Improved methods for making organoid compositions

Abstract

To provide improved organoid compositions that more closely resemble in vivo tissue, and to provide methods of making the organoid compositions that are more robust, scalable, faster, and cost-effective.SOLUTION: Provided is a method of producing one or more aggregated organoids, comprising: differentiating definitive endoderm to a gut endoderm monolayer and gut spheroids, the gut endoderm monolayer being adherent and the gut spheroids being detached and suspended in a growth medium; separating the gut endoderm monolayer from the gut spheroids; dissociating the gut endoderm monolayer to a 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 the one or more aggregated organoids.SELECTED DRAWING: Figure 9A

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 herein by reference in its entirety.

[0002] Aspects of the present disclosure generally relate 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 characteristics of functional intestinal tissue, including absorption, intestinal 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 into crypt-villus structures, including the adult stem cell activity / progenitor zone of the crypt, and mature epithelium, capable of functions such as nutrient absorption and brush border enzyme activity. Consequently, organoids derived from pluripotent stem cells are a physiologically relevant and powerful tool for studying intestinal development and disease and also provide a novel platform for drug development. Furthermore, given that induced pluripotent stem cells can be derived from any individual, including those with intestinal diseases, 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 more closely resemble in vivo tissues, and for methods to generate organoid compositions that are more robust, scalable, faster, and cost-effective. 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 the gut endoderm monolayer from the gut spheroids; dissociating the gut endoderm monolayer into a 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 one or more gut endoderm aggregates to produce one or more aggregated organoids.In some embodiments, the gut endoderm monolayer is adherent, and the gut spheroids are separated and suspended in growth medium.In some embodiments, the definitive endoderm is differentiated from pluripotent stem cells.In some embodiments, the definitive endoderm is differentiated from embryonic stem cells or induced pluripotent stem cells.In some embodiments, the definitive endoderm is human definitive endoderm.In some embodiments, the separating 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 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, 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 foregoing cell numbers. In some embodiments, the culturing step comprises contacting the one or more enteric endoderm aggregates with an extracellular matrix, or a mimetic or derivative thereof. In some embodiments, the extracellular matrix, or a mimetic or derivative thereof comprises Matrigel.

[0005] In any of the embodiments disclosed herein, intestinal endoderm monolayer is foregut endoderm monolayer, and intestinal spheroid is 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, one or more FGF signaling pathway activators comprise FGF4, one or more Wnt signaling pathway activators comprise CHIR99021, or one or more BMP signaling pathway inhibitors comprise Noggin, or any combination thereof.In some embodiments, 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 antrum gastric organoids.In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated antrum 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 intestinal endoderm monolayer is a hindgut endoderm monolayer, and the intestinal 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, one or more FGF signaling pathway activators comprise FGF4, or one or more Wnt signaling pathway activators comprise CHIR99021, or both.In some embodiments, one or more aggregated organoids are 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, 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 intestinal endoderm aggregates to form one or more aggregated colon 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.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 enteric 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 10,000 enteric endoderm aggregates, or any number of enteric endoderm aggregates within a number defined by any two of the foregoing enteric 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 aforementioned 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 aforementioned volumes, or both.

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

[0009] Also disclosed herein is any one or more aggregated organoid 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 ±10%, ±9%, ±8%, ±10% or ±10% of the average diameter of the plurality of intestinal endoderm aggregates. 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% of the average volume of the plurality of enteric endoderm aggregates, or any volume within a range defined by any two of the aforementioned volumes, or both. In some embodiments, the plurality of enteric 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 enteric endoderm aggregates according to claim 38 or 39, each of the plurality of microwells containing a single enteric endoderm aggregate from the plurality of enteric endoderm aggregates. In some embodiments, for any one of the plurality of enteric endoderm aggregates disclosed herein, or any one of the forming plates disclosed herein, the plurality of enteric 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 hindgut endoderm (HGE) to form HGE-derived single cell populations;

[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 of 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 of 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 cell population. [Brief explanation 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 to HIOs. [Figure 2A-C] 1 shows an embodiment of the preparation of an intestinal endoderm monolayer 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 component thereof. [Figure 7A] Shown is a schematic embodiment of an existing spheroid production protocol for human intestinal organoid (HIO) generation. [Figure 7B] 1 shows an embodiment of the inter-experimental variability of spheroid production using an embodiment of an existing protocol. [Figure 7C] 10 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] Figure 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] 10 shows an embodiment of an image demonstrating a greatly increased yield of uniform spheroids produced by the aggregation method. [Figure 9D] 1 shows an embodiment in which the yield of spheroids per well from HGE aggregation is significantly 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] 1 shows an embodiment of an image demonstrating that the growth and morphology of aggHIOs are indistinguishable from spontaneous HIOs. [Figure 11B] 1 shows an embodiment of an image showing that AggHIOs contain CDX2+ intestinal epithelium and Emilin1+ mesenchyme. [Figure 11C] 1 shows an embodiment of an image showing that both spontaneously isolated 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 after in vivo transplantation. [Figure 12B] 1 shows an embodiment of immunofluorescence analysis of mature small intestinal 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 aHGOs derived from condensed foregut endoderm are morphologically indistinguishable from spontaneous aHGOs. [Figure 13C] 10 shows an embodiment of images demonstrating that the expression of gastric epithelial markers cannot distinguish between spontaneous and aggregated aHGOs. [Figure 14A] FIG. 1 shows an embodiment of a schematic for the generation of human colon organoids (HCOs) by aggregation. [Figure 14B] 10 shows an embodiment of an image demonstrating that HCOs derived from aggregated hindgut endoderm spheroids are morphologically indistinguishable from HCOs derived from spontaneous spheroids. [Figure 14C] 10 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 (HLOs) 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 proportion of mesoderm and definitive endoderm populations in the cultures of FIG. 16A. [Figure 16C] 1 shows an embodiment of an image showing the population density 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 and day 7 hindgut endoderm cultures. DETAILED DESCRIPTION OF THE INVENTION

[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 (approximately 7 days of culture). During this time, spontaneous morphogenesis occurs, resulting in the formation and separation of three-dimensional spheroids resembling the embryonic gut. These spheroids are embedded in an extracellular matrix, or its mimic or derivative (e.g., Matrigel), and cultured in a medium that promotes proliferation and organ differentiation. After approximately 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 growth and maturation.

[0028] However, current organoid generation methods have several limitations. In particular, there is significant variability in the efficiency of spontaneous spheroid production and isolation between different hPSC strains. Even among strains known to have a strong ability to produce spontaneous spheroids, spontaneous spheroid production and isolation vary greatly from experiment to experiment. Within a given organoid generation experiment, there is also significant variability in the efficiency of spontaneous spheroid production and well-to-well isolation. Spontaneous spheroid formation often occurs in large "chains" of multiple attached spheroids. The size of spontaneous spheroids generated between strains can vary greatly. Therefore, 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 the improved method for producing organoid or its composition, which overcomes one or more limitations of existing method.In some embodiments, the disclosed method eliminates or reduces the low efficiency of spontaneous spheroid production, which is associated with the variability between strains, between experiments and between wells, and provides a method for improving scalability.

[0030] The methods disclosed herein take advantage of the ability of hPSC-derived cells to self-organize upon aggregation. In the case of gastrointestinal organoids, hPSCs are differentiated into DE, followed by FGE or HGE dissociation into single cells using standard methods, with the cells remaining attached to the cell culture plate, including cases where spontaneous morphogenesis and spheroid formation and separation are not detected. 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 differences 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 in conjunction with the methods described herein.

[0031] Disclosed herein is aggregated organoid and its composition, and the method for producing it, which involves aggregating a single cell suspension of intestinal endoderm monolayer (as opposed to intestinal spheroid) and then culturing the aggregate to form aggregated organoid.The method disclosed herein produces organoid with higher reliability and reproducibility than the conventional method known in the art, and affects 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, autotransplantation or allogeneic transplantation into subjects such as humans or other mammals, or xenotransplantation into immunodeficient animals.In some embodiments, the aggregated organoid is liver, stomach, antrum stomach, fundus of the stomach, intestine or colon organoid.In some embodiments, the aggregated organoid is 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, 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 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 generally identify like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are 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 stated steps or elements or group of steps or elements, but not the exclusion of any other steps or elements or group of steps or elements. "Consisting of" means including everything that the phrase "consisting of" follows. Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of" means the inclusion of all elements listed before this phrase, limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed 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 is understood that such terms are used for illustrative purposes only and are not intended to be limiting or absolute. In that regard, it will be understood that the instruments as disclosed herein can be used in a variety of orientations and positions that are not limited to those shown and described herein.

[0038] The terms "individual," "subject," or "patient" as used herein have their common and usual meanings 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 terms "effective amount" or "effective dose" as used herein have their common and ordinary meaning as understood in light of the specification and refer to that amount of a 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 amount of the active composition or compound effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend on various factors, including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

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

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

[0042] As used herein, the term "isolated" has its common and ordinary meaning as understood in light of the specification and refers to a substance and / or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (in nature and / or in an experimental setting) and / or (2) has been separated from at least some of the components with which it was associated when produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or equal to, about, at least about, less than, or about less than 100% (or ranges including and / or spanning the foregoing values) of other components with which they were originally associated. In some embodiments, an isolated agent is equal to, about, 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 from other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0043] As used herein, "in vivo" is given its common 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 common and ordinary meaning as understood in light of the specification and refers to the performance of a 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 a method outside biological conditions, e.g., in a petri dish or test tube.

[0046] As used herein, the terms "nucleic acid" or "nucleic acid molecule" have their common and ordinary meaning as understood in the context of this specification and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those occurring naturally in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have alterations in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotide" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The nucleic acid(s) can be contained in a nucleic acid vector or construct (e.g., a plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used to amplify and / or express 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 with extra nucleic acid, for example, between linkers, repeats, or restriction enzyme sites, or any other sequence 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, 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. The term "downstream" as used herein with respect to a nucleic acid has its common and ordinary meaning as understood in light of the specification, and refers to the sequence after the 3' end of the preceding sequence on the strand containing the coding sequence (sense strand) when the nucleic acid is double-stranded. The term "upstream" as used herein with respect to a nucleic acid has its common and ordinary meaning as understood in light of the specification, and refers to the sequence before the 5' end of the succeeding sequence on the strand containing the coding sequence (sense strand) when 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 light of the present specification and refers to two or more sequences that occur in close proximity to any other sequence, but generally not between sequences that encode functional or catalytic polypeptides, proteins, or protein domains, either directly or with extra nucleic acid, for example, between linkers, repeats, or restriction enzyme sites, or that are at, about, at least about, less than, or about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within a range defined by any two of the foregoing lengths.

[0048] The nucleic acid described herein comprises nucleobases.The primary, standard, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil.Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobase, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl base, dye-labeled base, fluorescent base or biotin-labeled base.

[0049] As used herein, the terms "peptide," "polypeptide," and "protein" have their common and ordinary meanings as understood in light of this specification and refer to polymers 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 functions. 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 nucleic acid templates, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions 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, for example, between linkers, repeats, epitopes, or tags, or any other sequence that is, about, at least, at least about, less than, or about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within a range defined by any two of the aforementioned lengths. As used herein, the term "downstream" with respect to a polypeptide has its common and ordinary meaning as understood in light of the specification, and refers to sequences that follow the C-terminus of the preceding sequence. The term "upstream" as used herein in reference to a polypeptide has its common and ordinary meaning as understood in light of the specification, and refers to sequences that precede the N-terminus of a subsequent sequence.

[0050] As used herein, the term "purity" of any given substance, compound, or material has its common and ordinary meaning as understood in light of specifications and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimal points therebetween. Purity may be affected by unwanted impurities, including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious 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 light of specifications 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 about, less than, or equal to 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 reaction or process efficiency, undesired side reactions, decomposition, the quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material at any step in the production.

[0052] The term "w / w%" or "weight / weight %" as used herein has its ordinary and ordinary meaning as understood in the context of the present 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 the present 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 building complete, viable organisms. These cells are produced from the fusion of egg and sperm cells. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0054] As used herein, the term "embryonic stem cells (ESCs), commonly abbreviated as ES cells, as used herein, has its plain and ordinary meaning as understood in light of the present specification and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of 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 this specification and encompasses any cell that can differentiate into almost any cell type of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs may be the progeny of inner cell mass cells of a preimplantation blastocyst or may be obtained by the induction of non-pluripotent stem cells, e.g., adult somatic cells, by forcing the expression of specific genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0056] As used herein, the term "induced pluripotent stem cells (iPSCs)" has its plain and ordinary meaning as understood in light of the specification. These terms are commonly abbreviated as iPSCs and refer to a type of pluripotent stem cell artificially induced from normally non-pluripotent cells, such as adult somatic cells, by inducing the "forced" expression of specific genes. hiPSCs refer to human iPSCs. In several methods known in the art, iPSCs can be derived by transfecting specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes can include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes can also improve the efficiency of induction. After 3–4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically and are typically isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include first-generation iPSCs, second-generation iPSCs in mice, and human induced pluripotent stem cells.In some methods, retrovirus system is used to transform human fibroblasts into pluripotent stem cells using four essential 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.Examples of 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., Sox1, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klf1, 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, progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent stem cells (induced or non-induced), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be derived from embryos, infants, children, or adults. In some embodiments, progenitor cells can be somatic cells that have been subjected to treatment to confer pluripotency via genetic manipulation or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

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

[0059] Additional stem cells that can be used in embodiments according to the present disclosure include, but are not limited to, those obtained by and described in databases hosted by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC cell Bank at the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, Calif), Cellartis AB (Göteborg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present 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 original 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. Because adenovirus does not combine its own genes with the target host, the risk of tumor formation is eliminated. In some embodiments, non-viral techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system at all, albeit with very low efficiency. In other embodiments, direct protein delivery is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, mouse iPSCs can be generated using a similar methodology. Repeated treatment of cells with specific proteins delivered to the cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0062] The term "feeder cells," as used herein, has its common and ordinary 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 culture medium or displaying them on their surface. Feeder cells are generally adherent cells and may be growth-arrested. For example, feeder cells may be growth-arrested by irradiation (e.g., gamma rays), 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 culture medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human forehead fibroblasts, human skin fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, the conditioned medium prepared from feeder cells is used instead of or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the proliferation of target stem cells.

[0063] The term "extracellular matrix" as used herein has its plain and ordinary meaning in the context of this 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 mimetics or derivatives thereof, can be used in the methods disclosed herein. Some examples of extracellular matrices, or mimetics 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 membranes, Matrigel, hydrogels, 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 pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0065] As used herein, "pharmaceutically acceptable" has its plain and ordinary meaning as understood in light of the specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed to the cells or mammals at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" has its plain and ordinary meaning as understood in light of the specification and is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are approved by a regulatory agency of the federal government, state government, or other regulatory body, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, including humans and non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous solutions of dextrose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers may also include one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt formation inhibitors such as sodium; non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, as desired. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation should be compatible with the method of administration.

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

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

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

[0069] Appropriate formulations vary depending on the selected route of administration. Techniques for the formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques for administering compounds exist in the art, including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intraarterial, intravenous), intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[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 bodily organs.

[0071] As used herein, the term "diluent" has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It can also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline, which mimics the composition of human blood.

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

[0073] PSC differentiation In some embodiments, PSCs, such as ESCs and iPSCs, undergo stepwise directed differentiation, first to the definitive endoderm (DE), then to the foregut or hindgut lineages, and then to gastrointestinal tissue. In some non-limiting embodiments, the 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) to promote DE formation and molecules for subsequent tissue formation are simultaneously added. 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, the Wnt signaling pathway, the Wnt / APC signaling pathway, the FGF signaling pathway, the TGF-β signaling pathway, the BMP signaling pathway, the Notch signaling pathway, the Hedgehog signaling pathway, the LKB signaling pathway, and the Par polarity signaling pathway. Each of the signaling pathways listed 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. The posterior DE forms the midgut and hindgut, the small and large intestines, and parts of the urogenital system. Studies using mouse, chick, and frog embryos suggest that establishing an anterior-posterior pattern of the DE at the gastrula stage is a prerequisite for subsequent foregut and hindgut development. Wnt and FGF signaling pathways are important for promoting the posterior endoderm / hindgut or anterior endoderm / foregut fates. In the hindgut, a simple cuboidal epithelium first develops into a pseudostratified columnar epithelium, which then develops into a polarized columnar epithelium and a villi containing a proliferative zone at the base of the villi, corresponding to the presumptive progenitor region.

[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 the gonadal ridges of an embryo. Embryonic stem cells or germ cells can be derived from various animal species, including, but not limited to, various mammalian species, including humans. In some embodiments, human embryonic stem cells are used to 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 differentiating into definitive endoderm. In some embodiments, PSCs are genetically modified to express an exogenous nucleic acid or protein before differentiating into definitive endoderm.

[0076] In some embodiments, embryonic stem cells, germ cells, or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a period of time that is, is about, is at least, is at least about, is less than, or is about less than 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, or 300 hours, or any period of time within a range defined by any two of the aforementioned periods, for example, 6 hours to 300 hours, 24 hours to 120 hours, 48 ​​hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, 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, embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / mL, 10000 ng / mL or or at a concentration of about, at least about, at most, or at most about, or less than 15,000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, e.g., 10 ng / mL to 15,000 ng / mL, 100 ng / mL to 5,000 ng / mL, 500 ng / mL to 2,000 ng / mL, 10 ng / mL to 2,000 ng / mL, or 1000 ng / mL to 15,000 ng / mL. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout treatment. In some embodiments, the concentration of one or more small molecule compounds, activators, inhibitors, or growth factors is varied over the course of treatment. In some embodiments, multiple small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the concentrations of the multiple small molecule compounds, activators, inhibitors, or growth factors may vary.

[0078] In some embodiments, ESCs, germ cells, or iPSCs are cultured in a growth medium that supports stem cell proliferation. In some embodiments, ESCs, germ cells, or iPSCs are cultured in a stem cell growth medium. In some embodiments, the stem cell growth medium is RPMI 1640, DMEM, DMEM / F12, mTeSR1, mTeSR Plus, DE differentiation, hindgut endoderm differentiation, gut-based, or complete Sato medium. In some embodiments, the stem cell growth medium contains fetal bovine serum (FBS). In some embodiments, the stem cell growth medium contains FBS at a concentration that is, about, 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%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the foregoing 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, a cell population enriched in definitive endoderm cells is used. 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, 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 committed to differentiate in vitro into gastrointestinal epithelium or mesenchyme, including secretory, endocrine, and absorptive cell types. It will be understood that molecules such as growth factors can be added at any developmental stage to promote the formation of specific types of intestinal tissue.

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

[0083] Those skilled in the art will understand that altering the concentration, expression, or function of one or more Wnt signaling proteins in combination with altering the concentration, expression, or function of one or more FGF proteins can result in directed differentiation according to the present disclosure. In some embodiments, cellular components associated with the Wnt and / or FGF signaling pathway, such as natural inhibitors, antagonists, activators, or agonists of the pathway, can be used to inhibit or activate the Wnt and / or FGF signaling pathway. In some embodiments, siRNA and / or shRNA targeting cellular components associated with the Wnt and / or FGF signaling pathway are used to inhibit or activate these pathways.

[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 their interaction with cell surface-associated heparan sulfate proteoglycans has been shown to be essential for FGF signaling. FGFs play important roles in the proliferation and differentiation 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 fibroblast growth factor receptors (FGFRs). 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 share striking sequence similarity, they do not bind to FGFRs and are involved in intracellular processes independent of FGFs. This group is also known as "iFGFs." Members FGF15 through FGF23 are newer and less well characterized. FGF15 is the mouse ortholog of human FGF19 (hence, there is no human FGF15). Human FGF20 was identified based on its homology to Xenopus FGF-20 (XFGF-20). In contrast to the local activity of other FGFs, FGF15 / FGF19, FGF21, and FGF23 have more systemic effects.

[0085] It will be understood by those skilled in the art that in some embodiments, any FGF can be used in combination with a protein 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 subsequently into various intermediate mature gastrointestinal cell types can be determined by the presence of stage-specific cellular markers. In some embodiments, the expression of representative cellular components is used to determine DE formation. Representative cellular components include CMKOR1, CXCR4, GPR37, RTN4RL1, SLC5A9, SLC40A1, TRPA1, AGPAT3, APOA2, C20orf56, C21orf129, CALCR, CCL2, CER1, CMKOR1, CRIP1, CXCR4, CXorf1, DIO3, DIO30S, EB-1, EHHADH, ELOVL2, EPSTI1, FGF17, FLJ10970, FLJ21195, FLJ22471, FLJ23514, and FOXA2. , FOXQ1, GATA4, GPR37, GSC, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SOX17, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706, or AW772192, or any combination thereof. In some embodiments, the absence of cellular components such as the foregut marker Pdx1 and albumin can be used to reveal directed hindgut formation. In some embodiments, one or more (e.g., at least one or three) intestinal transcription factors CDX2, KLF5, or SOX9 can be used to represent intestinal development. In some embodiments, one or more of GATA4 or GATA6 protein expression can be used to represent intestinal development.

[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 intestinal 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, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 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. In some embodiments, a highly complex epithelium surrounded by mesenchymal cells can be observed. In some embodiments, gastrointestinal organoids, epithelium, polarized columnar epithelium, mesenchyme, neural cells, or smooth muscle cells can be observed at, 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, 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 directing differentiation of 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, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, PBMCs are grown on 0.1% gelatin.

[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, about, at least about, less than, or 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, 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 an extracellular matrix, or a mimetic 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 onto Matrigel-coated plates 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 the 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, PSCs are contacted with activin A or one or more BMP signaling pathway activators at a concentration that is, about, at least about, less than, or 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) 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, lungs, thyroid gland, 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 is 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, the gut endoderm monolayer is attached to, for example, a tissue culture plate or an embodiment of the formation plate disclosed herein, and the 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 the definitive endoderm that have undergone patterning into the 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 the small intestine and large intestine organ lineages.During the differentiation of definitive endoderm into gut endoderm, gut spheroids spontaneously form and are separated from the gut endoderm monolayer as floating cell masses. These intestinal spheroids exhibit early characteristics of organoids, particularly the heterogeneity of 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, although 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. Previously described methods for differentiating definitive endoderm into gut spheroids can be considered synonymous with differentiating definitive endoderm into both gut endoderm monolayers and gut spheroids, as the production of a gut endoderm monolayer usually results in the simultaneous production of a gut endoderm monolayer.

[0095] In some embodiments, definitive endoderm is differentiated into gut endoderm monolayers and gut spheroids by 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 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 inhibitor 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 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, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 70000, 80000, 90000, 10000, 1000 The contact may be at a concentration that is, about, at least about, less than, or equal to 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, if provided, is contacted at a concentration that is, about, at least about, less than, or about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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 a foregut endoderm monolayer and a foregut spheroid. In some embodiments, the gut endoderm monolayer is a foregut endoderm monolayer, and the gut spheroids are foregut spheroids. In some embodiments, differentiating the definitive endoderm into a foregut endoderm monolayer and a foregut spheroid 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 hindgut spheroids. In some embodiments, the gut endoderm monolayer is a hindgut endoderm monolayer, and the gut spheroids are hindgut spheroids. In some embodiments, differentiating the definitive endoderm into a hindgut endoderm monolayer and 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. 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 intestinal spheroids 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, gut endoderm monolayers produced by any of the methods disclosed herein are 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 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 population, or a percentage of the total cell population that is about 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 foregoing percentages, e.g., 1%-20%, 1%-10%, 10%-20%, or 5%-15% mesoderm and / or mesenchyme. In some embodiments, the intestinal endoderm monolayer comprises more mesoderm and / or mesenchyme than the intestinal spheroids at the same stage of culture. In some embodiments, the intestinal endoderm monolayer comprises some mesoderm and / or mesenchyme, i.e., 1, 2, 3, 4, or 5 times, or about, or at least about, or less than, or about less than the number of mesoderm and / or mesenchyme found in the intestinal spheroids, or any multiple within a range defined by any two of the aforementioned multiples.

[0099] Isolation and dissociation of gut endoderm After differentiating the definitive endoderm into a 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 known in the art for separating adherent cells (e.g., gut endoderm monolayer) and floating cells (e.g., gut spheroids) can be used. For example, as a non-limiting example, the growth medium and suspended gut spheroids are aspirated, leaving behind 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 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 wash 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, 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 enzymatically dissociated 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, less than, or about less than 10,000 enteric endoderm cells, or any number of enteric endoderm cells within a range defined by any two of the foregoing 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 contains 500 ng / mL or about 500 ng / mL of recombinant human EGF, 100 ng / mL or about 100 ng / mL of recombinant human Noggin, or 500 ng / mL or about 500 ng / mL of 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 10 μM or about 10 μM.

[0102] In some embodiments of any of the methods disclosed herein, the intestinal endoderm cells are aggregated using an orbital shaker. In some embodiments, the suspension of intestinal 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 form 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 to 24 hours, 24 to 48 hours, or 12 to 36 hours.

[0103] In some embodiments of any of the methods disclosed herein, the intestinal 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 includes spotting a droplet of gut endoderm cells suspended in growth medium upside down onto a surface (e.g., a cell culture plate) and allowing the cells to sink to the bottom of the droplet 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 the gut endoderm monolayer and / or gut spheroids is contemplated. In some embodiments, the gut endoderm monolayer adheres 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 intestinal endoderm monolayer is separated from the intestinal spheroids by aspirating the growth medium and suspended intestinal spheroids from the biocompatible container (16). In some embodiments, the isolated intestinal endoderm monolayer is then dissociated into a single-cell suspension of intestinal 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 intestinal endoderm aggregates (20). In some embodiments, one or more intestinal endoderm aggregates (20) are placed in the same or different biocompatible container (16), and the one or more intestinal endoderm aggregates are cultured into one or more aggregated organoids.

[0107] 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 FIG. 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 about, less than, or equal to 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm, or any length within a range defined by any two of the foregoing 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 to be, about, at least about, less than, or equal to 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 about, less than, or equal to 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μm, or any depth within a range defined by any two of the foregoing 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 the opening (40) in the top surface (42) of the bottom (28) and the floor (44) of the bottom (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 forming plate (12) is not intended to be unnecessarily limited to the specific number, arrangement, or size of wells (24) and microwells (26) shown and described in any of the examples provided herein. In some embodiments, the forming 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, opposing longitudinal sidewalls (36) taper toward each other from the opening (40) to the floor (44), while opposing lateral sidewalls (38) similarly taper toward each other from the opening (40) to the floor (44). In some embodiments, gravity forces the single cells in suspension downward in a transverse direction, while the recoil forces exerted on the cells by the longitudinal and lateral sidewalls (36, 38) force the cells inward toward each other, effectively gathering and agglomerating the single cells together. In some embodiments, such tapering allows for aggregation of the cells into three-dimensional aggregates, which can be further enhanced by centrifugation. In some embodiments, each microwell (26) receives, or is about, or at least about, or less than, or about 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 identical dimensions, defining a void within the microwell (26) having the shape of an inverted pyramid. In some embodiments, the longitudinal and lateral sidewalls (36) and (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 understood that one of skill in the art can determine acceptable shapes for the microwells (26), including, 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 specific populations 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 specific shapes and dimensions of the formation plate (12) and / or microwells (26) shown in the figures or for use with the specific 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, 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 cell attachment 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 Figures 3A-6. In some embodiments, the formation plate (12) is formed from multiple components, and at least the surfaces of the microwells (26) are fabricated from a biocompatible material. In some embodiments, the biocompatible material comprises, consists essentially of, or consists of stainless steel, titanium, polymeric organosilicone compounds, polydimethylsiloxane (PDMS), glass, plastic, PVC, PE, PP, PMMA, PS, PTFE, nylon, polyurethane, PET, PES, hyaluronan, chitosan, sugar, ceramic, alumina, zirconia, bioglass, hydroxyapatite, or any combination thereof, or other biocompatible materials known in the art. In some embodiments, the formation plate (12) is sterile, resistant to adhesion by tissues and / or cells, comprises a hydrophobic surface, comprises features that improve the 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, gastric fundus organoids, antral gastric organoids, liver 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 gastric fundus organoids (HFGO), human antral gastric organoids (HAGO), human liver organoids (HHO), human intestinal organoids (HIO) or human colonic organoids (HCO), or any combination thereof. In some embodiments, after the single-cell suspension of enteric endoderm cells is aggregated into one or more enteric endoderm aggregates, the one or more enteric endoderm aggregates are cultured for a short period of time that is, is about, is at least about, is less than, or is about less than 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 periods, e.g., 1-50 hours, 10-40 hours, 20-30 hours, 1-30 hours, or 24-50 hours, to allow collection, recovery, and / or coalescence. In some embodiments, the one or more enteric endoderm aggregates are removed from the aggregation medium or resuspended. For example, in some embodiments, when one or more gut endoderm aggregates are aggregated in 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., by 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, such as complete Sato medium or other biocompatible aqueous solution, to ensure that all aggregates are collected. In some embodiments, 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 one or more gut endoderm aggregates because it may cause the aggregates to fuse together. In some embodiments, after sedimentation, one or more gut endoderm aggregates are cultured under conditions for differentiating the one or more gut endoderm aggregates into one or more aggregated organoids. For example, in some embodiments, after sedimentation, all remaining growth medium is removed. In some embodiments, 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 intestinal 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 intestinal endoderm aggregates into one or more aggregated organoids.Although the conditions for differentiating one or more intestinal endoderm aggregates into various different aggregated organoids are provided herein, other methods previously known for differentiating intestinal spheroids (such as foregut spheroids and / or hindgut spheroids) into respective organoids can be used to differentiate one or more intestinal 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, 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 herein by reference in its entirety.

[0113] In some embodiments, when the enteric endoderm cells are foregut endoderm cells, the one or more enteric endoderm aggregates are foregut endoderm aggregates, and the one or more enteric endoderm aggregates are differentiated into one or more aggregated organoids of the 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 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 (for example, at least 1, 2, 3, 4, 5, 6).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, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5500, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 55000, 60000, 70000, 70000, 70000, 80000 , 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or a concentration that is about, 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, 1 to 1000 ng / mL, 50 to 500 ng / mL, 500 to 1000 ng / mL, or 1 to 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, when provided, is contacted at a concentration that is, about, at least about, less than, or 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, one or more aggregated organoids are or comprise one or more aggregated gastric organoids.In some embodiments, one or more aggregated gastric organoids are or comprise one or more aggregated fundus organoids or one or more aggregated antrum gastric organoids, or both.In some embodiments, culturing one or more intestinal endoderm aggregates to form one or more aggregated antrum gastric organoids comprises contacting one or more intestinal endoderm aggregates with one or more (for example, at least 1, 2, or 3) of EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, or any combination thereof.In some embodiments, one or more BMP signaling pathway inhibitors comprise Noggin.In some embodiments, 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 EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, if provided, 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, The contact is performed at a concentration of, about, at least about, less than, or equal to 900, 950, or 1000 ng / mL, 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 EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, if provided, is contacted at a concentration that is, about, at least about, less than, or about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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 enteric endoderm cells are hindgut endoderm cells, the one or more enteric endoderm aggregates are or comprise one or more hindgut endoderm aggregates, and the one or more enteric endoderm aggregates are differentiated into one or more aggregated organoids of the hindgut lineage.

[0119] In some embodiments, one or more aggregated organoids are or comprise 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 one or more (for example, 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, 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 intestinal organoids comprise intestinal epithelium and intestinal mesenchyme.In some embodiments, the intestinal epithelium of one or more aggregated intestinal organoids is CDH1+, CDX2+, E-cad+, or any combination thereof.In some embodiments, the intestinal mesenchyme of 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, one or more mature aggregated intestinal organoids comprise intestinal cell types.In some embodiments, 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 intestinal endoderm aggregates to form one or more aggregated colon organoids comprises contacting one or more intestinal 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 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 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, 7500, 8000, 9000, 10 ... The contact may be at a concentration of, about, at least about, less than, or equal to 50, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10-1000 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, or 10-200 ng / mL. In some embodiments, 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, are each 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 a concentration of 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 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, the one or more gut endoderm aggregates are cultured for, about, at least about, less than, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, or any number of days within a range defined by any two of the foregoing 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 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 aggregated organoids comprise functional lumen.In some embodiments, one or more aggregated organoids have the ability to further differentiate when transplanted.In some embodiments, one or more 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 intestinal endoderm aggregates and / or resulting aggregated organoids.In some embodiments, the method and use of aggregation medium (for example, any one of the formation plates disclosed herein) allows the formation of multiple intestinal endoderm aggregates. In some embodiments, the plurality of enteric endoderm aggregates comprises, or is about, or at least about, or less than, or about or less than 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 enteric endoderm aggregates, or any number of enteric endoderm aggregates within a number defined by any two of the foregoing numbers of enteric endoderm aggregates, e.g., 1,000-1,000,000 enteric endoderm aggregates, 5,000-100,000 enteric endoderm aggregates, 1,000-10,000 enteric endoderm aggregates, or 10,000-1,000,000 enteric endoderm aggregates. In some embodiments, the formation of homogeneous or nearly homogeneous gut endoderm aggregates and / or resulting aggregated organoids is defined by the fact that the variation in at least one spatial dimension of a plurality of gut endoderm aggregates and / or resulting aggregated organoids is reduced compared with the organoids that are produced from gut endoderm spheroids and / or 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, the 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 dispersion in at least one spatial dimension comprises 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 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 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 a range defined by any two of the aforementioned diameters. In some embodiments, the reduction in the variance in at least one spatial dimension comprises the volume of the average volume of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids that is within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%, or any volume within the 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 the volume of the average volume of the plurality of enteric endoderm aggregates and / or resulting aggregated organoids that is within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%, or any volume within the 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 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 enteric endoderm aggregates and / or resulting aggregated organoids, the multiple enteric 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 human. In some embodiments, the subject has a disease, previously has a disease, 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 enteric endoderm aggregates and / or resulting aggregated organoids derived from a subject can be used for genetic testing or drug screening purposes. In some embodiments, the multiple enteric endoderm aggregates and / or resulting aggregated organoids derived from a subject can be used in large-scale drug screening to identify effective therapies for alleviating, ameliorating, or treating a disease in the subject. In some embodiments, the large-scale drug screening comprises testing multiple compounds, each of which has a subpopulation of multiple enteric endoderm aggregates and / or resulting aggregated organoids.

[0126] Also disclosed herein are embodiments of an aggregation medium comprising a plurality of microwells and a plurality of gut endoderm aggregates. In some embodiments, the aggregation medium 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 are 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 are 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 Methods 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 human.In some embodiments, the recipient subject is the subject from which definitive endoderm or precursor pluripotent stem cells are derived.In some embodiments, 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, one or more aggregated organoids show better engraftment, maturation, proliferation, or any combination thereof compared with 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, or 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, for example, 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, dog, hamster, or 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 who has been 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 that needs an organ transplant. In some embodiments, the recipient subject is a human that needs an organ transplant.

[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 are in recipient subject, or are about these days, or are at least these days, or are at least about these days, or are less than these days, or are less than these days. In some embodiments, one or more aggregated organoids grow larger or mature faster than the in vitro aggregated organoids that are prepared at the same time.In some embodiments, one or more aggregated organoids show the 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 harmful 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 stomach organoids, one or more aggregated fundus organoids, one or more aggregated antral stomach organoids, one or more aggregated liver organoids, one or more aggregated small intestine (intestinal) 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 obtained from the subject or prepared from the induced pluripotent cells derived from the subject.In some embodiments, the subject needs 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, one or more aggregated organoids are used for the object that needs to restore organoid function.In some embodiments, one or more aggregated organoids are one or more aggregated organoids as described herein.In some embodiments, 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 the gut endoderm monolayer from the gut spheroid; dissociating the gut endoderm monolayer into a 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 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 separating step comprises aspirating growth medium and suspended gut spheroids from the gut endoderm monolayer.In some embodiments, the separating 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 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 organoids may comprise, at or about, at least, at least about, less than, or 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, the culturing step comprises contacting one or more enteric endoderm aggregates with an extracellular matrix, or a mimetic or derivative thereof. In some embodiments, the extracellular matrix, or a mimetic or derivative thereof, comprises Matrigel. In some embodiments, the enteric endoderm monolayer is a foregut endoderm monolayer, and the enteric spheroids are foregut spheroids. 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 antrum stomach organoids.In some embodiments, intestinal endoderm monolayer is hindgut endoderm monolayer, and 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 colon 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 the gut endoderm monolayer from the gut spheroids; dissociating the gut endoderm monolayer into a 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 separating step comprises aspirating growth medium and suspended gut spheroids from the gut endoderm monolayer.In some embodiments, the dissociating step comprises enzymatically dissociating the gut endoderm monolayer.In some embodiments, the gut endoderm monolayer is enzymatically dissociated by Accutase.In some embodiments, the aggregating 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 enteric endoderm aggregates comprises, or is about, or at least about, or less than, or about less than 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 enteric endoderm cells, or any number of enteric endoderm cells within a range defined by any two of the foregoing numbers, e.g., 1,000-5,000 cells, 2,000-4,000 cells, 1,000-3,000 cells, or 3,000-5,000 enteric endoderm cells. In some embodiments, the culturing step comprises contacting the one or more enteric endoderm aggregates with Matrigel. In some embodiments, intestinal endoderm monolayer is foregut endoderm monolayer, and intestinal spheroid is foregut spheroid.In some embodiments, one or more aggregated organoid is or comprises one or more aggregated liver organoid.In some embodiments, one or more aggregated organoids are or comprise one or more aggregated gastric organoids.In some embodiments, one or more gastric organoids are or comprise one or more aggregated antrum gastric organoids.In some embodiments, intestinal endoderm monolayer is hindgut endoderm monolayer, and 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.

[0134] In some embodiments of any of the methods disclosed herein, the gut endoderm monolayer is a foregut endoderm monolayer, and the gut spheroids are foregut spheroids. In some embodiments, differentiating the definitive endoderm into a foregut endoderm monolayer and a foregut spheroid 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. 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, the one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors are each contacted at a concentration that is, about, at least about, less than, or 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, one or more Wnt signaling pathway activators include CHIR99021. In some embodiments, CHIR99021 is provided at a concentration of 3 μM or about 3 μM. In some embodiments, 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, 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, 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 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 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, 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 a concentration that is about, 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, 1 to 1000 ng / mL, 50 to 500 ng / mL, 500 to 1000 ng / mL, or 1 to 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, when provided, is contacted at a concentration that is, about, at least about, less than, or 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 antrum gastric organoids.In some embodiments, culturing one or more enteric endoderm aggregates to form one or more aggregated antrum 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 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 1000 ng / mL, or about 1000 ng / mL, or at least about 1000 ng / mL, or at 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, EGF, retinoic acid, or one or more BMP signaling pathway inhibitors are each 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 a concentration of 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, 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 spheroids are foregut spheroids. 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, each of the one or more FGF signaling pathway activators, or one or more Wnt signaling pathway activators, or both, 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, 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, 11000, 12000, 13000, 14000, 150 The contact may be at a concentration of, about, at least about, less than, or equal to 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, one or more FGF signaling pathway activators or one or more Wnt signaling pathway activators, or both, are 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 1 μM, or at least about 1 μM, or at most about 1 μM, or at 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 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 the 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 colon organoids, or both.

[0138] In some embodiments of any of the methods disclosed herein, one or more aggregated organoids are or comprise 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 contact is performed at a concentration of, about, at least about, less than, or equal to 900, 950, or 1000 ng / mL, 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, EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway inhibitors are each 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 a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, 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 comprise one or more aggregated colonic organoids.In some embodiments, culturing one or more intestinal endoderm aggregates to form one or more aggregated intestinal organoids comprises making one or more intestinal endoderm aggregates contact 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 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, 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, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, , 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, EGF, one or more Wnt signaling pathway activators, or one or more BMP signaling pathway activators are each 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 1 μM, or at least about 1 μM, or at most about 1 μM, 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 comprise BMP2. In some embodiments, BMP2 is provided at a concentration of 100 ng / mL or about 100 ng / mL.

[0141] In some embodiments of any of the methods disclosed herein, the intestinal spheroids are dissociated and suspended in a growth medium. In some embodiments of any of the methods disclosed herein, the definitive endoderm is differentiated from pluripotent stem cells. In some embodiments of any of the methods disclosed herein, the definitive endoderm is differentiated from embryonic stem cells or induced pluripotent stem cells. 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, method further comprises transplanting one or more aggregated organoids into recipient subject.In some embodiments, recipient subject is mammal.In some embodiments, recipient subject is human.

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

[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 recognize that many other embodiments are also within the scope of the disclosure, as described herein and in the claims.

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

[0146] hPSCs were cultured two days (day -2) before differentiation into definitive endoderm. 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 differentiated areas were removed from undifferentiated hPSCs. The medium from each well containing hPSCs was carefully aspirated. 1 mL of prewarmed dispase solution was added to each well containing hPSCs, and the cells were incubated at 37°C until the edges of the colonies appeared slightly folded back. After approximately 4 minutes of dispase incubation, the edges of the hPSC colonies were observed to have begun to lift from the wells. If the edges of the colonies had not lifted, 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. The Matrigel-coated dishes were not allowed to dry out at any time. As needed, 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 prewarmed DMEM-F12 medium. It was important to prevent the plate from drying out and to avoid dislodging colonies 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 separated 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 dispensed into each well of a Matrigel-coated 24-well plate. The newly 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 day 0 of culture, plated hPSCs (e.g., as described in Example 1) were uniformly distributed at approximately 60-70% confluence and were confirmed to exhibit typical undifferentiated morphology before initiating differentiation. Taking care not to allow the cells to dry out, the mTeSR1 medium in each well was aspirated. 0.5 mL of day 1 DE differentiation medium (Table 1) was added per well. The cells were incubated at 37°C and 5% CO2 for 24 hours.

[0148] On day 1 of culture, the 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% CO for 24 hours.

[0149] On day 2 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. Cells were incubated at 37°C and 5% CO for 24 hours.

[0150] The recipe for DE differentiation medium provided in Table 1 is for making 1 mL of medium. The volume 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 it is preferable to prepare them 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 presence of a flat, homogenous cell 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 hours.

[0152] On day 4 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% CO 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 at 37°C and 5% CO2 for 24 hours. At this stage, the beginning of morphogenesis can be observed. There may be several isolated spheroids in the well. Care was taken to avoid aspirating 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 hours. At this stage, clear morphogenesis was observed. There may have been some isolated spheroids in the well. Care was taken to avoid aspirating 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. The volume can be scaled up as needed. [Table 2]

[0157] Example 4. Existing spheroid protocols introduce variability Figure 7A shows a schematic diagram of the existing spheroid production directed toward the formation of HIOs, as generally described in Examples 1-3. Briefly, 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 and spontaneous spheroid production occur. On day 7, dissociated spheroids are collected from the HGE monolayer, embedded in Matrigel, and cultured for 28 days in 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin. 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 evaluated as successful (more than 50 isolated spheroids / well), intermediate (fewer than 50 isolated spheroids / well), or unsuccessful (no isolated spheroids). Scoring was performed by a single individual. As shown in Figure 7B, there is a large variability in spheroid formation 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 evaluated using existing protocols. Eight wells per cell line were plated and subjected to differentiation simultaneously. On day 7, the number of dissociated spheroids per well was counted and images of each well were captured. As shown in Figure 7C, spheroid production varied among PSC cell lines, impacting personalized medicine applications. Example images of spheroid production using different PSC lines are shown in Figure 7D. The number of dissociated spheroids in each well is indicated in the upper left corner of each condition. Strong morphogenesis was observed, along with line-to-line variability in the number of dissociated spheroids, although a lack of spheroid dissociation was observed in some wells.

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

[0161] Aggrewell 400 plates were prepared. Each well of the 24-well Aggrewell 400 plate can produce up to 1200 aggregates. The following is for a single well of an Aggrewell 400 plate. The amounts can be scaled up to prepare a sufficient number of wells / aggregates. 500 μL of anti-adhesion rinse (StemCell Technologies) was added to the wells of the Aggrewell plate. The plate was centrifuged at 1300 × g for 5 minutes 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 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 (Table 4) supplemented with 10 μM Y-27632 was added to the well. The Aggrewell was stored in a 37°C incubator until further use.

[0162] A single-cell suspension of HGE cells was prepared. The medium and spheroids isolated from the HGE endoderm tissue culture were discarded. 0.5 mL of prewarmed 37°C Accutase was added to each well, and the plate was incubated at 37°C for approximately 5–10 minutes. The plate was monitored under a microscope to ensure 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 replaced 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 as single cells to a 15 mL centrifuge tube. 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 The desired number of cells was transferred to a new centrifuge tube and centrifuged at 300 × g for 5 minutes. The supernatant was evacuated, and the cell pellet was resuspended in 1 mL of prewarmed 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 evenly distribute the cells throughout the wells. The Aggrewell plate was centrifuged at 100 × g for 3 minutes to capture the cells within the microwells. The plate was examined under a microscope to ensure that the cells were evenly distributed throughout the microwells. The Aggrewell plate was returned to the incubator overnight.

[0163] The recipes for intestine-based medium and complete Sato medium provided in Tables 3 and 4 are for making 50 mL of medium. Volumes can be scaled up as needed. Intestine-based medium 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 pipette tip. The collected aggregates were transferred to a sterile 15 mL centrifuge tube. To remove any remaining aggregates in the well, 1 mL of preheated 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, as 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 solution was gently pipetted up and down, avoiding the formation of air bubbles, to uniformly resuspend and mix the aggregates. 50 μL of the Matrigel / aggregate mixture was added to the center of each well of a 24-well tissue culture-treated plate. Care was taken to keep the Matrigel in a single drop to prevent it from touching the sides of the well, which could cause the Matrigel to flatten and result in spheroids adhering to the plastic. This plating process was repeated until the entire Matrigel / aggregate volume was plated. To prevent the spheroids from settling on the surface of the tissue culture plate, the plate was quickly but carefully inverted upside down. To promote Matrigel polymerization, the plate was transferred to a 37°C incubator for 20 minutes. Subsequently, 0.5 mL of prewarmed 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 tissue As a control, 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. Briefly, human pluripotent stem cells were first exposed to 100 ng / mL activin A for 3 days to produce DE. The DE was 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 occurred. On day 7, a single-cell suspension of HGE was prepared, regardless of the presence of isolated spontaneous spheroids, and subjected to 24 hours of aggregation using Aggrewell plates. The aggregates were then harvested from the microwells, embedded in Matrigel, and cultured for 28 days in 500 ng / mL EGF, 100 ng / mL Noggin, and 500 ng / mL R-spondin. On day 35, aggregated human intestinal organoids (aggHIOs) were 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 magnification (left column, scale bar = 500 µm) and 200x magnification (right column, scale bar = 100 µm). As seen in Figure 9B, uniform aggregation of gut endoderm cells is achievable in Aggrewell formation plates.

[0168] H1 hESCs were differentiated using a conventional non-aggregation protocol. After 7 days of differentiation, spontaneously generated 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 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 generated from the non-dissociated monolayer using the aggregation method were observed.

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

[0170] Spontaneously separated (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 intestinal endoderm aggregates exhibit 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 HGEs. On day 7, dissociated spontaneous spheroids were directly embedded in Matrigel, and undissociated cells were allowed to aggregate for 24 hours 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 evaluated. As shown in Figure 10A, both conditions were able to generate organoid precursors that proliferated and matured appropriately.

[0172] Spontaneously separated 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, while non-dissociated HGE were allowed to dissociate and aggregate for 24 hours before being embedded in Matrigel. On days 18, 25, and 35, morphological analysis of the organoids showed that HGEs arising from both dissociated spheroids and aggregated HGEs exhibited 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 resulted in 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 the engrafted AggHIOs were excised and subjected to histological analysis by hematoxylin / eosin (H&E) staining. As shown in Figure 12A, the aggregated intestinal organoids were amenable to transplantation and underwent 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 of the formation of aggregated antral gastric organoids. A comparison of existing methods with the aggregation method is provided. Figure 13A, top: A method that relies on the spontaneous formation of antral organoids (e.g., as seen in McCracken et al. Nature. (2014) 516(7531):400-4). Figure 13A, bottom: An 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 the gastric epithelial markers CLDN18 and MUC5AC. The 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 colon organoids Figure 14A shows a schematic diagram of the formation of aggregated colon organoids. A comparison of existing methods with the aggregation method is provided. Top of Figure 14A: A method that relies on the spontaneous formation of colon organoids. Bottom of Figure 14A: An 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 toward 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. The CDH1+ epithelium of HCOs generated from spontaneous and aggregated hindgut endoderm displayed similar numbers of SATB2-positive cells (Figure 14C).

[0184] Example 10. Formation of aggregated liver organoids Figure 15 shows a schematic diagram for the formation of aggregated liver organoids. HLOs are generated from spontaneously isolated 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 Because conventional protocols for differentiating gut endoderm involve in vitro culture of definitive endoderm with minimal growth factors, 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. Therefore, 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 the mesoderm marker Brachyury (T) and the definitive endoderm marker FOXA2 (Figure 16A). The images show that the monolayers are largely composed of definitive endoderm, with little mesoderm. The mesoderm and definitive endoderm populations of these gut endoderm monolayers were quantified (Figure 16B). On average, both the foregut and hindgut endoderm monolayers contained 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. Because these cultures had progressed further in differentiation and gut patterning (including spontaneous spheroid formation), we examined mesenchymal cells rather than mesodermal cells. The monolayers were stained with the mesenchymal marker FOXF1 and the endodermal marker FOXA2, and a general increase in the mesenchymal fraction was observed (Figure 16C). The mesenchymal and endoderm populations were quantified (Figure 16D). The foregut endoderm monolayer contained 3% mesenchyme and 91% endoderm, while the hindgut endoderm monolayer contained 11% mesenchyme and 86% endoderm. As observed, there is a significant increase in the mesodermal / mesenchymal lineage after culturing the hindgut endoderm monolayer.

[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 the day 7 hindgut endoderm monolayer cultures compared to the 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 variations 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 ordinary 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, various singular / plural permutations may be expressly set forth 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 introduced claim recitations is intended, such intent will be expressly recited in the claim, and that in the absence of such recitation, 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 claim recitations. However, the use of such phrases should not be construed as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" means that any particular claim that includes the so-introduced claim recitation is limited 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"); the same applies to the use of definite articles used to introduce claim recitations. Additionally, those skilled in the art will recognize that when a specific number of introduced claim recitations is explicitly recited, such recitations should be construed to mean at least the recited number (e.g., the mere recitation of "two recitations" without any other modifiers means at least two recitations or more than two recitations).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such syntax is typically intended to mean how one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such syntax is typically intended to mean how one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). 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" would be understood to include the possibilities of "A" or "B" or "A and B."

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

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

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

[0195] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification supersedes and / or is intended to supersede 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; Culturing the one or more enteric endoderm aggregates to produce the one or more aggregated organoids.

2. The method of claim 1 , wherein the definitive endoderm is differentiated from a pluripotent stem cell.

3. The method of claim 1 or 2, wherein the definitive endoderm is differentiated from an embryonic stem cell or an induced pluripotent stem cell.

4. 10. The method of any one of the preceding claims, wherein said definitive endoderm is human definitive endoderm.

5. 10. The method of any one of the preceding claims, wherein the separating step comprises aspirating the growth medium and suspended intestinal spheroids from the intestinal endoderm monolayer.

6. 10. The method of any one of the preceding claims, wherein the dissociation step comprises enzymatically dissociating the intestinal endoderm monolayer.

7. 7. The method of claim 6, wherein the intestinal endoderm monolayer is enzymatically dissociated using Accutase, Accumax, trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express, or TrypLE Select, or any combination thereof.

8. 10. The method of any one of the preceding claims, 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.

9. The method of claim 8 , wherein the forming plate is an Aggrewell plate.

10. 10. The method of any one of the preceding claims, wherein 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 aforesaid cell numbers.

11. 10. The method of any one of the preceding claims, wherein the culturing step comprises contacting the one or more gut endoderm aggregates with an extracellular matrix, or a mimetic or derivative thereof.

12. 12. The method of claim 11, wherein the extracellular matrix, or a mimetic or derivative thereof, comprises Matrigel.

13. The method according to any one of claims 1 to 12, wherein the intestinal endoderm monolayer is a foregut endoderm monolayer and the intestinal spheroids are foregut spheroids.

14. 14. The method of claim 13, 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.

15. 15. The method of claim 14, wherein 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.

16. The method according to any one of claims 13 to 15, wherein the one or more aggregated organoids are aggregated liver organoids.

17. 17. The method of claim 16, 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.

18. 18. The method of claim 17, wherein the one or more FGF signaling pathway activators include FGF2, or the one or more BMP signaling pathway activators include BMP4, or both.

19. The method of any one of claims 13 to 15, wherein the one or more aggregated organoids are aggregated gastric organoids.

20. 20. The method of claim 19, wherein said one or more aggregated gastric organoids are aggregated antral gastric organoids.

21. 21. The method of claim 20, wherein culturing the one or more intestinal endoderm aggregates to form the one or more aggregate antral gastric organoids comprises contacting the one or more intestinal endoderm aggregates with EGF, retinoic acid, or one or more BMP signaling pathway inhibitors, or any combination thereof.

22. 22. The method of claim 21, wherein the one or more BMP signaling pathway inhibitors include Noggin.

23. The method according to any one of claims 1 to 12, wherein the intestinal endoderm monolayer is a hindgut endoderm monolayer and the intestinal spheroids are hindgut spheroids.

24. 24. The method of claim 23, 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.

25. 25. The method of claim 24, wherein the one or more FGF signaling pathway activators comprise FGF4, or the one or more Wnt signaling pathway activators comprise CHIR99021, or both.

26. 26. The method of any one of claims 23 to 25, wherein the one or more aggregated organoids are aggregated intestinal organoids.

27. 27. The method of claim 26, 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.

28. 28. The method of claim 27, wherein the one or more Wnt signaling pathway activators comprise R-spondin, or the one or more BMP signaling pathway inhibitors comprise Noggin, or both.

29. 26. The method of any one of claims 23 to 25, wherein the one or more aggregated organoids are aggregated colonic organoids.

30. 30. The method of claim 29, 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.

31. 31. The method of claim 30, wherein the one or more Wnt signaling pathway activators comprise R-spondin, or the one or more BMP signaling pathway activators comprise BMP2, or any combination thereof.

32. 10. The method of any one of the preceding claims, wherein the one or more intestinal 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 10,000 intestinal endoderm aggregates, or any number of intestinal endoderm aggregates within a number defined by any two of the aforesaid numbers of intestinal endoderm aggregates.

33. each of the one or more gut endoderm aggregates 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 any diameter within a range defined by any two of the foregoing diameters; or 10. The method of any one of the preceding claims, comprising 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, or any volume within a range defined by any two of the foregoing volumes, or both.

34. 10. The method of any one of the preceding claims, further comprising transplanting the one or more aggregated organoids into a recipient subject.

35. 35. The method of claim 34, wherein the recipient subject is a mammal.

36. 36. The method of claim 34 or 35, wherein the recipient subject is a human.

37. 37. One or more aggregated organoids produced by any one of claims 1 to 36.

38. 1. A plurality of enteric endoderm aggregates comprising at least 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 enteric endoderm aggregates, or any number within a number defined by any two of the aforementioned enteric endoderm aggregate numbers, wherein each of the plurality of enteric endoderm aggregates comprises: a diameter within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average diameter of said plurality of gut endoderm aggregates, or any diameter within a range defined by any two of the foregoing diameters; or A plurality of intestinal endoderm aggregates, comprising a volume within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the average volume of the plurality of intestinal endoderm aggregates, or any volume within a range defined by any two of the aforementioned volumes, or both.

39. 39. The multiple intestinal endoderm aggregates of claim 38, wherein the multiple intestinal endoderm aggregates are derived from the same subject.

40. A formation plate comprising a plurality of microwells and a plurality of intestinal endoderm aggregates as described in claim 38 or 39, wherein each of the plurality of microwells contains a single intestinal endoderm aggregate from the plurality of intestinal endoderm aggregates.

41. A plurality of intestinal endoderm aggregates according to claim 38 or a formation plate according to claim 40, wherein the plurality of intestinal endoderm aggregates are produced according to a method according to any one of claims 1 to 36.

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