JPWO2021030373A5 - - Google Patents

Description

Human pluripotent stem cells (hPSCs; including both embryonic and induced pluripotent stem cells) form human three-dimensional gastrointestinal tissues (e.g., human intestinal organoids) organized into distinct epithelial and mesenchymal layers; Represents a renewable resource for producing HIO). For example, in HIO, the epithelium contains all known intestinal epithelial cell types and exhibits several properties of functional intestinal tissue, including absorption, intestinal hormone synthesis, and mucosal secretion. After implantation into an experimental animal model, HIO undergoes significant proliferation and maturation to resemble the postnatal human intestine, including the mucosa, submucosa, and muscularis propria. Epithelial cells are arranged in the crypt villus structure, which contains the adult stem cell active/progenitor zone of the crypt, and in the mature epithelium capable of functions such as nutrient uptake and brush border enzymatic activity. As a result, pluripotent stem cell-derived organoids are physiologically relevant and powerful tools 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 disease, to generate disease/patient-specific organoids such as HIOs for personalized medical applications. Is possible. There is a continuing need for improved organoid compositions that more closely resemble in vivo tissue and methods of making organoid compositions that are more robust, scalable, faster and cost effective.
Prior art document information related to the invention of this application includes the following (including documents cited in the international phase after the international filing date and documents cited in the national phase of other countries).
(Prior art document)
(Patent document)
(Patent Document 1) US Patent Application Publication No. 2019/0093076
(Patent Document 2) US Patent Application Publication No. 2018/0258400
(Patent Document 3) US Patent Application Publication No. 2018/0043357
(Patent Document 4) US Patent Application Publication No. 2017/0202885
(Non-Patent Literature)
(Non-Patent Document 1) ARORA et al. "A process engineering approach to increase organoid yield," Development, 15 March 2017 (15.03.2017). Vol. 144, No. 6, Pgs. 1128-1136. whole document
(Non-Patent Document 2) MILLER et al. "Generation of lung organoids from human pluripotent stem cells in vitro," Nature Protocols. 28 February 2019 (28.02.2019), Vol. 14, No. 2, Pgs. 518-540. whole document

Claims (24)

A method of producing one or more aggregated organoids, comprising:
Differentiating definitive endoderm into an intestinal endoderm monolayer and intestinal spheroids,
differentiating, wherein said intestinal endoderm monolayer is adherent and said intestinal spheroids are separated and suspended in 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 intestinal endoderm cells into one or more intestinal endoderm aggregates;
culturing the one or more intestinal endoderm aggregates to produce the one or more aggregated organoids. 2. The method of claim 1 , wherein said separating step comprises aspirating said growth medium and suspended intestinal spheroids from said intestinal endoderm monolayer. 3. The method of claim 1 or 2 , wherein said dissociating step comprises enzymatically dissociating said intestinal endoderm monolayer. the aggregating step comprises aggregating the single cell suspension in a hanging drop; centrifuging the single cell suspension in a 'v' or 'u' bottomed microwell culture plate; orbital shaker. or centrifuging the single cell suspension in a forming plate, or any combination thereof. or the method described in paragraph 1. 5. The method of any one of claims 1-4 , wherein said culturing step comprises contacting said one or more intestinal endoderm aggregates with an extracellular matrix, or a mimetic or derivative thereof. 6. The method of any one of claims 1-5 , wherein the intestinal endoderm monolayer is a foregut endoderm monolayer and the intestinal spheroids are foregut spheroids. Differentiating said definitive endoderm into said foregut endoderm monolayer and said foregut spheroids comprises activating said definitive endoderm with one or more FGF signaling pathway activators, one or more Wnt signaling pathway activators 7. The method of claim 6 , comprising contacting with a factor or one or more BMP signaling pathway inhibitors, or any combination thereof. 8. The method of claim 6 or 7 , wherein the one or more aggregated organoids are aggregated liver organoids. Culturing said one or more gut endoderm aggregates to form said one or more aggregated liver organoids comprises treating said one or more gut endoderm aggregates with one or more FGF signaling pathway activators. , one or more BMP signaling pathway activators, retinoic acid, hepatocyte growth factor, dexamethasone, or oncostatin M, or any combination thereof. 8. The method of claim 6 or 7 , wherein the one or more aggregated organoids are aggregated gastric organoids. 11. The method of claim 10 , wherein the one or more aggregated gastric organoids are aggregated sinus gastric organoids. Culturing said one or more intestinal endoderm aggregates to form said one or more aggregated sinus gastric organoids comprises treating said one or more intestinal endoderm aggregates with EGF, retinoic acid, or one or more 12. The method of claim 11 , comprising contacting with a BMP signaling pathway inhibitor, or any combination thereof. 6. The method of any one of claims 1-5 , wherein the gut endoderm monolayer is a hindgut endoderm monolayer and the gut spheroids are hindgut spheroids. Differentiating said definitive endoderm into said hindgut endoderm monolayer and said hindgut spheroids is characterized by: 14. The method of claim 13 , comprising contacting with a pharmacolytic factor, or both. 15. The method of claim 13 or 14 , wherein the one or more aggregated organoids are aggregated intestinal organoids. Culturing said one or more gut endoderm aggregates to form said one or more aggregated intestinal organoids comprises culturing said one or more gut endoderm aggregates with EGF, one or more Wnt signaling pathway activity 16. The method of claim 15 , comprising contacting with a inhibitor, or one or more BMP signaling pathway inhibitors, or any combination thereof. 15. The method of claim 13 or 14 , wherein the one or more aggregated organoids are aggregated colon organoids. Culturing said one or more intestinal endoderm aggregates to form said one or more aggregated colon organoids comprises treating said one or more intestinal endoderm aggregates with EGF, one or more Wnt signaling pathway activity 18. The method of claim 17 , comprising contacting with a activator, or one or more BMP signaling pathway activators, or any combination thereof. said one or more gut endoderm aggregates is at least 100 , 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000; 9000 or 10000 gut endoderm aggregates , each of said one or more gut endoderm aggregates comprising :
±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% from the average diameter of said one or more gut endoderm aggregates, or within ±1% of the diameter , or
±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% from the mean volume of said one or more gut endoderm aggregates, or volume within ±1%, or
including both
A method according to any one of claims 1-18 . One or more aggregated organoids produced by any one of claims 1-19 . a plurality of gut endoderm aggregates comprising at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 gut endoderm aggregates, said plurality of gut endoderm aggregates each of the
±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ± from the mean diameter of said plurality of gut endoderm aggregates diameter within 1%, or ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3 from the mean volume of said plurality of gut endoderm aggregates A plurality of intestinal endoderm aggregates comprising %, volume within ±2%, or ±1%, or both. 22. The plurality of gut endoderm aggregates of claim 21 , wherein said plurality of gut endoderm aggregates are derived from the same subject. 23. A forming plate comprising a plurality of microwells and a plurality of gut endoderm aggregates according to claim 21 or 22 , wherein each of said plurality of microwells comprises a single of said plurality of gut endoderm aggregates. A forming plate containing intestinal endoderm aggregates. The plurality of gut endoderm aggregates of claim 21 or the formation of claim 23 , wherein said plurality of gut endoderm aggregates are produced according to the method of any one of claims 1-19 . plate.