL or about 500ng / mL recombinant human R-spondin, or any combination thereof. In some embodiments, any of the media disclosed herein (e.g., complete Sato medium) can be supplemented with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor is supplemented at 10μM or about 10μM.

[0102] In some embodiments of any of the methods disclosed herein, the gut endoderm cells are aggregated using an orbital shaker. In some embodiments, a suspension of gut endoderm cells is placed on an orbital shaker in an incubator at 37° C. The motion imparted to the suspension by the shaker causes the cells to contact each other and form aggregates. In some embodiments, gut endoderm cell aggregates are formed within 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, or 48 hours of shaking, or any time within a range defined by any two of the foregoing times, e.g., 1-24 hours, 24-48 hours, or 12-36 hours.

[0103] In some embodiments of any of the methods disclosed herein, the gut endoderm cells are aggregated by allowing the cells to settle out of suspension by gravity.

[0104] In some embodiments of any of the methods disclosed herein, the gut endoderm cells are aggregated by the hanging drop method, which in some embodiments includes spotting a drop of gut endoderm cells suspended in growth medium upside down on a surface (e.g., a cell culture plate) and allowing the cells to sink to the bottom of the drop and aggregate.

[0105] Forming Plate 2A-C show an embodiment of an exemplary gut endoderm monolayer preparation for single cell dissociation and aggregation. In some embodiments, aggregation is performed in a formation plate, a microwell culture plate, a "v" bottom microwell culture plate, a "u" bottom microwell culture plate, or using an orbital shaker, or any combination thereof. In some embodiments, the formation plate is an Aggrewell plate (StemCell Technologies), or generally any other plate for aggregating cells according to the methods described herein.

[0106] 2A and 2B, in some embodiments, a plurality of induced pluripotent stem cells (14) are cultured in a biocompatible container (16) under conditions described herein or known in the art to form definitive endoderm (18). In some embodiments, the definitive endoderm (18) continues to be cultured under conditions described herein or known in the art to differentiate into a gut endoderm monolayer and gut spheroids in the biocompatible container (16). In some embodiments, the gut endoderm monolayer is a foregut endoderm monolayer or a hindgut endoderm monolayer, and the gut spheroids are foregut spheroids or hindgut spheroids, although any variation of gut endoderm monolayer and / or gut spheroids is contemplated. In some embodiments, the gut endoderm monolayer is attached to the biocompatible container (16), while the gut spheroids are dissociated and suspended in growth medium contained within the biocompatible container (16). In some embodiments, the gut endoderm monolayer is separated from the gut spheroids by aspirating the growth medium and the suspended gut spheroids from the biocompatible container (16). In some embodiments, the isolated gut endoderm monolayer is then dissociated into a single cell suspension of gut endoderm cells (10) as described herein and shown in FIG. 2C. In some embodiments, the single cell suspension (10) is collected and subjected to aggregation according to any of the methods disclosed herein or known in the art to form one or more gut endoderm aggregates (20). In some embodiments, the one or more gut endoderm aggregates (20) are placed in the same or different biocompatible container (16) to culture the one or more gut endoderm aggregates into one or more aggregated organoids.

[0107] Figures 3A-6 show embodiments of a forming plate (12). In some embodiments, the forming plate (12) has a base (22) and a plurality of wells (24). The exemplary plate shown in Figure 4 has six wells, but it will be understood that any number of wells (e.g., at least 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, 100, 500, 1000, 2000, 5000, or more) can be used as well. In some embodiments, each well (24) of the formation plate (12) includes a plurality of microwells (26) along its bottom (28) configured to receive a single cell suspension of gut endoderm cells (10) and aggregate the single cell suspension of gut endoderm cells (10) into a plurality of gut endoderm aggregates (20). With reference to Figure 6, in some embodiments, each microwell (26) includes a length (30), a width (32), and a depth (34). In some embodiments, the length (30) extends longitudinally at, about, at least, at least about, less than, or about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm, or any length within a range defined by any two of the aforementioned lengths, e.g., 100-1000 μm, 100-500 μm, 500-1000 μm, or 300-600 μm. In some embodiments, the length is defined between opposing longitudinal sidewalls (36) of the microwell (26). In some embodiments, the width (32) extends laterally that is, about, at least, at least about, less than, or about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm, or any width within a range defined by any two of the foregoing widths, e.g., 100-1000 μm, 100-500 μm, 500-1000 μm, or 300-600 μm.In some embodiments, the width is defined between opposing lateral sidewalls (38) of the microwell (26). In some embodiments, the depth (34) is, about, at least, at least about, less than, or about less than 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μm, or any depth within a range defined by any two of the aforementioned depths, e.g., 50-500 μm, 50-300 μm, 300-500 μm, or 100-400 μm extending perpendicular to the transverse longitudinal and lateral directions. In some embodiments, the depth (34) is defined between an opening (40) in a top surface (42) of the base (28) and a floor (44) of the base (28). In some embodiments, each microwell (26) is defined between a respective longitudinal sidewall (36), lateral sidewall (38), opening (40), and floor (44). In some embodiments, a lid may be included in the plate (12) and configured to cover the wells (24) so ​​that they are encapsulated rather than open. In some embodiments, the formation plate (12) is not intended to be unnecessarily limited to the particular number, arrangement, or size of wells (24) and microwells (26) shown and described in any of the examples provided herein. In some embodiments, the formation plate is an Aggrewell plate (StemCell Technologies). In some embodiments, the Aggrewell plate is an Aggrewell 400 or Aggrewell 800 plate.

[0108] In some embodiments, to aggregate the single cell suspension (10), the microwells (26) taper together from a relatively wide opening (40) to a relatively narrow floor (44). In some embodiments, as shown in FIG. 6, the opposing longitudinal sidewalls (36) taper toward one another from the opening (40) to the floor (44), while the opposing lateral sidewalls (38) similarly taper toward one another from the opening (40) to the floor (44). In some embodiments, gravity forces the single cells in suspension laterally downward, while the recoil forces exerted on the cells by the longitudinal and lateral sidewalls (36, 38) force the cells inward toward one another, effectively gathering and agglomerating the single cells together. In some embodiments, such tapering allows for aggregation of the cells into three-dimensional aggregates, which may be further facilitated in a centrifuge. In some embodiments, each microwell (26) receives, or is about, or is at least about, or is less than, or is less than, 50, 100, 200, 400, 600, 800, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, or 10000 single cells, or any number of cells within a range defined by any two of the foregoing numbers, e.g., 50-10000 cells, 50-4000 cells, 1000-10000 cells, or 1000-5000 cells.

[0109] In some embodiments, the longitudinal and lateral sidewalls (36, 38) have the same dimensions, defining a void within the microwell (26) having the shape of an inverted pyramid. In some embodiments, the longitudinal sidewalls (36) and lateral sidewalls (38) are planar and taper together toward the floor (44), which is essentially the inverted tip of the pyramid shape. In some embodiments, one or more of the longitudinal sidewalls (36), lateral sidewalls (38), and floor (44) are continuous surfaces rather than intersecting at various edges. In some embodiments, the various sidewalls (36, 38) and floor (44) of the microwell (26) are not intended to be unnecessarily limited to the discontinuous intersecting surfaces shown in some of the examples herein. In some embodiments, the void within the microwell (26) is shaped in other shapes that allow for collection and / or coalescence of contained cells. It will be appreciated that one of skill in the art can determine acceptable shapes for the microwells (26), including, for example, but not limited to, conical, dome-shaped, concave, elliptical, parabolic, and / or hyperbolic shapes. In some embodiments, the shape and size of the microwells are varied to be specifically configured for more effective growth of a particular population of single cells, such as endoderm or progenitor cells associated with other tissues. Thus, in some embodiments, the present invention is not intended to be unnecessarily limited to the particular shapes and dimensions of the formation plate (12) and / or microwells (26) shown in the figures, or for use with the particular cells discussed herein.

[0110] In some embodiments, each microwell (26) of any one of the formation plates described herein receives, or is about, or is at least about, or is less than, or is less than, 50, 100, 200, 400, 600, 800, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, or 10000 single cells, or any number of cells within a range defined by any two of the foregoing numbers, e.g., 50-10000 cells, 50-4000 cells, 1000-10000 cells, or 1000-5000 cells.

[0111] In some embodiments, the formation plate (12) has a single, monolithic structure fabricated from a biocompatible material that inhibits attachment of cells to the formation plate (12) within the microwells (26), while allowing the development of single cells (10) into aggregates (20), as shown in the non-limiting examples of FIGS. 3A-6. In some embodiments, the formation plate (12) is formed from multiple components, with at least the surfaces of the microwells (26) being fabricated from a biocompatible material. In some embodiments, the biocompatible material comprises, consists essentially of, or consists of stainless steel, titanium, polymeric organosilicon 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 plate (12) is sterile, resistant to adhesion by tissues and / or cells, comprises a hydrophobic surface, comprises features that improve formation and subsequent removal and / or use of the disclosed tissues, or any combination thereof. In some embodiments, the formation plate (12) comprises 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 proliferation and / or differentiation.

[0112] Aggregated Organoids In some embodiments, the method disclosed herein further comprises culturing one or more intestinal endoderm aggregates to produce one or more aggregated organoids.In some embodiments, one or more aggregated organoids described herein are or comprise esophageal organoids, gastric organoids, fundic organoids, antral gastric organoids, hepatic organoids, intestinal organoids, or colonic organoids, or any combination thereof.In some embodiments, one or more aggregated organoids are or comprise human esophageal organoids (HEO), human gastric organoids (HGO), human fundic organoids (HFGO), human antral gastric organoids (HAGO), human hepatic organoids (HHO), human intestinal organoids (HIO), or human colonic organoids (HCO), or any combination thereof. In some embodiments, after the single cell suspension of gut endoderm cells is allowed to aggregate into one or more gut endoderm aggregates, the one or more gut endoderm aggregates are cultured for a short period of time that is, is, is about, is, is at least, is at least about, is less than, or is less than about, e.g., 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 hours, or any period within a range defined by any two of the foregoing times, e.g., 1-50 hours, 10-40 hours, 20-30 hours, 1-30 hours, or 24-50 hours, to be collected, recovered, and / or coalesced. In some embodiments, the one or more gut endoderm aggregates are removed from the aggregation medium or resuspended. For example, in some embodiments, when one or more gut endoderm aggregates are aggregated within a microwell culture plate, a "v" or "u" bottom microwell culture plate, or a formation plate, the one or more gut endoderm aggregates are removed from the microwells of the culture plate or formation plate (e.g., using a pipette to gently flow growth medium over the one or more gut endoderm aggregates and aspirating the aggregates into the tip of the pipette).In some embodiments, the aggregation medium is washed with fresh growth medium, e.g., complete Sato medium, or other biocompatible aqueous solution, to ensure that all aggregates are collected. In some embodiments, the one or more gut endoderm aggregates are collected in a container (e.g., a sterile tube) and allowed to settle by gravity. In some embodiments, centrifugation should not be used to collect the one or more gut endoderm aggregates, as this may cause the aggregates to fuse together. In some embodiments, after settling, the one or more gut endoderm aggregates are cultured under conditions to differentiate the one or more gut endoderm aggregates into one or more aggregated organoids. For example, in some embodiments, after settling, any remaining growth medium is removed. In some embodiments, the one or more gut endoderm aggregates are contacted with a basement membrane or extracellular matrix, or a mimic or derivative thereof. In some embodiments, the basement membrane or extracellular matrix, or a mimic or derivative thereof comprises Matrigel. In some embodiments, the remaining growth medium is removed to reduce the efficiency of polymerization of the basement membrane or extracellular matrix, or a mimic or derivative thereof. In some embodiments, one or more gut endoderm aggregates are contacted with one or more growth factors, nutrients, vitamins, sugars, proteins, small molecules, agonists, antagonists, cytokines, signal transduction pathway activators or signal transduction pathway inhibitors to induce the proliferation and maturation of one or more gut endoderm aggregates into one or more aggregated organoids.Although the conditions for differentiating one or more gut endoderm aggregates into various different aggregated organoids are provided herein, other methods previously known for differentiating gut spheroids (e.g., foregut spheroids and / or hindgut spheroids) into respective organoids can be used to differentiate one or more gut endoderm aggregates in the same or similar manner.Methods for organoid differentiation can be found, for example, in U.S. Patent Nos. 9,719,068 and 10,174,289, as well as PCT Publication Nos. WO2016 / 061464, WO2017 / 192997, WO2018 / 106628, WO2018 / 200481, WO2018 / 085615, WO2018 / 085622, WO2018 / 085623, WO2018 / 226267, and WO2020 / 023245, each of which is expressly incorporated herein by reference in its entirety.

[0113] In some embodiments, when the gut endoderm cells are foregut endoderm cells, the one or more gut endoderm aggregates are foregut endoderm aggregates, and the one or more gut endoderm aggregates are differentiated into one or more aggregated organoids of a foregut lineage.

[0114] In some embodiments, one or more aggregated organoids are or comprise one or more aggregated liver organoids.In some embodiments, culturing one or more gut endoderm aggregates to form one or more aggregated liver organoids comprises contacting one or more gut endoderm aggregates with one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, or any combination thereof.In some embodiments, one or more FGF signaling pathway activators comprise FGF2.In some embodiments, one or more BMP signaling pathway activators comprise BMP4.In some embodiments, one or more aggregated liver organoids comprise liver epithelium and liver mesenchyme.

[0115] In any of the provided embodiments, each of the one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, if provided, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450 , 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or about, at least, at least about, less than, or about, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 1-200 ng / mL. In some embodiments, each of the one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, as provided, is contacted at a concentration that is, is about, is at least about, is less than, or is about less than 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM.

[0116] In some embodiments, the one or more aggregated organoids are or comprise one or more aggregated gastric organoids.In some embodiments, the one or more aggregated gastric organoids are or comprise one or more aggregated fundus organoids or one or more aggregated antral gastric organoids, or both.In some embodiments, culturing one or more intestinal endoderm aggregates to form one or more aggregated antral gastric organoids comprises contacting one or more intestinal endoderm aggregates with one or more (e.g., at least one, two, or three) of EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, or any combination thereof.In some embodiments, the one or more BMP signaling pathway inhibitors comprise Noggin.In some embodiments, the one or more aggregated gastric organoids comprise gastric epithelium and gastric mesenchyme.In some embodiments, the gastric epithelium of one or more aggregated gastric organoids is CDH1+, CLDN18+, or MUC5AC+, or any combination thereof.

[0117] In any of the provided embodiments, each of the EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, if provided, is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, The antibody is contacted at a concentration that is, is about, is at least about, is at most, is less than, or is about equal to 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, e.g., 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of the EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, when provided, is contacted at a concentration that is, about, at least about, less than, or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM.

[0118] In some embodiments, when the gut endoderm cells are hindgut endoderm cells, the one or more gut endoderm aggregates are or comprise one or more hindgut endoderm aggregates, and the one or more gut endoderm aggregates are differentiated into one or more aggregated organoids of a hindgut lineage.

[0119] In some embodiments, the one or more aggregated organoids are or comprise one or more aggregated intestinal organoids.In some embodiments, culturing one or more gut endoderm aggregates to form one or more aggregated intestinal organoids comprises contacting one or more gut endoderm aggregates with one or more (e.g., at least 1, 2, or 3) of EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof.In some embodiments, the one or more Wnt signaling pathway activators comprise R-spondin, or the one or more BMP signaling pathway inhibitors comprise Noggin, or both.In some embodiments, the one or more aggregated intestinal organoids comprise intestinal epithelium and intestinal mesenchyme.In some embodiments, the intestinal epithelium of the one or more aggregated intestinal organoids is CDH1+, CDX2+, E-cad+, or any combination thereof.In some embodiments, the intestinal mesenchyme of the one or more aggregated intestinal organoids is FOXF1+, CDX2+, Emilin+, or any combination thereof. In some embodiments, the intestinal epithelium of one or more aggregated intestinal organoids shows proximal intestinal marker. In some embodiments, proximal intestinal marker comprises CDH17 or PDX1, or both. In some embodiments, one or more aggregated intestinal organoids are transplanted into recipient subject and undergo maturation. In some embodiments, the mature one or more aggregated intestinal organoids comprise intestinal cell types. In some embodiments, the intestinal cell types comprise epithelial cells, goblet cells, enteroendocrine cells, or Paneth cells, or any combination thereof. In some embodiments, epithelial cells are SI+, goblet cells are Muc2+, enteroendocrine cells are chromogranin A+, or Paneth cells are lysozyme+, or any combination thereof.

[0120] In some embodiments, one or more aggregated organoids are or comprise one or more aggregated colon organoids.In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated colon organoids comprises contacting one or more enteric endoderm aggregates with one or more (for example, at least 1, 2, 3) of EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators, or any combination thereof.In some embodiments, one or more Wnt signaling pathway activators comprise R-spondin, or one or more BMP signaling pathway activators comprise BMP2, or any combination thereof.In some embodiments, one or more aggregated colon organoids comprise colon epithelium and colon mesenchyme.In some embodiments, colon epithelium is CDH1+ or SATB2+, or both.

[0121] In any of the provided embodiments, each of the EGF, one or more Wnt signaling pathway activators, one or more BMP signaling pathway inhibitors, or one or more BMP signaling pathway activators, if provided, is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 7000, 8000, 9000, 10 ... The contact is at a concentration that is, about, at least, at least about, less than, or about 50, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, e.g., 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of the EGF, one or more Wnt signaling pathway activators, one or more BMP signaling pathway inhibitors, or one or more BMP signaling pathway activators, when provided, is contacted at a concentration that is, is about, is at least, is at least about, is less than, or is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, the gut endoderm aggregates are cultured in complete Sato medium. In some embodiments, the complete Sato medium is supplemented with a ROCK inhibitor. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the ROCK inhibitor is supplemented at or about 10 μM.

[0122] In some embodiments, one or more gut endoderm aggregates are cultured for, about, at least about, less than, or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or any number of days within a range defined by any two of the foregoing days, e.g., 1-50 days, 10-30 days, 20-40 days, 1-30 days, or 20-50 days, to form one or more aggregated organoids.

[0123] In some embodiments, one or more of the aggregated organoids obtained are used to study the function of esophagus, stomach, intestine or colon, including but not limited to drug screening, neurological function, microbiota interaction, or transplantation, or any combination thereof.In some embodiments, one or more of the aggregated organoids comprise functional lumen.In some embodiments, one or more of the aggregated organoids have the ability to further differentiate when transplanted.In some embodiments, one or more of the aggregated organoids grow in vitro to embryonic stage, and further differentiate when transplanted.

[0124] Uniformity and scalability of gut endoderm aggregates and aggregated organoids In some embodiments, the method disclosed herein allows the formation of many homogeneous or nearly homogeneous gut endoderm aggregates and / or resulting aggregated organoids.In some embodiments, the method and use of aggregation medium (e.g., any one of the formation plates disclosed herein) allows the formation of multiple gut endoderm aggregates. In some embodiments, the plurality of gut endoderm aggregates comprises, or is about, or at least, or at least about, or less than, or about or less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100000, 500000, or 1000000 gut endoderm aggregates, or any number of gut endoderm aggregates within a number defined by any two of the foregoing gut endoderm aggregate numbers, e.g., 1000-1000000 gut endoderm aggregates, 5000-100000 gut endoderm aggregates, 1000-10000 gut endoderm aggregates, or 10000-1000000 gut endoderm aggregates. In some embodiments, the formation of homogeneous or nearly homogeneous gut endoderm aggregates and / or resulting aggregated organoids is defined by a plurality of gut endoderm aggregates and / or resulting aggregated organoids that have reduced variation in at least one spatial dimension compared with gut endoderm spheroids and / or organoids produced from spheroids without aggregation.In some embodiments, at least one spatial dimension comprises length, width, depth, volume, or surface area, or any combination thereof.In some embodiments, gut endoderm aggregates and / or resulting aggregated organoids are spherical in shape, and at least one spatial dimension comprises radius, diameter, circumference, volume, or surface area, or any combination thereof.In some embodiments, the reduction in variance in at least one spatial dimension comprises a diameter within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids, or any diameter within a range defined by any two of the aforementioned diameters.In some embodiments, each of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids comprises a diameter within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids, or any diameter within a range defined by any two of the aforementioned diameters. In some embodiments, the reduction in variance in at least one spatial dimension comprises a volume that is within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids, or any volume within a range defined by any two of the aforementioned volumes.In some embodiments, each of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids comprises a volume that is within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids, or any volume within a range defined by any two of the aforementioned volumes.In some embodiments, each of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids comprises both a diameter that is within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids, or any diameter within the range defined by any two of the aforementioned diameters, and a volume that is within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids, or any volume within the range defined by any two of the aforementioned volumes.For some embodiments, the reduced dispersion (i.e., uniformity) of enteric endoderm aggregates compared to spontaneously formed spheroids can be seen in Figures 9B, 9C, and 9E.

[0125] In any of the embodiments of the multiple gut endoderm aggregates and / or resulting aggregated organoids, the multiple gut endoderm aggregates and / or resulting aggregated organoids are derived from the same subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a disease, has previously had a disease, or is at risk of having a disease, or any combination thereof. In some embodiments, the disease is a gastrointestinal disease. In some embodiments, the multiple gut endoderm aggregates and / or resulting aggregated organoids derived from the subject can be used for genetic testing or drug screening purposes. In some embodiments, the multiple gut endoderm aggregates and / or resulting aggregated organoids derived from the subject can be used in large-scale drug screening to identify effective therapies for reducing, improving, or treating the subject's disease. In some embodiments, the large-scale drug screening includes testing multiple compounds, each with a subpopulation of multiple gut endoderm aggregates and / or resulting aggregated organoids.

[0126] Also disclosed herein are embodiments of aggregation media comprising a plurality of microwells and a plurality of gut endoderm aggregates. In some embodiments, the aggregation media is a microwell culture plate, a "v" or "u" bottom microwell culture plate, or any one of the formation plates disclosed herein. In some embodiments, the plurality of gut endoderm aggregates is a plurality of gut endoderm aggregates disclosed herein, or any one of one or more gut endoderm aggregates disclosed herein. In some embodiments, the plurality of gut endoderm aggregates is a plurality of gut endoderm aggregates produced by any one of the methods disclosed herein, or any one of one or more gut endoderm aggregates produced by any one of the methods disclosed herein. In some embodiments, each of the plurality of microwells comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gut endoderm aggregates of the plurality of gut endoderm aggregates. In some embodiments, each of the plurality of microwells comprises a single gut endoderm aggregate of the plurality of gut endoderm aggregates.

[0127] Transplantation and Treatment In some embodiments, the method disclosed herein comprises the additional step of transplanting any one or more of the aggregated organoids disclosed herein into a recipient subject.In some embodiments, the recipient subject is a mammal.In some embodiments, the recipient subject is a human.In some embodiments, the recipient subject is the subject from which definitive endoderm or precursor pluripotent stem cells are derived.In some embodiments, the one or more aggregated organoids are derived from definitive endoderm or PSCs isolated from the recipient subject.In some embodiments, when transplanted into a recipient subject, the one or more aggregated organoids show better engraftment, maturation, proliferation, or any combination thereof, compared to non-aggregated organoids known in the art.

[0128] In some embodiments, one or more aggregated organoids described herein are transplanted into a recipient subject, for example, as a therapeutic or experimental model described herein. In some embodiments, transplantation occurs after culturing the organoids for, about, at least about, less than, or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or any number of days within a range defined by any two of the foregoing days, e.g., 1-50 days, 10-40 days, 20-30 days, 1-30 days, or 20-50 days. In some embodiments, one or more aggregated organoids are mature enough to be transplanted and / or studied several days before organoids prepared by other methods known in the art reach the same or similar maturity state, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, about, at least, at least about, less than, or about less than, or any number of days within a range defined by any two of the aforementioned days, such as 1-20 days, 5-15 days, 10-15 days, 1-15 days, or 10-20 days. In some embodiments, the recipient subject is a mammal. In some embodiments, the recipient subject is an immunodeficient mammal. In some embodiments, the recipient subject is an immunodeficient mouse. In some embodiments, the recipient subject is a monkey, a dog, a hamster, or a rat. In some embodiments, the recipient subject is an immunocompromised monkey, dog, hamster, or rat. In some embodiments, the recipient subject is a human. In some embodiments, the recipient subject is an immunocompromised human.In some embodiments, the recipient subject is an immunocompetent human. In some embodiments, the recipient subject is an immunocompetent human treated with an immunosuppressant. In some embodiments, the recipient subject is an immunocompetent human, and the aggregated organoid is autologous to the host organism. In some embodiments, the recipient subject is an immunocompetent human, and the aggregated organoid is allogeneic to the host organism. In some embodiments, the recipient subject is a mammal in need of organ transplantation. In some embodiments, the recipient subject is a human in need of organ transplantation.

[0129] In some embodiments, one or more aggregated organoids are transplanted into the appropriate region of recipient subject.In some embodiments, one or more aggregated organoids grow in recipient subject 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, or about those days, or at least those days, or at least about those days, or less than those days, or less than those days. In some embodiments, one or more aggregated organoids grow larger or mature faster than the in vitro aggregated organoids prepared at the same time.In some embodiments, one or more aggregated organoids show integration with recipient target tissue.In some embodiments, one or more aggregated organoids comprise gastrointestinal cell lineage.In some embodiments, one or more aggregated organoids spontaneously develop gastrointestinal cell lineage.

[0130] Described herein is a method for treating a subject with reduced organ function or for improving or inhibiting adverse organ damage in a subject in need thereof. In some embodiments, the method comprises transplanting or engrafting one or more aggregated organoids into a subject. In some embodiments, the one or more aggregated organoids are one or more aggregated organoids of any one of the methods described herein. In some embodiments, the one or more aggregated organoids are or comprise one or more aggregated esophageal organoids, one or more aggregated gastric organoids, one or more aggregated fundus organoids, one or more aggregated antral gastric organoids, one or more aggregated hepatic organoids, one or more aggregated small intestine (intestine) organoids, or one or more aggregated large intestine (colon) organoids, or any combination thereof. In some embodiments, the one or more aggregated organoids are autologous or allogeneic to the subject. In some embodiments, the one or more aggregated organoids are prepared from induced pluripotent cells obtained from or derived from the subject. In some embodiments, the subject is in need of organ transplantation. In some embodiments, one or more aggregated organoids are transplanted or engrafted as one or more complete aggregated organoids.In some embodiments, the transplantation site is organ tissue.

[0131] Also described herein is any one or more of the aggregated organoids produced by any one of the methods disclosed herein.Furthermore, in some embodiments, the one or more aggregated organoids are used in restoring organoid function in a subject that needs it.In some embodiments, the one or more aggregated organoids are one or more aggregated organoids described herein.In some embodiments, the one or more aggregated organoids are one or more aggregated organoids produced by any one of the methods described herein.

[0132] Non-limiting methods of producing aggregated organoids Disclosed herein is a method for producing one or more aggregated organoids.In some embodiments, the method comprises: differentiating definitive endoderm into gut endoderm monolayer and gut spheroid; separating gut endoderm monolayer from gut spheroid; dissociating gut endoderm monolayer into single cell suspension of gut endoderm cells; aggregating the single cell suspension of gut endoderm cells into one or more gut endoderm aggregates; and culturing the one or more gut endoderm aggregates to produce one or more aggregated organoids.In some embodiments, the gut endoderm monolayer is adherent.In some embodiments, the separation step comprises aspirating growth medium and suspended gut spheroids from the gut endoderm monolayer.In some embodiments, the dissociation step comprises enzymatically dissociating gut endoderm monolayer. In some embodiments, the gut endoderm monolayer is enzymatically dissociated using Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express, or TrypLE Select, or any combination thereof. In some embodiments, the aggregation step comprises aggregating the single cell suspension in hanging drops, centrifuging the single cell suspension in a "v" or "u" bottom microwell culture plate, aggregating the single cell suspension using an orbital shaker, or centrifuging the single cell suspension in a formation plate, or any combination thereof. In some embodiments, centrifugation of the single cell suspension may be replaced by allowing the single cell suspension to settle by gravity. In some embodiments, the formation plate is an Aggrewell plate.In some embodiments, each of the one or more gut endoderm aggregates is 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or The cell number may be, about, at least, at least about, or less than 10,000 gut endoderm cells, or any number of gut endoderm cells within a range defined by any two of the aforementioned numbers, such as 50-10,000 cells, 50-4,000 cells, 1,000-10,000 cells, or 1,000-5,000 gut endoderm cells. In some embodiments, the culturing step comprises contacting the one or more gut endoderm aggregates with an extracellular matrix, or a mimic or derivative thereof. In some embodiments, the extracellular matrix, or a mimic or derivative thereof comprises Matrigel. In some embodiments, the gut endoderm monolayer is a foregut endoderm monolayer and the gut spheroid is a foregut spheroid. In some embodiments, the one or more aggregated organoids are or comprise one or more aggregated liver organoids. In some embodiments, the one or more aggregated organoids are or comprise one or more aggregated stomach organoids. In some embodiments, one or more aggregated organoids are or comprise one or more aggregated antral stomach organoids.In some embodiments, the intestinal endoderm monolayer is hindgut endoderm monolayer, and the intestinal spheroid is hindgut spheroid.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated intestinal organoids.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated colonic organoids.

[0133] Disclosed herein is a method for producing one or more aggregated organoids. In some embodiments, the method comprises: differentiating definitive endoderm into gut endoderm monolayer and gut spheroids; separating gut endoderm monolayer from gut spheroids; dissociating gut endoderm monolayer into single cell suspension of gut endoderm cells; aggregating the single cell suspension of gut endoderm cells into one or more gut endoderm aggregates; and culturing the one or more gut endoderm aggregates to produce one or more aggregated organoids. In some embodiments, the gut endoderm monolayer is adherent. In some embodiments, the separation step comprises aspirating growth medium and suspended gut spheroids from the gut endoderm monolayer. In some embodiments, the dissociation step comprises enzymatically dissociating the gut endoderm monolayer. In some embodiments, the gut endoderm monolayer is enzymatically dissociated by Accutase. In some embodiments, the aggregation step comprises centrifuging the single cell suspension in the formation plate, or any combination thereof. In some embodiments, centrifugation of the single cell suspension may be replaced by allowing the single cell suspension to settle by gravity. In some embodiments, the formation plate is an Aggrewell plate. In some embodiments, each of the one or more gut endoderm aggregates comprises, about, at least, at least about, less than, or about 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 gut endoderm cells, or any number of gut endoderm cells within a range defined by any two of the foregoing numbers, e.g., 1000-5000 cells, 2000-4000 cells, 1000-3000 cells, or 3000-5000 gut endoderm cells. In some embodiments, the culturing step comprises contacting the one or more gut endoderm aggregates with Matrigel. In some embodiments, the intestinal endoderm monolayer is anterior endoderm monolayer, and the intestinal spheroid is anterior gut spheroid.In some embodiments, the one or more aggregated organoids are or comprise one or more aggregated liver organoids.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated stomach organoids.In some embodiments, one or more gastric organoids are or comprise one or more aggregated antral stomach organoids.In some embodiments, the intestinal endoderm monolayer is a hindgut endoderm monolayer, and the intestinal spheroid is a hindgut spheroid.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated intestinal organoids.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated colonic organoids.

[0134] In some embodiments of any of the methods disclosed herein, the gut endoderm monolayer is a foregut endoderm monolayer, and the gut spheroid is a foregut spheroid.In some embodiments, differentiating definitive endoderm into foregut endoderm monolayer and foregut spheroid comprises contacting definitive endoderm with one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof. In some embodiments, each of the one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors is administered in an amount of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, 950, or 1000 ng / mL, or about, at least, at least about, less than, or about less than, or any concentration within a range defined by any two of the foregoing concentrations, for example, 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of the one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors is contacted at a concentration that is, is about, is at least about, is less than, or is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, the one or more FGF signaling pathway activators include FGF4.In some embodiments, FGF4 is provided at a concentration of 500ng / mL or about 500ng / mL. In some embodiments, the one or more Wnt signaling pathway activators include CHIR99021. In some embodiments, CHIR99021 is provided at a concentration of 3μM or about 3μM. In some embodiments, the one or more BMP signaling pathway inhibitors include Noggin. In some embodiments, Noggin is provided at a concentration of 200ng / mL or about 200ng / mL. In some embodiments, the foregut endoderm monolayer is dissociated into a single cell suspension of foregut endoderm cells. In some embodiments, the single cell suspension of foregut endoderm cells is aggregated into one or more foregut endoderm aggregates. In some embodiments, the one or more foregut endoderm aggregates are cultured to produce one or more aggregated liver organoids, or one or more aggregated stomach organoids, or both.

[0135] In some embodiments of any of the methods disclosed herein, one or more aggregated organoids are or comprise one or more aggregated liver organoids.In some embodiments, one or more foregut endoderm aggregates are cultured to produce one or more aggregated liver organoids.In some embodiments, culturing one or more gut endoderm aggregates to form one or more aggregated liver organoids comprises contacting one or more gut endoderm aggregates with one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone or oncostatin M, or any combination thereof. In some embodiments, each of the one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, when provided, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500 , 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or about, at least, at least about, less than, or about, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 1-200 ng / mL.In some embodiments, each of the one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, as provided, is contacted at a concentration that is, is about, is at least about, is less than, or is about less than 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.01-20 μM, 0.01-10 μM, 1-15 μM, or 10-20 μM.

[0136] In some embodiments of any of the methods disclosed herein, one or more aggregated organoids are or comprise one or more aggregated gastric organoids.In some embodiments, one or more foregut endoderm aggregates are cultured to produce one or more aggregated gastric organoids.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated antral gastric organoids.In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated antral gastric organoids comprises contacting one or more enteric endoderm aggregates with EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, or any combination thereof. In some embodiments, each of the EGF, retinoic acid, or one or more BMP signaling pathway inhibitors is administered at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or at a concentration that is about, at least, at least about, or less than about, or less than about, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of EGF, retinoic acid, or one or more BMP signaling pathway inhibitors is contacted at a concentration that is, is about, is at least, is at least about, is less than, or is about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, EGF is provided at a concentration of 100 ng / mL or about 100 ng / mL.In some embodiments, retinoic acid is provided at a concentration of 2 μM or about 2 μM. In some embodiments, the one or more BMP signaling pathway inhibitors include Noggin. In some embodiments, Noggin is provided at a concentration of 200 ng / mL or about 200 ng / mL.

[0137] In some embodiments of any of the methods disclosed herein, the gut endoderm monolayer is a foregut endoderm monolayer, and the gut spheroid is a foregut spheroid.In some embodiments, differentiating the definitive endoderm into a hindgut endoderm monolayer and a hindgut spheroid comprises contacting the definitive endoderm with one or more FGF signaling pathway activators, or one or more Wnt signaling pathway activators, or both.In some embodiments, the one or more FGF signaling pathway activators, or one or more Wnt signaling pathway activators, or both, are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000, 10000, 10000, 10000, 10000, 10000, The contact is at a concentration that is, is about, is at least about, is at most, is less than, or is about less than 0, 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of the one or more FGF signaling pathway activators or the one or more Wnt signaling pathway activators, or both, are contacted at a concentration that is, is about, is at least, is at least about, is less than, or is less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, the one or more FGF signaling pathway activators include FGF4. In some embodiments, FGF4 is provided at a concentration of 500 ng / mL or about 500 ng / mL. In some embodiments, the one or more Wnt signaling pathway activators include CHIR99021.In some embodiments, hindgut endoderm monolayer is dissociated into single cell suspension of hindgut endoderm cells.In some embodiments, the single cell suspension of hindgut endoderm cells is aggregated into one or more hindgut endoderm aggregates.In some embodiments, one or more hindgut endoderm aggregates are cultured to produce one or more aggregated intestinal organoids, or one or more aggregated colonic organoids, or both.

[0138] In some embodiments of any of the methods disclosed herein, one or more aggregated organoids are or include one or more aggregated intestinal organoids.In some embodiments, culturing one or more intestinal endoderm aggregates to form one or more aggregated intestinal organoids comprises contacting one or more intestinal endoderm aggregates with EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof.In some embodiments, each of EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, The antibody is contacted at a concentration that is, is about, is at least about, is at most, is less than, or is about equal to 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, e.g., 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors is contacted at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at least about, less than, or less than about, or at any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, EGF is provided at a concentration of 500 ng / mL or about 500 ng / mL. In some embodiments, the one or more Wnt signaling pathway activators include R-spondin.In some embodiments, R-spondin is provided at a concentration of 500ng / mL or about 500ng / mL.In some embodiments, one or more BMP signaling pathway inhibitors include Noggin.In some embodiments, Noggin is provided at a concentration of 100ng / mL or about 100ng / mL.

[0139] In some embodiments of any of the methods disclosed herein, one or more aggregated organoids are or include one or more aggregated colon organoids.In some embodiments, culturing one or more intestinal endoderm aggregates to form one or more aggregated intestinal organoids comprises contacting one or more intestinal endoderm aggregates with EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators, or any combination thereof.

[0140] In some embodiments, each of EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 950, or 1000 ng / mL, or about, at least, at least about, less than, or about less than, or any concentration within a range defined by any two of the aforementioned concentrations, for example, 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, each of EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators is contacted at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or at about, at least, at least about, at most, or at most, or at most, or at most, or at most, or at most, or at any concentration within a range defined by any two of the aforementioned concentrations, for example, 1-20 μM, 1-10 μM, 5-15 μM, or 10-20 μM. In some embodiments, EGF is provided at a concentration of 500 ng / mL or about 500 ng / mL. In some embodiments, the one or more Wnt signaling pathway activators include R-spondin. In some embodiments, R-spondin is provided at a concentration of 500 ng / mL or about 500 ng / mL. In some embodiments, the one or more BMP signaling pathway activators include BMP2. In some embodiments, BMP2 is provided at a concentration of or about 100 ng / mL.

[0141] In some embodiments of any of the methods disclosed herein, the intestinal spheroids are separated and suspended in a growth medium. In some embodiments of any of the methods disclosed herein, the definitive endoderm is differentiated from a pluripotent stem cell. In some embodiments of any of the methods disclosed herein, the definitive endoderm is differentiated from an embryonic stem cell or an induced pluripotent stem cell. In some embodiments of any of the methods disclosed herein, the definitive endoderm is human definitive endoderm.

[0142] In any embodiment of the method disclosed herein, the method further comprises transplanting one or more aggregated organoids into recipient subject.In some embodiments, the recipient subject is a mammal.In some embodiments, the recipient subject is human.

[0143] Also described herein are one or more aggregated organoids produced by any one of the methods disclosed herein. EXAMPLES

[0144] Some aspects of the embodiments discussed herein are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will appreciate that many other embodiments are also within the scope of the disclosure, as described and claimed herein.

[0145] Example 1. Cultivation of human pluripotent stem cells (hPSCs) An exemplary schematic for the formation of aggregate organoids, such as aggregate human intestinal organoids (AggHIOs), is provided in FIG. 1.

[0146] Two days (day -2) before differentiating hPSCs into definitive endoderm, hPSCs were cultured. Matrigel-coated 24-well culture dishes were prepared. 5 mL of dispase solution (1 mg / mL, StemCell Technologies) was warmed to 37 °C. If necessary, any differentiation areas were removed from the undifferentiated hPSCs. The medium from each well containing hPSCs was carefully aspirated. 1 mL of pre-warmed dispase solution was added to each well containing hPSCs, and the cells were incubated at 37 °C until the edges of the colonies appeared to be slightly folded back. After approximately 4 minutes of dispase incubation, it was observed that the edges of the hPSC colonies had begun to lift off the wells. If the edges of the colonies had not lifted off, the cells were incubated for a few more minutes, checking periodically until lifting was observed. During the dispase incubation, Matrigel-coated dishes were prepared by aspirating the Matrigel solution and adding 0.5 mL of fresh mTeSR1 medium (StemCell Technologies) to each well. Matrigel-coated dishes were not allowed to dry out at any time. When necessary, wells were aspirated and replenished with mTeSR1, one row at a time, rather than all at once. The hPSC plate was removed from the incubator. The dispase solution was gently aspirated and the wells were washed at least three times with 2 mL of pre-warmed DMEM-F12 medium. It was important not to allow the plate to dry out and to prevent colonies from becoming dislodged during pipetting. The medium was gently dispensed around the edge of each well. The DMEM-F12 wash solution was aspirated from each well and 2 mL of warm mTeSR1 medium was added to each well. Colonies were carefully detached using a sterile disposable cell scraper. The mTeSR1 and cell aggregates were combined into a single well, taking care not to overly disrupt the cell aggregates. Cells were triturated by carefully pipetting up and down once. If large aggregates were visible after one trituration, pipetting was repeated, checking the size of the aggregates after each trituration. If necessary, a large bore micropipette (e.g., p1000) was used to break up only large aggregates.The hPSC aggregates were gently dispersed, and 0.5 mL of the cell suspension was distributed into each well of a Matrigel-coated 24-well plate. The freshly plated cells were gently shaken back and forth to disperse the cells, and then transferred to an incubator.

[0147] Example 2. Definitive Endoderm (DE) Differentiation On culture day 0, we confirmed that plated hPSCs (e.g., as described in Example 1) were uniformly distributed at approximately 60-70% confluence and that the cells exhibited standard undifferentiated morphology before initiating differentiation. Aspirated the mTeSR1 medium in each well, taking care not to allow the cells to dry out. Added 0.5 mL of day 1 DE differentiation medium (Table 1) per well. Cells were incubated at 37 °C and 5% CO2 for 24 h.

[0148] On day 1 of culture, day 1 DE differentiation medium was discarded and 0.5 mL of day 2 DE differentiation medium (Table 1) was added per well. Cells were incubated at 37° C. and 5% CO2 for 24 h.

[0149] On the second day of culture, the day 2 DE differentiation medium was discarded and 0.5 mL of day 3 DE differentiation medium (Table 1) was added per well. The cells were incubated at 37 °C and 5% CO2 for 24 h.

[0150] The recipe for DE differentiation medium provided in Table 1 is for making 1 mL of medium. Volumes can be scaled up as needed. The final Activin A concentration each day is 100 ng / mL. These media can be prepared in advance and stored at 4°C, but are preferably prepared on the day of use. [Table 1]

[0151] Example 3. Patterning of the hindgut endoderm On day 3 of culture, the quality of differentiated definitive endoderm was assessed based on the cells being in a flat, homogenous monolayer and / or the expression of definitive endoderm markers (e.g., Sox17, FoxA2, ​​and / or CXCR4). DE differentiation medium was aspirated from each well, taking care not to allow the cells to dry out. 0.5 mL of hindgut endoderm differentiation medium (Table 2) was added per well. Cells were incubated at 37 °C and 5% CO2 for 24 h.

[0152] On the fourth day of culture, the previous hindgut endoderm differentiation medium was discarded and 0.5 mL of fresh hindgut endoderm differentiation medium was added per well. The cells were incubated at 37° C. and 5% CO2 for 24 hours.

[0153] On day 5 of culture, the previous hindgut endoderm differentiation medium was discarded and 0.5 mL of fresh hindgut endoderm differentiation medium was added per well. The cells were incubated for 24 h at 37 °C and 5% CO2. At this stage, the beginning of morphogenesis can be observed. There may be some detached spheroids in the well. Care was taken not to aspirate these spheroids by tilting the plate while aspirating, leaving a small amount of medium containing the spheroids in the well.

[0154] On day 6 of culture, the previous hindgut endoderm differentiation medium was discarded and 0.5 mL of fresh hindgut endoderm differentiation medium was added per well. The cells were incubated at 37°C and 5% CO2 for 24 h. At this stage, clear morphogenesis was observed. There could be some detached spheroids in the well. Care was taken not to aspirate these spheroids by tilting the plate while aspirating, leaving a small amount of medium containing the spheroids in the well.

[0155] By day 7 of culture (4 days of exposure to CHIR99021 / FGF4), extensive morphogenesis was observed with many separate spheroids. Cells were processed for spheroid analysis as described in Example 4 or for aggregation as described in Example 5.

[0156] The recipe for hindgut endoderm differentiation medium provided in Table 2 is for making 1 mL of medium. Volumes can be scaled up as needed. [Table 2]

[0157] Example 4. Existing spheroid protocols introduce variability Figure 7A shows a schematic diagram for pre-existing spheroid production directed to the formation of HIOs, as generally described in Examples 1-3. In summary, human pluripotent stem cells are first exposed to 100 ng / mL Activin A for 3 days to produce definitive endoderm (DE). The DE is then exposed to a combination of 3 μM CHIR99021 and 500 ng / mL FGF4 for 4 days, during which patterning and morphogenesis into hindgut endoderm, as well as spontaneous spheroid production, occurs. On day 7, dissociated spheroids are collected from the HGE monolayer, embedded in Matrigel, and cultured with 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin for 28 days. On day 35, HIOs are harvested and used for subsequent experiments.

[0158] Spheroid production in multiple HIO generation experiments (n=96) using H1 human embryonic stem cells was assessed as successful (>50 isolated spheroids / well), intermediate (<50 isolated spheroids / well), or failed (no spheroid isolation). Scoring was performed by a single individual. As shown in Figure 7B, there is a large variability in the formation of spheroids in separate replicates of the same protocol.

[0159] Spheroid production from H1 hESCs and three human iPSC lines (iPSC72_3, iPSC75_1, and iPSC285_1) was assessed using existing protocols. Eight wells per cell line were plated and subjected to differentiation simultaneously. On day 7, the number of detached spheroids per well was counted and images of each well were captured. As shown in Figure 7C, spheroid production differed between PSC cell lines, impacting personalized medical applications. Exemplary images of spheroid production with different PSC lines are shown in Figure 7D. The number of detached spheroids in each well is indicated in the top left corner of each condition. Robust morphogenesis was observed along with line-to-line variability in the number of detached spheroids, although a lack of spheroid detachment was observed in some wells.

[0160] Example 5. Condensation of hindgut endoderm Several approaches can be taken to aggregate hPSCs and cell derivatives (e.g., foregut or hindgut endoderm). These include, but are not limited to, generation of hanging drops, centrifugation into 96-well or 384-well "v" or "u" bottom microwell culture plates, and aggregation of cells using an orbital shaker. In this example, the use of Aggrewell (StemCell Technologies) is described.

[0161] Aggrewell 400 plates were prepared. Each well of the 24-well sized Aggrewell 400 plate can produce up to 1200 aggregates. The following is for a single well of an Aggrewell 400 plate. Amounts can be scaled up to prepare a sufficient number of wells / aggregates. 500 μL of anti-adhesion rinse solution (StemCell Technologies) was added to the wells of the Aggrewell plate. The plate was centrifuged for 5 minutes at 1300×g in a swinging bucket rotor with a plate holder attachment. The plate was observed under a microscope to ensure that air bubbles had been removed from the microwells. If air bubbles remained, the centrifugation step was repeated. The anti-adhesion rinse solution was discarded. The wells were rinsed with 2 mL of pre-warmed 37°C intestine-based medium (Table 3). The intestine-based medium was discarded. 1 mL of pre-warmed 37°C complete Sato medium supplemented with 10 μM Y-27632 (Table 4) was added to the wells. The Aggrewells were stored in a 37° C. incubator until further use.

[0162] A single cell suspension of HGE cells was prepared. The medium and spheroids detached from the HGE endoderm tissue culture were discarded. 0.5 mL of pre-warmed 37 °C Accutase was added to each well and the plate was incubated at 37 °C for approximately 5-10 min. The plate was monitored under a microscope to ensure that the cells had detached from the plate. If necessary, the cells can be incubated at 37 °C for an additional period of time. Accutase can be substituted with other enzymatic dissociation reagents known in the art, such as trypsin, EDTA, TrypLE Express (Thermo Fisher) or TrypLE Select (Thermo Fisher) to prepare single cells. 0.5 mL of complete Sato medium supplemented with 10 μM Y-27632 was added to each well. The cells were gently dispersed with a pipette and transferred to a 15 mL centrifuge tube as single cells. The concentration of cells in the suspension was measured and the number of cells used was calculated according to the following ratio: 1.2 x 10 cells needed per well of an Aggrewell 400 plate to form aggregates of 1,000 cells. 6 cells. This desired number of cells was transferred to a new centrifuge tube and centrifuged at 300 × g for 5 min. The supernatant was evacuated and the cell pellet was resuspended in 1 mL of pre-warmed 37 °C complete Sato medium supplemented with 10 μM Y-27632. The previously prepared Aggrewell 400 plate was removed from the incubator. The resuspended cells were transferred to the Aggrewell wells without removing the medium already in the plate. The cells were immediately mixed with a pipette to distribute the cells evenly throughout the wells. The Aggrewell plate was centrifuged at 100 × g for 3 min to capture the cells within the microwells. The plate was examined under a microscope to ensure that the cells are evenly distributed in the microwells. The Aggrewell plate was returned to the incubator overnight.

[0163] The recipes for Intestine-based and Complete Sato media provided in Tables 3 and 4 are for making 50 mL of medium. Volumes can be scaled up as needed. Intestine-based media can be stored at 4 °C for up to 2 weeks. For Complete Sato medium, EGF, Noggin, and R-spondin are added immediately before use. [Table 3] [Table 4]

[0164] Example 6. Harvesting and embedding of aggregates On day 8 of culture, for each well containing aggregates, the aggregates were gently removed from the microwells by pipetting and aspirated into the tip of the pipette. The collected aggregates were transferred to a sterile 15 mL centrifuge tube. To remove any aggregates remaining in the wells, 1 mL of pre-warmed 37 °C complete Sato medium (without ROCK inhibitor) was added and the collection process was repeated. This volume was combined with the previously collected aggregates. The collected aggregates (approximately 1200 per well of the Aggrewell 400) were allowed to settle to the bottom of the tube by gravity. After settling, as much of the supernatant as possible was removed from the aggregates. This removal step is important because any remaining liquid will compromise the integrity of the Matrigel matrix upon resuspension. 200 μL of ice-cold Matrigel was added to the tube containing the aggregates. The aggregates were resuspended and mixed evenly by gently pipetting the solution up and down, avoiding the formation of air bubbles. 50 μL of the Matrigel / aggregate mixture was added to the center of a well in a 24-well tissue culture-treated plate. If the Matrigel touches the side of the well, it may flatten and cause the spheroids to adhere to the plastic, so care was taken to keep the Matrigel in a single drop to prevent this from happening. This plating process was repeated until all the Matrigel / aggregate volume was plated. The plate was quickly but carefully inverted upside down to prevent the spheroids from settling on the surface of the tissue culture plate. The plate was transferred to a 37 °C incubator for 20 min to promote polymerization of the Matrigel. Subsequently, 0.5 mL of pre-warmed 37 °C complete Sato medium was added to each well. The medium was changed every 3-4 days. Developing organoids should be passaged if the pH indicator changes rapidly or if the organoids appear very dense.

[0165] Example 7. Aggregated intestinal organoids resemble highly reproducible in vivo tissues As controls, H1 hESCs and four human iPSC lines (iPSC72_3, iPSC75_1, iPSC115_1, and iPSC285_1) were subjected to differentiation. On day 7, HGE formation was assessed by immunofluorescence for CDX2 (HGE marker) and DAPI (nuclei). Tile scans of four randomly selected well areas from each cell line are shown. As shown in Figure 8, uniform expression of CDX2 was observed, indicating robust and efficient HGE production in all hPSC cell lines tested.

[0166] Figure 9A shows a schematic diagram for the production of gut endoderm aggregates and aggregated organoids, which is generally described in Examples 5-6. In summary, human pluripotent stem cells are first exposed to 100 ng / mL activin A for 3 days to produce DE. The DE is then exposed to a combination of 3 μM CHIR99021 and 500 ng / mL FGF4 for 4 days, during which patterning into hindgut endoderm and spontaneous spheroid production occurs. On day 7, a single cell suspension of HGE is prepared, regardless of the presence of isolated spontaneous spheroids, and subjected to 24 hours of aggregation using Aggrewell plates. The aggregates are then harvested from the microwells, embedded in Matrigel, and cultured in 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin for 28 days. On day 35, aggregated human intestinal organoids (aggHIOs) are harvested and used for subsequent experiments.

[0167] H1 hESCs and four human iPSC lines (iPSC72_3, iPSC75_1, iPSC115_1, and iPSC285_1) were subjected to differentiation using the aggregation protocol. After 7 days of differentiation, HGEs were dissociated into single cells using Accutase and allowed to aggregate for 24 hours in medium containing 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin. After aggregation, images of cells in each Aggrewell were captured and shown at 50x (left column, scale bar = 500 μm) and 200x (right column, scale bar = 100 μm). As seen in Figure 9B, uniform aggregation of gut endoderm cells is achievable in the Aggrewell formation plate.

[0168] H1 hESCs were subjected to differentiation using the existing non-aggregation protocol. After 7 days of differentiation, spontaneously produced dissociated spheroids were harvested, counted, and imaged using a Keyence BZ-X800 system. The remaining HGE (i.e., non-dissociated material) was then dissociated into single cells using Accutase and allowed to aggregate for 24 hours in medium containing 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin. The aggregates were then also counted and imaged using a Keyence BZ-X800 system. Representative images are shown in Figure 9C. Both an increase in the number of spheroids and a more uniform size of spheroids produced from the non-dissociated monolayer using the aggregation method were observed.

[0169] The number of spheroids after culturing gut endoderm aggregates in formation plates was quantified and is shown in Figure 9D. The number of dissociated spheroids produced per well using the existing non-aggregation protocol was scored for H1 hESC and four human iPSC lines (iPSC72_3, iPSC75_1, iPSC115_1, and iPSC285_1). Additionally, the average number of formed aggregates per well from dissociated cells obtained from non-aggregated HGE material in each experiment was determined. N=4 experiments.

[0170] Spontaneously detached (day 7) or aggregated (day 8) spheroids were fixed and subjected to immunostaining to identify intestinal epithelial cells (CDH1+ / CDX2+) and intestinal mesenchymal cells (FoxF1 / CDX2+). Figure 9E shows representative images captured by confocal microscopy. Spheroids cultured from gut endoderm aggregates show robust patterning of intestinal epithelial and mesenchymal cells with more uniform morphology.

[0171] H1 hESCs and four human iPSC lines (iPSC72_3, iPSC75_1, iPSC115_1, and iPSC285_1) were subjected to differentiation into HGE. On day 7, dissociated spontaneous spheroids were embedded directly into Matrigel, and non-dissociated cells were allowed to aggregate for 24 h before being embedded in Matrigel. After 3 days of culture in medium containing 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin, the overall morphology of the embedded spheroids was assessed. As shown in Figure 10A, both conditions were able to generate organoid precursors that proliferated and matured appropriately.

[0172] Spontaneously detached or aggregated spheroids were embedded in Matrigel. After 3 days of culture, the spheroids were fixed and subjected to whole-mount immunostaining to identify intestinal epithelial cells (CDH1+ / CDX2+) and intestinal mesenchymal cells (FoxF1 / CDX2+). As shown in Figure 10B, both conditions resulted in organoids containing intact intestinal epithelium and mesenchyme.

[0173] H1 hESCs were subjected to differentiation into HGE. On day 7, dissociated spontaneous spheroids were embedded in Matrigel, and non-dissociated HGE were allowed to dissociate and aggregate for 24 h, and then embedded in Matrigel. On days 18, 25, and 35, morphological analysis of the organoids showed that HIOs arising from both dissociated spheroids and aggregated HGE showed similar organoid growth and contained distinct epithelial and mesenchymal layers (Figure 11A).

[0174] On day 35, H1-derived HIOs and AggHIOs were harvested and subjected to co-immunofluorescence analysis for the presence of intestinal epithelium (CDX2+ / E-cad+) and mesenchymal cells (Emilin1). As shown in Figure 11B, both conditions resulted in well-formed CDX2+ / E-cad+ epithelium and Emilin1+ mesenchyme.

[0175] On day 35, H1-derived HIOs and AggHIOs were harvested and subjected to immunofluorescence analysis for the presence of proximal intestinal markers CDH17 and PDX1. All CDX2+ epithelial cells were positive for both CDH17 and PDX1, indicating patterning of the proximal small intestine. As shown in Figure 11C, both conditions led to patterning of CDH17+ / PDX1+ proximal small intestinal tissue.

[0176] On day 35, AggHIOs were harvested and engrafted into the kidney capsule of immunodeficient mice. After 6 weeks, mice were euthanized and engrafted AggHIOs were excised and subjected to histological analysis by hematoxylin / eosin (H&E) staining. As shown in Figure 12A, the aggregated intestinal organoids are amenable to transplantation and undergo robust proliferation and maturation.

[0177] Transplanted AggHIOs were sectioned and subjected to immunofluorescence analysis of mature intestinal markers sucrase-isomaltase (SI, epithelium), Muc2 (goblet cells), chromogranin A (enteroendocrine cells), and lysozyme (Paneth cells). As shown in Figure 12B, transplanted aggregated intestinal organoids were positive for all intestinal cell markers tested.

[0178] Example 8. Formation of aggregated gastric organoids Figure 13A shows a schematic diagram for the formation of aggregated antral gastric organoids. A comparison of existing methods with the aggregation method is provided. Top of Figure 13A: Method that relies on the spontaneous formation of antral organoids (e.g., as seen in McCracken et al. Nature. (2014) 516 (7531): 400-4). Bottom of Figure 13A: Improved protocol incorporating steps for aggregation of foregut endoderm.

[0179] Antral organoids were generated from spontaneously dissociated spheroids and from spheroids generated from aggregated foregut endoderm. Representative images of aHGO morphology were taken at day 35 (Figure 13B).

[0180] On day 35, aHGOs generated from either spontaneous spheroids or aggregated foregut endoderm were fixed, sectioned, and subjected to immunostaining for gastric epithelial markers CLDN18 and MUC5AC. Epithelial cells of aHGOs derived from both spontaneous and aggregated cells were uniformly Cdh1 / CLDN18 / MUC5AC positive (Figure S1C).

[0181] Example 9. Formation of aggregated colonic organoids Figure 14A shows a schematic diagram for the formation of aggregated colonic organoids. A comparison of existing methods with the aggregation method is provided. Top of Figure 14A: Method that relies on spontaneous formation of colonic organoids. Bottom of Figure 14A: Improved protocol incorporating a step for aggregating spheroids before posteriorization with BMP2.

[0182] HCOs were generated from spontaneously dissociated spheroids and from spheroids generated from aggregated hindgut endoderm. Spheroids were then patterned into a posterior fate by exposure to BMP2 for 3 days. After 32 days, representative images of HCO morphology were captured (Figure 14B).

[0183] On day 35, HCOs generated from either spontaneous spheroids or aggregated hindgut endoderm were fixed, sectioned, and subjected to immunostaining for the colonic epithelial marker SATB2. CDH1+ epithelium of HCOs generated from spontaneous and aggregated hindgut endoderm displayed similar numbers of SATB2-positive cells (Figure S14C).

[0184] Example 10. Formation of aggregated liver organoids Figure 15 shows a schematic for the formation of aggregated liver organoids. HLOs are generated from spontaneously dissociated spheroids and from spheroids generated from aggregated hindgut endoderm. The spheroids are then patterned into liver organoids by exposure to FGF2 (10-100 ng / mL), BMP4 (10-100 ng / mL), retinoic acid (2 µM), hepatocyte growth factor (10-20 ng / mL), dexamethasone (0.1 µM), and oncostatin M (10-100 ng / mL).

[0185] Example 11. Culturing gut endoderm monolayers increases mesoderm populations Conventional protocols for differentiating gut endoderm involve in vitro culture of definitive endoderm with minimal growth factors, and therefore the resulting gut endoderm and downstream spheroids / organoids contain less representative (i.e., less) proportions of other important cell types such as mesoderm and mesenchyme than in vivo tissues. In normal development, mesoderm and associated mesenchyme are critical for proper cellular organization and tissue maturation. Thus, there is a need to increase the mesoderm / mesenchyme population in organoid compositions.

[0186] Day 3 foregut and hindgut endoderm monolayers were prepared according to the procedures discussed in Examples 1-5 and 8. The monolayers were examined by immunofluorescence imaging staining for mesoderm marker Brachyury (T) and definitive endoderm marker FOXA2 (Figure 16A). The images show that the monolayers are composed mostly of definitive endoderm with little mesoderm. The mesoderm and definitive endoderm populations of these gut endoderm monolayers were quantified (Figure 16B). On average, both foregut and hindgut endoderm monolayers had only 3% and 1% mesoderm cells, respectively, and 94% and 87% definitive endoderm cells, respectively.

[0187] Similar to the day 3 monolayer cultures, day 7 foregut and hindgut endoderm monolayer cultures were prepared. As these cultures had progressed further in differentiation and gut patterning (including spontaneous spheroid formation), we examined mesenchymal cells rather than mesodermal cells. Monolayers were stained with the mesenchymal marker FOXF1 and the endoderm marker FOXA2, and a general increase in the mesenchymal fraction was observed (Figure 16C). The mesenchymal and endoderm populations were quantified (Figure 16D). Foregut endoderm monolayers contained 3% mesenchyme and 91% endoderm, while hindgut endoderm monolayers contained 11% mesenchyme and 86% endoderm. As observed, there is a significant increase in the mesodermal / mesenchymal lineage following culture of hindgut endoderm monolayers.

[0188] Quantification of the hindgut endoderm monolayer was repeated in an independent experiment. As seen in Figure 16E, there is again an increase in mesodermal / mesenchymal lineages in day 7 hindgut endoderm monolayer cultures compared to parental definitive endoderm cultures. Quantification was performed by staining cells with the mesodermal marker T and the endodermal marker FOXA2 compared to the proliferative cell marker Ki67.

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

[0190] With respect to the use of substantially all plural and / or singular terms herein, those of skill 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, the various singular / plural permutations may be expressly described herein.

[0191] Those skilled in the art will generally understand that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "include" should be interpreted as "including but not limited to," etc.). Those skilled in the art will further understand that where a specific number of claim recitations to be introduced are intended, such intent is expressly recited in this claim, and that in the absence of such recitations, no such intent exists. For example, as an aid to understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitations. However, the use of such phrases should not be construed as meaning that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes the claim recitation so introduced to embodiments that include only one such recitation, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce a claim recitation. In addition, those skilled in the art will recognize that if a specific number of claim recitations that are introduced are explicitly recited, such recitations should be construed to mean at least the number recited (e.g., the mere recitation of "two recitations" without any other modifiers means at least two recitations or more than two recitations).Furthermore, where a convention similar to "at least one of A, B, and C, etc." is used, such syntax is typically intended to mean as one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where a convention similar to "at least one of A, B, or C, etc." is used, such syntax is typically intended to mean as one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

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

[0193] As would be understood by one of ordinary skill in the art, for all purposes, including in terms of written description, all ranges disclosed herein encompass all possible subranges and combinations of these subranges. Any range listed can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least 2, 3, 4, 5, 10, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As would be understood by one of ordinary skill in the art, all words such as "up to," "at least," "greater than," "less than," etc. refer to ranges that include the recited numbers and that can subsequently be broken down into subranges as discussed above. Finally, as would be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5, etc. items, and so forth.

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

[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 supersedes and / or is intended to take precedence over such conflicting material.

Claims

1. 1. A method for producing one or more aggregated organoids, comprising: Differentiating definitive endoderm into gut endoderm monolayers and gut spheroids, Differentiating the intestinal endoderm monolayer, wherein the intestinal spheroids are dissociated and suspended in a growth medium; Separating the intestinal endoderm monolayer from the intestinal spheroids; dissociating the intestinal endoderm monolayer into a single cell suspension of intestinal endoderm cells; aggregating the single cell suspension of gut endoderm cells into one or more gut endoderm aggregates; and culturing said one or more gut endoderm aggregates to produce said one or more aggregated organoids.

2. 2. The method of claim 1, wherein the isolating step comprises aspirating the growth medium and suspended intestinal spheroids from the intestinal endoderm monolayer.

3. The method of claim 1 or 2, wherein the dissociation step comprises enzymatically dissociating the intestinal endoderm monolayer.

4. 4. The method of any one of claims 1 to 3, wherein the aggregation step comprises aggregating the single cell suspension in hanging drops, centrifuging the single cell suspension in a "v" or "u" bottom microwell culture plate, aggregating the single cell suspension using an orbital shaker, or centrifuging the single cell suspension in a formation plate, or any combination thereof.

5. The method of any one of claims 1 to 4, wherein the culturing step comprises contacting the one or more gut endoderm aggregates with an extracellular matrix, or a mimetic or derivative thereof.

6. The method according to any one of claims 1 to 5, wherein the intestinal endoderm monolayer is a foregut endoderm monolayer and the intestinal spheroids are foregut spheroids.

7. 7. The method of claim 6, wherein differentiating the definitive endoderm into the foregut endoderm monolayer and the foregut spheroids comprises contacting the definitive endoderm with one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof.

8. The method of claim 6 or 7, wherein the one or more aggregated organoids are aggregated liver organoids.

9. The method of claim 8, wherein culturing the one or more enteric endoderm aggregates to form the one or more aggregated liver organoids comprises contacting the one or more enteric endoderm aggregates with one or more FGF signaling pathway activators, one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, or any combination thereof.

10. The method of claim 6 or 7, wherein the one or more aggregated organoids are aggregated gastric organoids.

11. 11. The method of claim 10, wherein the one or more aggregated gastric organoids are aggregated antral gastric organoids.

12. 12. The method of claim 11, wherein culturing the one or more enteric endoderm aggregates to form the one or more aggregated antral gastric organoids comprises contacting the one or more enteric endoderm aggregates with EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, or any combination thereof.

13. The method according to any one of claims 1 to 5, wherein the intestinal endoderm monolayer is a hindgut endoderm monolayer and the intestinal spheroids are hindgut spheroids.

14. 14. The method of claim 13, wherein differentiating the definitive endoderm into the hindgut endoderm monolayer and the hindgut spheroids comprises contacting the definitive endoderm with one or more FGF signaling pathway activators, or one or more Wnt signaling pathway activators, or both.

15. The method of claim 13 or 14, wherein the one or more aggregated organoids are aggregated intestinal organoids.

16. 16. The method of claim 15, wherein culturing the one or more enteric endoderm aggregates to form the one or more aggregated intestinal organoids comprises contacting the one or more enteric endoderm aggregates with EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors, or any combination thereof.

17. The method of claim 13 or 14, wherein the one or more aggregated organoids are aggregated colonic organoids.

18. 18. The method of claim 17, wherein culturing the one or more enteric endoderm aggregates to form the one or more aggregated colon organoids comprises contacting the one or more enteric endoderm aggregates with EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators, or any combination thereof.

19. the one or more gut endoderm aggregates comprise at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 gut endoderm aggregates, each of the one or more gut endoderm aggregates comprising: a diameter within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of the one or more gut endoderm aggregates; or or a volume within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the one or more gut endoderm aggregates; Including both, 19. The method according to any one of claims 1 to 18.

20. 20. One or more aggregated organoids produced by any one of claims 1 to 19.

21. 1. A plurality of gut endoderm aggregates comprising at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 gut endoderm aggregates, each of said plurality of gut endoderm aggregates comprising: a diameter within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of the plurality of gut endoderm aggregates; or a volume within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the plurality of gut endoderm aggregates; or Multiple gut endoderm aggregates, including both.

22. The multiple intestinal endoderm aggregates of claim 21 , wherein the multiple intestinal endoderm aggregates are derived from the same subject.

23. A formation plate comprising a plurality of microwells and a plurality of gut endoderm aggregates as described in claim 21 or 22, wherein each of the plurality of microwells comprises a single gut endoderm aggregate among the plurality of gut endoderm aggregates.

24. A plurality of intestinal endoderm aggregates according to claim 21 or a formation plate according to claim 23, wherein the plurality of intestinal endoderm aggregates are produced according to a method according to any one of claims 1 to 19.

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