Method for efficiently producing stomach organoid with self-organizing asymmetric pattern

Abstract

The invention relates to a method for efficiently producing a stomach-like organ with a self-organizing asymmetric pattern, and belongs to the technical field of organ culture. The invention provides a new application of the nerve cell precursor cell, the nerve cell precursor cell can promote posterior anterior intestine sphere differentiation to obtain a stomach organ with a self-organizing stomach fundus-antral pattern (free-antral pattern) characteristic, the structure and the cell lineage of the stomach organ are similar to those of an in-vivo stomach organ, and the stomach organ has better fidelity. The method provided by the invention is suitable for various human pluripotent stem cells, and can be used for efficiently producing asymmetric stomach organs.

Description

Technical Field

[0001] The present invention relates to a method for efficiently producing gastric organoids with self-organized asymmetric patterns, belonging to the technical field of organ culture. Background Art

[0002] One of the main methods for producing gastrointestinal organoids is to produce gastrointestinal organoids by directing differentiation of human pluripotent stem cells (hPSC) in vitro. In recent years, many articles, such as those cited in References 1 to 3, have reported on methods for producing gastric organoids. Figure 1 ) : hPSCs are first differentiated into definitive endoderm (DE) cells in a two-dimensional environment. These cells then bud to form endoderm spheroids associated with specific locations in the gastrointestinal tract. These spheroids are then cultured in a specific three-dimensional environment to form gastric organoids. However, existing methods cannot form gastric organoids with self-organizing asymmetric patterns and can only form fundic-like gastric organoids (FGO) or antral-like gastric organoids (AGO).

[0003] Human pluripotent stem cells can be differentiated into endoderm cells through in vitro culture, further budding to form spherical organoid precursors, and then, after three-dimensional culture, can form a variety of gastrointestinal organoids. Existing methods for producing gastrointestinal organoids only produce simple PFGs during the budding step, which can only produce fundus organoids (FGOs) or antral organoids (AGOs), but cannot produce asymmetric gastric organoids. Their fidelity is poor and they cannot effectively simulate gastric development in vivo.

[0004] References:

[0005] [1] KWMcCracken, EMCatá, CMCrawford, KLSinagoga, M.Schumacher, BERockich, Y.-H.Tsai, CNMayhew, JRSpence, Y.Zavros, JMWells. Modeling human development and disease in pluripotent stem-cell-derived gastricorganoids. Nature 2014, 516, 400.

[0006] [2] KWMcCracken, E.Aihara, B.Martin, CMCrawford, T.Broda, J.Treguier,

[0007] [3]TRBroda,KWMcCracken,JMWells.Generation of human antral and fundic gastric organoids from pluripotent stem cells.Nat.Protoc.2019,14,28. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] The purpose of the present invention is to provide a method for efficiently producing a gastric organoid model with a self-organized asymmetric pattern, specifically using neural cell precursor cells to promote the differentiation of gastric organoid precursor spheres into asymmetric gastric organoids, or using suspension culture to perform embryoid body culture to construct gastric organoid precursor spheres containing non-endodermal lineage-derived cells, and further induce their differentiation and maturation to obtain a new gastric organoid model with self-organized fundus-antral patterning characteristics.

[0010] Solutions for solving problems

[0011] [1] Use of neural cell precursor cells in the in vitro generation of asymmetric gastric organoids, wherein the neural cell precursor cells are used to promote the formation of asymmetric gastric organoids from gastric organoid precursors;

[0012] The asymmetric gastric organoid comprises gastric fundus tissue and gastric antrum tissue.

[0013] [2] The use according to [1], wherein the gastric organoid precursors are differentiated from pluripotent stem cells;

[0014] Optionally, the gastric organoid precursor comprises a spherical gastric organoid precursor; preferably, the spherical gastric organoid precursor comprises a posterior foregut spheroid;

[0015] Optionally, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells.

[0016] [3] The use according to [1] or [2], wherein the neural cell precursor cells can be added exogenously or generated endogenously;

[0017] Optionally, the neural cell precursor cells include neuroepithelial cells, neural crest cells, or multi-lineage cells having the potential to differentiate into neuroepithelial cells and / or neural crest cells.

[0018] [4] The use according to [3], wherein the exogenous addition is to contact neural cell precursor cells generated by in vitro culture with gastric organoid precursors.

[0019] [5] The use according to [3], wherein the endogenous generation is through suspension culture of pluripotent stem cells to generate neural cell precursors within differentiated gastric organoid precursors.

[0020] [6] A method for generating asymmetric gastric organoids in vitro, wherein the formation of asymmetric gastric organoids from gastric organoid precursors is promoted by neural cell precursor cells;

[0021] The asymmetric gastric organoid has gastric fundus tissue and gastric antrum tissue.

[0022] [7] The method according to [6], wherein the gastric organoid precursors are differentiated from pluripotent stem cells;

[0023] Optionally, the gastric organoid precursor comprises a spherical gastric organoid precursor; preferably, the spherical gastric organoid precursor comprises a posterior foregut spheroid;

[0024] Optionally, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells;

[0025] Optionally, the neural cell precursor cells include neuroepithelial cells, neural crest cells, or multi-lineage cells having the potential to differentiate into neuroepithelial cells and / or neural crest cells.

[0026] [8] The method according to [6] or [7], wherein the method comprises:

[0027] (A) culturing pluripotent stem cells adherently and differentiating them into gastric organoid precursors, and then contacting neural cell precursors with the gastric organoid precursors to form asymmetric gastric organoids; or,

[0028] (B) culturing the pluripotent stem cells in suspension in a growth medium to differentiate into gastric organoid precursors, wherein the gastric organoid precursors contain neural cell precursor cells;

[0029] Optionally, the suspension culture comprises culturing in a U-shaped well plate or a low-adhesion cell culture dish.

[0030] [9] The method according to [7] or [8], wherein the pluripotent stem cells are contacted with one or more of activin A, an agent that inhibits the BMP signaling pathway, an agent that activates the FGF signaling pathway, an agent that activates the WNT signaling pathway, and retinoic acid to form gastric organoid precursors.

[0031]

[10] The method according to [8] or [9], wherein the gastric organoid precursor is contacted with neural cell precursor cells or neural cell precursor cells are generated inside the gastric organoid precursor, and then contacted with one or more of EGF, an agent that activates the FGF signaling pathway, Noggin, and retinoic acid to form an asymmetric gastric organoid.

[0032] Effects of the Invention

[0033] The present invention provides a new use of neural cell precursor cells, which can promote the differentiation of posterior foregut spheroids to obtain gastric organoids with self-organized fundal-antral patterning characteristics. Their structure and cell lineage are similar to those of in vivo gastric organs and have better fidelity.

[0034] The method for preparing asymmetric gastric organoids provided by the present invention is universal and applicable to a variety of human pluripotent stem cells, and can efficiently produce asymmetric gastric organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The prior art process for producing gastric organoids is shown.

[0036] Figure 2 The fabrication process and morphology of a high-throughput U-shaped orifice plate are shown.

[0037] Figure 3 Demonstrates the establishment of a gastric organoid construction system with self-organizing fundus-antrum patterning.

[0038] (a) Schematic diagram of the asymmetric anterior-posterior structure of the early gastrointestinal tract during development; posterior foregut (PFG); fundus; antrum. (b) A workflow for constructing gastric organoids (gastroids) characterized by self-organizing fundal-antral patterning. (c) Brightfield images from the gastroid construction workflow, showing the morphological changes of the organoids during differentiation. I: Columnar epithelial spheres with large cavities; II: Pseudostratified epithelium with small cavities; III: Mesenchymal-like cells.

[0039] Figure 4Immunofluorescence staining of gastric development-specific markers was used to identify gastric organoids with fundus-antrum pattern characteristics.

[0040] Immunofluorescence staining of the collected gastric organoids for gastric development-specific markers (GATA4, SOX2, and PDX1) revealed that the gastoroids obtained using the embryoid body method expressed GATA4 and SOX2, and their PDX1 expression was asymmetric along the anterior-posterior axis, indicating a fundus-antrum pattern. Traditional methods can only produce FGOs (GATA4+ / SOX2+ / PDX1-) or AGOs (GATA4+ / SOX2+ / PDX1+).

[0041] Figure 5 Cell lineage identification of gastric organoids with fundus-antrum patterning characteristics.

[0042] Immunofluorescence staining of the collected gastric organoids revealed that in addition to gastric epithelium, gastorids also contained neural epithelium (NE), neural crest cells (ENCC) (a) and interstitial cells (b).

[0043] Figure 6 To construct gastric organoids with fundus-antrum patterning characteristics using different cell lines.

[0044] (a) shows that the embryoid body method can generate gastric organoids (gastroids) for both H1 and iPSB1 cell lines. Immunofluorescence staining identifies the asymmetry of the gastric epithelium, and (b) and (c) show that the generated gastroids also contain neural epithelium and neural crest cells.

[0045] Figure 7 To construct an assembly using PFG and NE recombination experiments.

[0046] (a) Schematic diagram of the gastric assembloid construction process using PFG and NE recombination. (b) Brightfield images of the gastric assembloid construction process, showing the morphological changes of the organoid during differentiation. (c) Immunofluorescence staining of a gastric assembloid on day 16 of differentiation, showing anterior-posterior asymmetry of PDX1 expression.

[0047] Figure 8 The mesogastroduodenal precursors generated by traditional technology were compared with those constructed by suspension culture. The gastric organoid precursor spheres constructed by suspension culture contained an inner cell mass that could differentiate into non-endodermal cell lineages.

[0048] Figure 9 The cell lineages of multi-lineage gastric organoids are shown. DETAILED DESCRIPTION

[0049] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0050] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0051] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0052] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0053] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0054] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0055] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0056] As used herein, the term "differentiation" describes the process by which a less specialized cell becomes a specific specialized target cell type.

[0057] The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or change the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0058] As used herein, "in vitro" is given its ordinary and customary meaning as understood in light of this specification and refers to methods performed outside of biological conditions, such as in a culture dish or test tube.

[0059] In this specification, embryoid bodies (EBs) are three-dimensional cell aggregates spontaneously formed by pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells, iPSCs) under in vitro culture conditions.

[0060] In this specification, the term "multi-lineage cell" refers to a cell population with the ability to differentiate into multiple different lineage cell types. Such cells can differentiate into cell types of multiple lineages (such as mesoderm, endoderm and ectoderm) under appropriate induction conditions. In some embodiments, the multi-lineage cells can differentiate into mesodermal cell lineages and / or ectoderm cell lineages. In some exemplary embodiments, the ectoderm cell lineage includes cell types such as neural stem cells, epithelial cells, brain, spinal cord, peripheral nerves. In other exemplary embodiments, the mesodermal cell lineage includes cell types such as mesenchymal stem cells, fat cells, muscle cells, bone cells and hematopoietic stem cells.

[0061] The scheme of the present invention is described in detail below:

[0062] The inventors unexpectedly discovered that after suspension culture of pluripotent stem cells, such as human embryonic stem cells, the posterior foregut spheres (PFGs) into which pluripotent stem cells, such as human embryonic stem cells, differentiated after suspension culture contained multi-lineage cells (i.e., inner cell mass) that could differentiate into non-endodermal cell lineages including neural cell precursor cells. Further differentiation and culture of the posterior foregut spheres (PFGs) containing these multi-lineage cells could form asymmetric gastric organoids comprising fundus tissue and antrum tissue. Based on this, the inventors further generated PFGs using traditional culture methods and cultured them together with neural cell precursor cells to generate asymmetric gastric organoids. This further confirmed that neural cell precursor cells play a crucial role in the formation of asymmetric gastric organoids in vitro.

[0063] <First Aspect>

[0064] Based on the above, a first aspect of the present invention provides the use of neural cell precursor cells in generating asymmetric gastric organoids in vitro; specifically, neural cell precursor cells are used to promote gastric organoid precursors to form asymmetric gastric organoids.

[0065] In some embodiments, the asymmetric gastric organoid comprises fundus tissue and antrum tissue. The asymmetric gastric organoid expresses GATA4 and SOX2, and also has PDX1 asymmetrically expressed along the anterior-posterior axis.

[0066] Neural progenitor cells

[0067] In some embodiments, the neural cell precursor cells are cells with differentiation potential that can differentiate into a variety of neural cell types. These precursor cells play a key role in the development of the nervous system and provide a basis for the generation of neurons and glial cells. In the present invention, the neural cell precursor cells include neuroepithelial cells (NE) and neural crest cells (ENCC).

[0068] Neuroepithelial cells are the earliest cells in the development of the nervous system, originating from the ectoderm of the embryo. These cells proliferate and differentiate to form the walls of the neural tube and are the precursors of the central nervous system (CNS).

[0069] The term "neural crest cells" refers to a type of migratory, multipotent stem cell unique to vertebrates that can differentiate into a variety of cell types, including melanocytes, craniofacial cells, chondrocytes, osteocytes, smooth muscle cells, central and peripheral neurons, and glial cells. These cells are formed during embryonic development and are located between the neural tube and the epidermis as a longitudinal band of cells. They have a high differentiation potential and can differentiate into multiple cell types to form various tissues and organs of the body. In some specific embodiments of the present invention, the neural crest cells include enteric neural crest cells.

[0070] In other embodiments, the neural cell precursor cells further include multi-lineage cells having the potential to differentiate into neuroepithelial cells and / or neural crest cells.

[0071] In some optional embodiments, the neural cell precursor cells have the function of promoting the differentiation of gastric organoid precursors into asymmetric gastric organoids, and all neural cell precursor cells with the same or similar functions can also be included in the scope of protection of the present invention.

[0072] In some embodiments, the neural cell precursor cells can be added exogenously or generated endogenously.

[0073] In some exemplary embodiments, the exogenous addition is to culture neural cell precursor cells in vitro, and then contact the neural cell precursor cells with the gastric organoid precursors.

[0074] In other exemplary embodiments, the endogenous generation refers to culturing pluripotent stem cells in suspension to generate neural cell precursors within differentiated gastric organoid precursors.

[0075] Gastric organoid precursors

[0076] In some embodiments, the gastric organoid precursors are differentiated from pluripotent stem cells.

[0077] "Pluripotent stem cells (PSCs)" include any cell that can differentiate into almost any cell type of the body, i.e., cells derived from any of the three germ layers (germline epithelium), including the endoderm (interior stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital) and ectoderm (epidermal tissue and nervous system). PSCs can be descendants of inner cell mass cells of a primplantation blastocyst, or obtained by inducing non-pluripotent cells (e.g., adult somatic cells) by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source, as will be readily understood by those skilled in the art. Examples of pluripotent stem cell sources include mammalian sources, including humans, rodents, pigs, cattle, but are not limited thereto. Pluripotent stem cells typically include embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells.

[0078] "Embryonic stem cells (ESCs), also often abbreviated as ES cells, refer to cells that are pluripotent and derived from the inner cell mass of a blastocyst (early embryo). In some exemplary embodiments, the embryonic stem cells include human embryonic stem cells, such as the H1 cell line.

[0079] "Induced pluripotent stem cells (iPSCs), also commonly abbreviated as iPS cells, have their simple and common meaning as understood in the specification, and are pluripotent stem cells with similar embryonic stem cell properties that are converted into differentiated somatic cells (such as skin cells, blood cells, etc.) through genetic reprogramming technology. In some methods known in the art, iPSCs are obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Induced pluripotent stem cells include, for example, the iPS18 cell line and the iPSB1 cell line.

[0080] In some embodiments, the gastric organoid precursor comprises a spheroid gastric organoid precursor; preferably, the spheroid gastric organoid precursor comprises a posterior foregut spheroid. In some alternative embodiments, the foregut spheroid comprises an inner cell mass of a non-endodermal cell lineage, wherein the non-endodermal cell lineage comprises a mesodermal cell lineage and / or an ectodermal cell lineage.

[0081] <Second Aspect>

[0082] A second aspect of the present invention provides a method for generating asymmetric gastric organoids in vitro, wherein the method comprises promoting gastric organoid precursors to form asymmetric gastric organoids by using neural cell precursor cells.

[0083] In some embodiments, the asymmetric gastric organoid comprises gastric fundus tissue and gastric antrum tissue.The asymmetric gastric organoid expresses GATA4 and SOX2, and also has PDX1 asymmetrically expressed along the anterior-posterior axis.

[0084] In some embodiments, the gastric organoid precursor is differentiated from a pluripotent stem cell. In some optional embodiments, the gastric organoid precursor comprises a spheroid gastric organoid precursor; and in some preferred embodiments, the spheroid gastric organoid precursor comprises a posterior foregut spheroid.

[0085] In some embodiments, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells.

[0086] In some specific embodiments, the method comprises: culturing pluripotent stem cells adherently and differentiating them into gastric organoid precursors, and then contacting the gastric organoid precursors with neural progenitor cells to form asymmetric gastric organoids. Exemplarily, the neural progenitor cells include neuroepithelial cells and / or neural crest cells.

[0087] In other specific embodiments, the method comprises: culturing pluripotent stem cells in suspension in a growth medium to differentiate into gastric organoid precursors, wherein the gastric organoid precursors contain neural progenitor cells. Exemplarily, the neural progenitor cells include multi-lineage cells with the potential to differentiate into neuroepithelial cells and / or neural crest cells.

[0088] The culture dishes used for suspension culture can be commercial culture dishes commonly used in the art or homemade culture dishes, as long as the cells can be suspended. In some exemplary embodiments, the suspension culture can be cultured in U-shaped well plates or low-adsorption cell culture dishes, such as commercial ultra-low adsorption culture dishes (such as Corning, Greiner, BeyoGold TM ), or using PDMS or agarose homemade low-adsorption surfaces for suspension culture.

[0089] In some embodiments, embryonic stem cells or pluripotent stem cells are contacted with one or more of activin A, an agent that inhibits the BMP signaling pathway, an agent that activates the FGF signaling pathway, an agent that activates the WNT signaling pathway, and retinoic acid (RA) to form gastric organoid precursors.

[0090] The agent for inhibiting the BMP signaling pathway includes BMP4 and / or Noggin; the BMP4 is provided at a concentration of 20 to 100 ng / mL; and the Noggin is provided at a concentration of 100 to 500 ng / mL.

[0091] The agent that activates the FGF signaling pathway includes FGF4 and is provided at a concentration of 200 to 800 ng / mL.

[0092] The agent that activates the WNT signal transduction pathway includes CHIR99021 and is provided at a concentration of 1 to 5 μM.

[0093] The RA is provided at a concentration of 1 to 5 μM, and the activin A is provided at a concentration of 50 to 150 ng / mL.

[0094] In some embodiments, the neural cell precursor cells are differentiated from stem cells after contact with LDN193189, an agent that inhibits the BMP signaling pathway, and SB431542, an agent that inhibits the TGF-β signaling pathway.

[0095] The agent that inhibits the BMP signaling pathway includes LDN193189 and is provided at a concentration of 0.01 to 0.5 μM. The agent that inhibits the TGF-β signaling pathway includes SB431542 and is provided at a concentration of 5 to 15 μM.

[0096] Furthermore, after the gastric organoid precursors are contacted with neural cell precursor cells, they are contacted with one or more of EGF, an agent that activates the FGF signaling pathway, Noggin, and retinoic acid (RA) to form asymmetric gastric organoids.

[0097] In some embodiments, EGF is provided at a concentration of 50-150 ng / mL, Noggin is provided at a concentration of 100-500 ng / mL, and RA is provided at a concentration of 1-5 μM. In some embodiments, the agent that activates the FGF signaling pathway includes FGF10 and is provided at a concentration of 20-100 ng / mL.

[0098] Example

[0099] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0100] Experimental Materials:

[0101] mTeSR1 was purchased from Stem Cell, catalog number 85850

[0102] Y-27632 was purchased from TargetMol, product number T1870

[0103] RPMI1640 was purchased from Gibco, catalog number 11875093

[0104] NEAA (Non-Essential Amino Acids) was purchased from Gibco, catalog number 11140050

[0105] Activin A was purchased from Peprotech, product number 120-14E

[0106] BMP4 (Bone Morphogenetic Protein 4) was purchased from Peprotech, catalog number 120-05ET

[0107] SB431542 was purchased from selleck, item number S1067

[0108] LDN193189 was purchased from selleck, item number S2618

[0109] dFBS (Defined Fetal Bovine Serum) was purchased from Hyclone, catalog number SH30070

[0110] FGF4 (Fibroblast growth factor 4) was purchased from Peprotech, catalog number 100-31CHIR99021 was purchased from Sigma, catalog number SML1046-5MG

[0111] Noggin was purchased from Peprotech, product number 120-10C

[0112] RA (Retinoic acid) was purchased from Sigma, product number R2625

[0113] EGF (Epidermal growth factor) was purchased from Peprotech, catalog number AF-100-15

[0114] FGF10 (Fibroblast growth factor 10) was purchased from Peprotech, catalog number 100-26

[0115] N2 culture supplement was purchased from INVITROGEN, product number 17502048

[0116] B27 culture supplement was purchased from INVITROGEN, product number 17504044

[0117] GlutaMAX supplement was purchased from Gibco, catalog number 35050061

[0118] Penicillin–streptomycin (penicillin–streptomycin mixture) was purchased from Gibco, product number 15070063

[0119] DMEM / F-12 was purchased from Gibco, catalog number C11330500BT.

[0120] Advanced DMEM / F-12 was purchased from Gibco, catalog number 12634010

[0121] Neurobasal medium was purchased from Gibco, catalog number 21103049

[0122] HEPES buffer was purchased from Gibco, catalog number 5630080

[0123] Dispase was purchased from Stem Cell, product number 7923

[0124] Accutase was purchased from Gibco, product number A1110501

[0125] Geltrex was purchased from Gibco, catalog number A1413302

[0126] Cells used in subsequent examples:

[0127] Human embryonic stem cells H1 (WA01; WiCell; NIH registration number: 0043) were purchased from ATCC

[0128] Induced human pluripotent stem cells iPS-18 (Y00300) were purchased from TaKaRa, and iPS-B1 (CA4025106) were purchased from CELLapy

[0129] 1. High-throughput U-shaped well plate production

[0130] (1) Mold preparation. An aluminum mold consisting of an array of hemispherical micropillars was carved using a high-precision lathe. The diameter and height of the micropillars were both 400 μm.

[0131] (2) Preparation of U-shaped well plates. Pour a 2% agarose solution (w / v, distilled water) into an aluminum mold, cool and solidify at room temperature, and then carefully peel it from the mold. Before use, cut the U-shaped bottom agarose microwell array into a size that fits the culture area of a 24-well plate. Sterilize the U-shaped bottom agarose microwell array in PBS with ultraviolet light for at least 30 minutes before seeding cells.

[0132] Preparation process as Figure 2 As shown in a, its shape is Figure 2 As shown in b.

[0133] 2. Culture medium required during the culture process:

[0134] Table 1

[0135]

[0136] *N2B27 (Neural Induction Medium (N2B27)): Advanced DMEM / F-12 (12634010, Gibco); Neural Basal Medium

[0137] (21103049, Gibco) (1:1), 0.5×N2 culture supplement, 0.5×B27 culture supplement, 1% non-essential amino acids, 2mM

[0138] GlutaMAX, and 0.1 mM β-mercaptoethanol.

[0139] *Gut medium: Advanced DMEM / F-12 + 1× N2 culture supplement + 1× B27 culture supplement (without vitamin A) + 2 mM GlutaMAX + 100 units / ml (1×) penicillin-streptomycin mixture + 15 mM HEPES.

[0140] Example 1

[0141] Taking asymmetric gastric organoids as an example, the construction process is as follows Figure 3 As shown in b, the culture medium used is shown in Table 1. After the cultured human pluripotent stem cells reached 80% confluence, the pluripotent stem cells were digested into single cells, resuspended in culture medium (the culture medium was mTeSR1, and 10 μM Y-27632 was added), and suspended in a U-shaped well plate. On the second day (after 24 hours of suspension culture), fresh culture medium was replaced for the endoderm cell differentiation process and Y-27632 was removed. On the third day (after 48 hours of suspension culture), the embryoid body suspension was added dropwise to a culture dish pre-coated with 1% Geltrex for adherent culture. On the eighth day, budding gastric organoid precursor spheres containing non-endodermal lineage-derived cells were obtained, and their differentiation and maturation were further induced to obtain asymmetric gastric organoids.

[0142] The specific steps are as follows:

[0143] Prior to day 0, human pluripotent stem cells (iPS18) were cultured using a feeder-free method. The culture medium used was mTeSR1. During passaging, cells were digested with Dispase and passaged as cell clumps. Culture plates were coated with 1% Geltrex solution (in DMEM / F-12 medium) at 37°C for 1 hour.

[0144] Day 0. Differentiation was initiated by digesting human pluripotent stem cells with Accutase into single cells, centrifuging and resuspending in mTeSR1 + 10 μM Y-27632 solution. 3×10 cells were plated per well. 5 The cells were added into the 24-well high-throughput U-shaped well plate prepared above.

[0145] Days 1-3: Differentiation toward endoderm. Change 500 μL of culture medium daily. On day 1, human pluripotent stem cells form EB spheres. Aspirate the culture medium from a 24-well high-throughput U-shaped plate and replace with Day 1 culture medium (RPMI1640 + NEAA + 100 ng / mL Activin A + 50 ng / mL BMP4). On day 2, coat the 24-well plate with 1% Geltrex solution (solvent: DMEM / F-12 medium) (37°C, 1 hour). Resuspend the EB spheres in 500 μL of Day 2 culture medium (RPMI1640 + NEAA + 100 ng / mL Activin A + 0.2% dFBS) per well and seed the plate into the coated 24-well plate. On day 3, replace with RPMI1640 + NEAA + 100 ng / mL Activin A + 2% dFBS.

[0146] Days 4-6: Further endoderm differentiation. Change 500 μL of culture medium daily. On days 4-5, change to RPMI1640 + NEAA + 2% dFBS + 200 ng / mL Noggin + 500 ng / mL FGF4 + 2 μM CHIR99021; on day 6, change to RPMI1640 + NEAA + 2% dFBS + 200 ng / mL Noggin + 500 ng / mL FGF4 + 2 μM CHIR99021 + 2 μM RA. On day 7, harvest the gastric organoid precursors. Aspirate the culture medium into a centrifuge tube and let it sit for 15 minutes. The organoid precursors will settle to the bottom of the tube. Carefully aspirate approximately 2 μL of the cell spheres and quickly mix with 18 μL of Geltrex. Drop the Geltrex mixed with cell spheres in the center of the bottom of a 48-well plate, let it stand at 37°C for 5-15 minutes to solidify, then add culture medium (basal intestinal culture medium + 100 ng / mL EGF + 50 ng / ml FGF10 + 200 ng / mL Noggin + 2 μM RA) and culture until the 10th day.

[0147] On days 10-12, the culture medium (basal intestinal medium + 100 ng / mL EGF + 50 ng / ml FGF10) was changed every 2-4 days.

[0148] From day 13 to day 15, the culture medium (basal intestinal medium + 100 ng / mL EGF) was changed every 2-4 days.

[0149] On the 7th day of culture, gastric organoid precursors were collected and immunofluorescence staining of endoderm-specific markers FOXA2 and GATA4 was performed. Figure 8 As shown, multi-lineage gastric organoid precursors have a FOXA2- / CDH1- inner cell mass compared with gastric organoid precursors obtained by traditional methods.

[0150] The morphology of gastric organoids during differentiation was observed on days 1, 2, 4, 7, 10, 13, and 16 of culture. Figure 3 As shown in c, multilineage gastric organoids gradually develop and contain stratified tissues (II) and mesenchymal-like cells (III) in addition to gastric epithelium (I), indicating that they have multiple cell lineages.

[0151] Gastric organoids were collected on day 16 and subjected to immunofluorescence staining for gastric development-specific markers (GATA4, SOX2, and PDX1). Figure 4 As shown in Figure 2, the gastric organoids generated by the method of this example expressed GATA4 and SOX2, and their PDX1 expression was characterized by anterior-posterior asymmetry, that is, they had a fundus-antral pattern. At the same time, it was also found that the gastric organoids had neuroepithelial, neural crest cells and mesenchymal cells ( Figure 5).

[0152] On day 16, gastric organoids were collected for single-cell sequencing analysis and compared with the relevant data in the previously published literature (Yu, Q. et al. Charting human development using a multi-endodermal organ atlas and organoid models. Cell. 2021 Jun 10; 184(12): 3281-3298.e22.). The results are as follows Figure 9 As shown, the cell lineages of multilineage gastric organoids are similar to those of in vivo gastric organoids.

[0153] Example 2

[0154] Taking the asymmetric gastric organoid assembly as an example, the construction process is as follows Figure 7 The culture medium used is shown in Table 1:

[0155] Prior to day 0, human pluripotent stem cells (iPS18) were cultured using a feeder-free method. The culture medium used was mTeSR1. During passaging, cells were digested with Dispase and passaged as cell clumps. Culture plates were coated with 1% Geltrex solution (in DMEM / F-12 medium) at 37°C for 1 hour.

[0156] First, differentiate PFG and NE separately:

[0157] ①PFG differentiation process:

[0158] Day 0. Coat a 24-well plate with 1% Geltrex solution (solvent: DMEM / F-12 medium) (37°C, 1 hour). Aspirate the Geltrex solution and add 500 μL of day 0 medium (mTeSR1 + 10 μM Y-27632). Digest human pluripotent stem cells with Accutase to single cells, centrifuge, and resuspend in mTeSR1 + 10 μM Y-27632 solution. 3 × 10 cells / well were plated. 5 Cells were added to a 24-well plate at a density of 10 cells.

[0159] On days 1-3, cells were differentiated into endoderm, with 500 μL of culture medium replaced daily. The following sequence was used: RPMI1640 + NEAA + 100 ng / mL Activin A + 50 ng / mL BMP4 on day 1, RPMI1640 + NEAA + 100 ng / mL Activin A + 0.2% dFBS on day 2, and RPMI1640 + NEAA + 100 ng / mL Activin A + 2% dFBS on day 3.

[0160] Days 4-6 are for further endoderm differentiation. Change 500 μL of culture medium daily. The following sequence is used: RPMI1640 + NEAA + 2% dFBS + 200 ng / mL Noggin + 500 ng / mL FGF4 + 2 μM CHIR99021 on days 4-5; and RPMI1640 + NEAA + 2% dFBS + 200 ng / mL Noggin + 500 ng / mL FGF4 + 2 μM CHIR99021 + 2 μM RA on day 6.

[0161] ② Differentiate NE while differentiating PFG. NE differentiation process:

[0162] Day 0. Coat a 24-well plate with 1% Geltrex solution (solvent: DMEM / F-12 medium) (37°C, 1 hour). Aspirate the Geltrex solution and add 500 μL of day 0 medium (mTeSR1 + 10 μM Y-27632). Digest human pluripotent stem cells with Accutase to single cells, centrifuge, and resuspend in mTeSR1 + 10 μM Y-27632 solution. 1×10 cells per well. 5 Cells were added to a 24-well plate at a density of 10 cells.

[0163] On days 1-5, cells were differentiated into NE and 500 μL of culture medium N2B27 + 10 μM SB431542 + 0.1 μM LDN193189 were replaced every day.

[0164] On the 6th day, NE was digested into single cells and aggregated into spheres in a 24-well high-throughput U-shaped plate at a cell density of approximately 5 × 10 4 cells / cm 2 , so that the size of NE spheres is similar to that of PFG, and the culture medium is N2B27+10μM Y-27632.

[0165] ③ Pre-assemble NE and PFG spheres and continue culturing to form asymmetric gastric organoid assemblies:

[0166] On the 7th day, the culture medium in the 24-well high-throughput U-shaped well plate was replaced with basal intestinal culture medium (culture medium for days 7-10) and the collected PFG was added (estimate the number of 24-well high-throughput U-shaped wells, and try to make the NE:PFG ratio 1:1). Let it stand for 24 hours to allow NE and PFG to pre-assemble.

[0167] On the 8th day, wells with both NE and PFG were selected, and the preassembled NE and PFG spheres were collected under a stereomicroscope and coated with 18 μL of Geltrex gel droplets. The number of assembled spheres in a single gel droplet was less than 10, and the culture medium was basal intestinal culture medium (culture medium on days 7-10).

[0168] On days 10-12, the culture medium (basal intestinal medium + 100 ng / mL EGF + 50 ng / ml FGF10) was changed every 2-4 days.

[0169] From day 13 to day 15, the culture medium (basal intestinal medium + 100 ng / mL EGF) was changed every 2-4 days.

[0170] The morphology of gastric organoids during differentiation was observed on the 7th, 8th, 10th, 13th, and 16th days of culture. Figure 7 As shown in b, neurospheres and epithelial spheres successfully assembled on day 8 and co-developed to form multi-lineage gastric organoids on day 16.

[0171] Gastric organoids were collected on day 16 and subjected to immunofluorescence staining for gastric development-specific markers (GATA4, SOX2, and PDX1). Figure 7 As shown in Figure c, the gastric organoids generated by the method of this example express GATA4 and SOX2, and their PDX1 expression has the characteristic of anterior-posterior asymmetry, that is, it has a fundus-antrum pattern feature.

[0172] Example 3

[0173] The specific implementation is the same as Example 1, except that on days 0-2, the 24-well high-throughput U-shaped well plate in Example 1 can be replaced with a low-attachment culture dish (such as Corning #3261) to allow cells to be cultured in suspension and to form EBs.

[0174] Taking asymmetric gastric organoids as an example, the culture medium used is shown in Table 1:

[0175] Prior to day 0, human pluripotent stem cells (iPS18) were cultured using a feeder-free method. The culture medium used was mTeSR1. During passaging, cells were digested with Dispase and passaged as cell clumps. Culture plates were coated with 1% Geltrex solution (in DMEM / F-12 medium) at 37°C for 1 hour.

[0176] Day 0. Stem cells were digested into single cells with Accutase, centrifuged and resuspended in mTeSR1 + 10 μM Y-27632 solution. 3×10 cells per well 5 Cells were added into 24-well low-attachment culture dishes at a density of 10 cells.

[0177] Days 1-3: Differentiation towards endoderm. Change 500 μL of culture medium daily. On day 1, stem cells form EB spheres. Aspirate the medium in the low-attachment culture dish and replace with the day 1 culture medium RPMI1640 + NEAA + 100 ng / mL Activin A + 50 ng / mL BMP4. On day 2, coat a 24-well plate with 1% Geltrex solution (solvent is DMEM / F-12 medium) (37°C, 1 hour). Resuspend the EB spheres in 500 μL of day 2 culture medium RPMI1640 + NEAA + 100 ng / mL Activin A + 0.2% dFBS per well and seed the plate into the coated 24-well plate. On day 3, replace with RPMI1640 + NEAA + 100 ng / mL Activin A + 2% dFBS.

[0178] Days 4-6: Further endoderm differentiation. Change 500 μL of culture medium daily. On days 4-5, change to RPMI1640 + NEAA + 2% dFBS + 200 ng / mL Noggin + 500 ng / mL FGF4 + 2 μM CHIR99021; on day 6, change to RPMI1640 + NEAA + 2% dFBS + 200 ng / mL Noggin + 500 ng / mL FGF4 + 2 μM CHIR99021 + 2 μM RA. Collect gastric organoid precursors on day 7. Aspirate the culture medium into a centrifuge tube and let it sit for 15 minutes. The organoid precursors will settle to the bottom of the tube. Carefully aspirate approximately 2 μL of the cell spheres and quickly mix with 18 μL of Geltrex. Drop the Geltrex mixed with cell spheres in the center of the bottom of a 48-well plate, let it stand at 37°C for 5-15 minutes to solidify, then add culture medium (basal intestinal culture medium + 100 ng / mL EGF + 50 ng / ml fibroblast growth factor 10 + 200 ng / mL Noggin + 2 μM RA) and culture until the 10th day.

[0179] On days 10-12, the culture medium (basal intestinal medium + 100 ng / mL EGF + 50 ng / ml fibroblast growth factor 10) was changed every 2-4 days.

[0180] From day 13 to day 15, the culture medium (basal intestinal medium + 100 ng / mL EGF) was changed every 2-4 days.

[0181] Example 4

[0182] The specific implementation method is the same as Example 1, except that the iPS18 cell line is replaced by the H1 or iPSB1 cell line. The results are as follows Figure 6As shown, according to the method of Example 1, the H1 or iPSB1 cell lines can also produce asymmetric gastric organoids, and the generated gastric organoids also have neuroepithelial and neural crest cells.

[0183] Comparative Example 1

[0184] According to the traditional culture method described in the article published by Broda et al. (TR Broda, KW McCracken, JM Wells. Generation of human antral and fundic gastric organoids from pluripotent stem cells. Nat. Protoc. 2019, 14, 28. It is incorporated herein by reference. Figure 1 On the 7th day of culture, gastric organoid precursors were collected and immunofluorescence staining of endoderm-specific markers FOXA2 and GATA4 was performed. Figure 8 (Left) As shown, gastric organoid precursors obtained by traditional methods only have a PFG epithelial cavity of a single cell lineage and lack an inner cell mass.

[0185] Gastric organoids were collected on the 16th day of culture and subjected to immunofluorescence staining for gastric-specific markers GATA4, SOX2, and PDX1. Figure 4 As shown in (below), gastric organoids obtained by traditional methods only have fundic organoids (GATA4+ / SOX2+ / PDX1-) or antral organoids (GATA4+ / SOX2+ / PDX1+) with a single cell lineage, and do not have the fundic-antral pattern with multiple cell lineages and asymmetric anterior-posterior axis of the stomach.

[0186] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0187] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Use of neural cell precursor cells in the generation of asymmetric gastric organoids in vitro, wherein: Using neural progenitor cells to promote the formation of asymmetric gastric organoids from gastric organoid precursors; The asymmetric gastric organoid comprises gastric fundus tissue and gastric antrum tissue.

2. The use according to claim 1, wherein The gastric organoid precursors are differentiated from pluripotent stem cells; Optionally, the gastric organoid precursor comprises a spherical gastric organoid precursor; preferably, the spherical gastric organoid precursor comprises a posterior foregut spheroid; Optionally, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells.

3. The use according to claim 1 or 2, wherein The neural cell precursor cells can be added exogenously or generated endogenously; Optionally, the neural cell precursor cells include neuroepithelial cells, neural crest cells, or multi-lineage cells having the potential to differentiate into neuroepithelial cells and / or neural crest cells.

4. The use according to claim 3, wherein The exogenous addition is to contact the neural cell precursor cells generated by in vitro culture with the gastric organoid precursors.

5. The use according to claim 3, wherein The endogenous generation is achieved by suspension culture of pluripotent stem cells to generate neural progenitor cells within differentiated gastric organoid precursors.

6. A method for generating asymmetric gastric organoids in vitro, wherein: Promoting the formation of asymmetric gastric organoids from gastric organoid precursors through neural progenitor cells; The asymmetric gastric organoid has gastric fundus tissue and gastric antrum tissue.

7. The method according to claim 6, wherein: The gastric organoid precursors are differentiated from pluripotent stem cells; Optionally, the gastric organoid precursor comprises a spherical gastric organoid precursor; preferably, the spherical gastric organoid precursor comprises a posterior foregut spheroid; Optionally, the pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells; Optionally, the neural cell precursor cells include neuroepithelial cells, neural crest cells, or multi-lineage cells having the potential to differentiate into neuroepithelial cells and / or neural crest cells.

8. The method according to claim 6 or 7, wherein: The method comprises: (A) culturing pluripotent stem cells adherently and differentiating them into gastric organoid precursors, and then contacting neural cell precursors with the gastric organoid precursors to form asymmetric gastric organoids; or, (B) culturing the pluripotent stem cells in suspension in a growth medium to differentiate into gastric organoid precursors, wherein the gastric organoid precursors contain neural cell precursor cells; Optionally, the suspension culture comprises culturing in a U-shaped well plate or a low-adhesion cell culture dish.

9. The method according to claim 7 or 8, wherein The pluripotent stem cells are contacted with one or more of activin A, an agent that inhibits the BMP signaling pathway, an agent that activates the FGF signaling pathway, an agent that activates the WNT signaling pathway, and retinoic acid to form gastric organoid precursors.

10. The method according to claim 8 or 9, wherein: Asymmetric gastric organoids are formed by contacting gastric organoid precursors with neural cell precursors or generating neural cell precursors within gastric organoid precursors and then contacting them with one or more of EGF, an agent that activates the FGF signaling pathway, Noggin, and retinoic acid.

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